SUSTAINABILITY

MARIA J. CASTRO1, CARLOS E. LÓPEZ1, RAJESWARI NARAYANASAMY2, JOLANTA E. MARSZALEK1,

MIRIAM P. LUEVANOS-ESCAREÑO1, GERARDO J. FAJARDO3, NAGAMANI BALAGURUSAMY1*
*Corresponding author
1. Laboratorio de Bioremediación, Facultad de Ciencias Biológicas, Universidad Autónoma de Coahuila,
Carretera Torreón-Matamoros km 7.5 Torreón Coahuila, México
2. Facultad de Ingeniería, Ciencias y Arquitectura de la Universidad Juárez del Estado de Durango
3. Facultad de Ingeniería Civil de la Universidad Autónoma de Nuevo León

Nagamani Balagurusamy

Potential of enzymes
(urease and carbonic anhydrase)
for a sustainable construction industry

KEYWORDS: Urease, carbonic anhydrase, biomineralization, bioconcrete, biocement, biodeposition.

Abstract Concrete is the most commonly used material of numerous structures around the world due to resistance,
durability and low cost compared with other construction materials. However, traditional construction
materials are susceptible to suffer deterioration by physical, chemical and biological factors that produce irreversible damage to their
structure, which requires a high-cost of repair and maintenance. Additionally, during production process cement industry emits about 0.73
– 0.99 t CO2/ t of cement produced. Bioconcrete is emerging as an ecological, economical and sustainable alternative for construction
industry. Biomineralization by bacteria facilitates the development of bioconcrete, wherein calcium carbonate is formed by the metabolic
activity of microorganisms, which involves a series of complex reactions directed mainly by urease and carbonic anhydrase enzymes. In
this review, biomineralization processes involved in bioconcrete formation and their potential use for the construction industry is discussed.

INTRODUCTION As a consequence of their self-repair character, CO2

emission could also be minimised. Bioconcrete production
Concrete is widely used as construction material around includes biomineralization process, which involves the
the world due to its resistance, durability and low cost formation of calcium carbonate by metabolic activity of
in comparison with other construction materials (1,2). microorganisms, where two enzymes; urease and carbonic
However, it is susceptible to undergo deteriorations due anhydrase play a vital role (8,9). Role of urease enzyme in
to diverse physico-chemical and biological factors (3,4). the biomineralization process is reviewed here.
These factors induce formation of cracks in internal
structure of concrete causing irreversible damage. It has
been estimated that the cost of repair and maintenance BIOMINERALIZATION
is around $147/m3 of concrete, despite the fact that cost
of concrete production ranges between $65 and $80/ Biomineralization is a complex process involving metabolic
m3 (5). Cement is the most important component of the activity of diverse organisms in the formation of minerals,
concrete since it provides properties of compaction, which are termed as “Biominerals”. Metabolic activity of
and with growth of modernization and industrialization microorganisms has been responsible in large part for the
demand for cement has been increasing. The demand for deposition of minerals throughout the history of the Earth
cement in 2006 was about 2540 million tons (Mt), which is (10, 11). At the end of the precambrian era and at the base
predicted to increase between 3680 (low estimate) and of the Cambrian era, about 540 million years ago, various
4380 Mt (high estimate) in 2050. This increase in activity microorganisms evolved with ability to generate most of the
of the construction industry has generated a negative 64 known minerals, and the composition of biominerals varied
impact on the environment. Concrete industry emits 0.73 according to the metabolic diversity of microorganisms (12).
– 0.99 t CO2/ t of cement produced (6), which accounts Carbonates, phosphates, silicates, sulphates, sulphides,
for about 5-7% of global CO2 emission (2, 7). Recently, civil oxides or hydroxides form biominerals along with variety of
and environmental engineering researchers are working cations such as calcium, iron, magnesium, and manganese.
on the development of sustainable building materials with Biominerals can also be formed by organic macromolecules;
extended useful life. Bioconcrete is being reported as such as proteins, polysaccharides, glycoproteins and
one of the novel materials showing increased resistance, proteoglycans, which also aid in structural formation,
durability, apart from self-repair properties. protection and sensory detection (13, 14).

56 Chimica Oggi - Chemistry Today - vol. 34(4) July/August 2016

Biomineralization of CO2 into magnesium- and/or calcium- UREASE (UE)
carbonate (15) is one of the most abundant biominerals in our
earth (16), and this is because calcium intervenes in various Urease (EC [Link]) is a metalloenzyme containing nickel
bacterial metabolic activities (12). Calcium biominerals belonging to the group of hydrolases classified as urea
group comprises 50% of the known calcite biominerals amidohydrolase. Urease plays an important role in nitrogen
(CaCO3), and constitute 4% of the earth crust (12, 17). requirements of various organisms including bacteria, fungi,
Biomineralization can be divided into two main algae and plants (23). Urease catalyses the hydrolysis of
mechanisms; biologically controlled mineralization (BCM) urea into ammonia and CO2. As a consequence, there
and biologically induced mineralization (BIM) (8). Another is considerable increase in pH and CO2 concentration in
mechanism, biologically influenced mineralization (BIFM) the microenvironment of the bacteria. One mole of urea is
was suggested in 2009 (18). BCM can occur by extracellular hydrolysed to 1 mole of carbamate and 1 mole of ammonia
(BCMe), intercellular (BCMi) and intracellular (BCMin) (eq.1) carbamate is spontaneously hydrolysed to form one
means, where microorganisms have control on grade of additional mole of ammonium and carbonic acid (eq.2),
nucleation, growth and deposition site of biominerals (8, 19). which are balanced in solution to form bicarbonate (eq.3),
Bacterial cells with BCMe mechanism produce a molecular 2 moles each of ammonia and hydroxide (eq.4) which
matrix of macromolecules such as proteins, glycoproteins, increases pH, and shifts the balance of bicarbonate into
proteoglycans and polysaccharides, which facilitates carbonate (eq.5). As a consequence, there is high influx
nucleation and deposition of biominerals (8, 11). In BCMi, of calcium ions and excessive expulsion of protons, which
the microorganisms coexist in biofilms, and the epithelial obligates the bacteria to export calcium outside the cell
surfaces of cells are used as the organic substrate leading and compensate for loss of protons for their survival. In this
to nucleation and formation of biominerals. On large areas process, there is availability of dissolved inorganic carbon
of cell surface exoskeleton is formed to protect structures. and calcium ions in the microenvironment, which results
BCMin occurs within specialized vesicles or vacuoles that in the precipitation of calcium carbonate outside the cell
lead directly to the nucleation of biominerals intracellularly. (eq.6 and 7) (Figure 1) (11).
In this mechanism, the microorganisms have a high degree
control over concentration of cations and anions that eq.1
constitute biominerals in a microenvironment in which
organic matrix is used as a site of nucleation. Mineralized eq.2
structures by this mechanism present intrinsic morphologies,

specific for each species (12). An example is formation of eq.3

nano-crystals ferromagnetic such as magnetite or greigite,
resultant product of interaction between biopolymers and eq.4
proteins. These minerals are connected to intracellular
structures known as magnetosomes that are synthesized eq.5
by a diverse group of bacteria involved in the iron cycle
called “magnetotactic bacteria” (19, 20). BIM occurs only eq.6
by extracellular means as a result of interaction between
metabolic activity of organisms and the environment. eq.7
In this process, microorganisms have limited control on
nucleation, growth and deposition of biominerals. These
biominerals are characterized by their wide range in size of
particulates, poor crystallinity and morphology (21).
This mechanism of biomineralization is favoured by
different biological surfaces such as membrane, cell walls,
exopolymers, biofilms and even inactive spores, which
function as a site of nucleation and growth of crystals
through ions adsorption (21, 22). Moreover, precipitation
of biominerals induced in some cases is dependent on
the chemistry of exterior environment of the cell (16, 22).
Microbially induced calcium-carbonate precipitation
(MICP) is widely present in bacteria and takes place in
different environments such as soil, fresh water, water and
marine sediments. Calcite, aragonite and vaterite are
some of the most representative biomineral polymorphs
with different types of crystallization such as rhombohedral,
orthorhombic and hexagonal (16). Urea hydrolysis,
photosynthesis, denitrification, sulphate reduction, Figure 1. Schematic illustration of MICP by urease activity.
oxidation of organic acids and methane oxidation are
the major metabolic pathways involved in MICP, and they
occur in interaction with calcium (5). Of these pathways, Among soil bacteria, urease activity is widely reported in
urea hydrolysis is less complex, where the precipitation of Sporosarcina pasteurii, previously known as Bacillus pasteurii
calcite occurs by production of carbonate ions in presence (11), Bacillus sphaericus and Bacillus cereus (24, 25). Apart
of ammonia through a series of complex biochemical from urease, carbonic anhydrase also plays a role in MICP,
reactions that are directed by the enzyme urease (17). although their role has not yet been studied in detail.

Chimica Oggi - Chemistry Today - vol. 34(4) July/August 2016 57

CARBONIC ANHYDRASE (CA) 1.125 and 1.018 mM [Link]-1 respectively (30).
CA and urease activity of B. megaterium was 115 [Link]-1
Carbonic anhydrase (EC.[Link]) is a metalloenzyme, with zinc and 690 [Link]-1 respectively (31). These data clearly
content that catalyses the inter-conversion of CO2 and HCO3– demonstrates the wide range in the metabolic activity
(eq.8). In this process, H+ ions are generated, which promotes of different bacteria, which depends on environmental
the precipitation of carbonate as calcium carbonate (eq.9) conditions and thus their potential use will have that
by the presence of calcium ions. dependency too.

eq.8 In general, the process of biocementation depends on six

fundamental concepts; (a) activity of urease, (b) activity
eq.9 of carbonic anhydrase, (c) concentration of calcium,
(d) concentration of dissolved inorganic carbon (DIC),
CA is also produced by prokaryotes and eukaryotes as it is (e) pH and (f) the availability of nucleation sites. Calcium
essential in many biological processes such as respiration, carbonate particles adhere to the cement, creating a
CO2 acquisition, ions transport, acid-base balance, solid mass, and in this process the biominerals can be
mineralization and photosynthesis. Activity of CA promotes deposited evenly in inter-granular spaces, reinforcing
the formation calcium carbonate (26). Role of carbonic properties of compaction (8). It is observed that the
anhydrase in biomineralization was reported by Hwang and cells tend to encapsulate as a means of protection
coworkers (27) and they observed that the morphology under unfavourable conditions due to increase in pH
of calcium carbonate due to microbial precipitation generated by concrete matrix. The encapsulation limits
is dependent on two parameters; constant pressure of the transfer of nutrients and therefore leads to inactivity
CO2 and addition of polymers involved in growth and and even death of bacteria (32, 33). Nonetheless, it is
nucleation (Figure 2). possible that cells can wake up from their vegetative
state and regain their metabolic and urease and CA
activities, as and when favourable conditions return in the
microenvironment. Bacterial spores were encapsulated
and their potential activity in self-healing of fractures
was studied (32). It was observed that self-healing was
48-80% higher than specimens without encapsulated
spores. They further observed that with adequate physico-
chemical conditions, bacterial spores germinated and
recorded urease activity. However the cell encapsulation
can also passively influence the biomineralization, as
microorganisms can serve as a nucleation site for the
deposition of calcite (9, 33). This passive process is known
as biologically influenced mineralization (18). Although,
this mechanism is similar to MICP, live bacteria is not
necessary for the formation and deposition of these
biominerals (18).

Figure 2. Schematic illustration of CaCO3 biomineralization by

carbonic anhydrase (27). BIODEPOSITION

The precipitation and biodeposition of calcium

BIOCEMENT carbonate has been applied as a novel technique for
the remediation of construction materials and is known as
MICP favors the formation of biocement, which is dependent “Calcite Bioconcept” (34). Calcite Bioconcept is based
on the activities of urease and carbonic anhydrase (eq.10). mainly on the ability of a bacterial cell to precipitate
Interdependence of both enzymes in this process can be calcium carbonate minerals on its cell surface and
explained by the fact that urease activity is dependent thereby heal the fractures formed in the structure of the
on the incorporation of nickel in their active site, which is costructions materials. This concept was first applied in
regulated by the interconversion of CO2/HCO3 catalyzed by 1990 for the protection of ornamental stones. However,
carbonic anhydrase (11, 28). the efficiency of this technique is widely being studied
under laboratory conditions in the last decade by way of
eq.10 incorporating different microorganisms (34, 35).
This technique was applied for the remediation and
It has been observed that different soil bacteria protection of Euville limestone material obtained from the
showed different kinetic values of urease and carbonic mixture of fossilized crinoids and equinoides, joined by
anhydrase activity. Km and Vmax of urease in free cells of crystals of calcium carbonate (36). Two ureolytic bacteria,
Sporosarcina pasteurii was 17.30 mM and 1.57 mM min−1. Bacillus sphaericus and Bacillus lentus were incorporated
mg−1 respectively and the immobilized cells recorded a into limestone cubes of various dimensions and were
Km of 22.3 mM with a Vmax of 0.73 mM min−[Link]−1 (29). studied for deposition of calcite on agar plates, capacity
Whereas, Km of free and immobilized, purified CA enzyme for urea hydrolysis, production of extracellular polymeric
from Bacillus pumilus was 1.211 and 4.547 mM with Vmax of substances, biofilm formation and ʆ- potential.

58 Chimica Oggi - Chemistry Today - vol. 34(4) July/August 2016

They reported that bacterial colonies of both tested strains 5. Seifan M., Samani A. K., & Berenjian A. Appl Microbiol
showed crystal aggregates in the range of 75 to 95% of Biotechnol, 100(6), 2591-2602 (2016).
surface, with urea hydrolysis in the range of 0.82 ± 0.04 g 6. Hasanbeigi A., Price L., & Lin E., Renew Sustainable Energy
Rev, 16(8), 6220-6238. (2012).
to 4.07 ± 0.15 g NH4- N l-1, production of extracellular
7. Ivanov V., Chu J., & Stabnikov V. Bacteria for concrete
polymeric substances in the range of 0.08 ± 0.01 to 0.12 ±
surface treatment, Chapter 15, in Handbook of Basics of
0.02, biofilm formation in the range of 1.4 ± 1.0. 102 to 3.2
construction microbial biotechnology, Edited by Pacheco F.,
± 0.6. 102 [Link]-2 and with ʆ- potential at pH 9 was in Labrincha J., Diamanti C., Yu C., Lee H., In Biotechnologies
the range of 13.53 ± 0.72 to 27.78 ± 1.73 mV. Further, they and biomimetics for civil engineering Springer International
concluded that Bacillus sphaericus demonstrated higher Publishing (2015).
ureolytic activity, in addition to decrease in capillary 8. Achal V., & Mukherjee A. Constr and Build Mater, 93, 1224-
water absorption by this building material than [Link]. 1235 (2015)
This group of ureolytic bacteria (Bacillus sphaericus) was 9. Bundur Z. B., Kirisits M. J., & Ferron R. D. Cem Concr Res, 67,
tested for biodeposition in the remediation of fractures, 237-245 (2015)
10. Achal V., Mukherjee A., Kumari D., & Zhang Q. Earth-Sci.
and was recorded that the incorporation of ureolytic
Rev,148,1–17 (2015).
bacteria [Link] in sol-gel with calcium (Cl 2Ca or
11. Jimenez-Lopez C., Jroundi F., Rodríguez-Gallego M., Arias J.
Ca (NO3) 2) showed effective results in the remediation
M., & Gonzalez-Muñoz M. T., Biomineralization Induced by
of cracks of 0.1 to 0.6 mm size and further reduced the Myxobacteria, in Communicating Current Research and
permeability (37). The effect of different temperatures Educational Topics and Trends in Applied Microbiology,
on limestone conservation and urea hydrolysis by Bacillus Formatex, Microbiology Series, 1, 1(2007)
sphaericus, Sporosarcina ureae, and Sporosarcina 12. Weiner S., & Dove P. M., Rev Mineral Geochem, 54(1), 1-29
psychrophila was studied (38). Among the strains tested, (2003).
[Link] demonstrated ureolytic activity at all 13. Ghosh S., Biswas M., Chattopadhyay B. D., & Mandal S.,
temperatures tested, while S. pasteurii showed highest Cement concrete comp, 31, 93–98 (2009).
14. Fu G., Valiyaveettil S.,Wopenka B., & Morse D. E.,
activity at 37 °C and S. psychrophila recorded high
Biomacromolecules, 6(3), 1289-1298 (2005)
activity at 10 and 20 °C. These authors concluded that
15. Kim I. G., Jo B. H., Kang D. G., Kim C. S., Choi Y. S., & Cha H. J.
[Link] is the most suitable ureolytic bacteria for
Chemosphere, 87(10), 1091-1096 (2012).
biodeposition applications under different temperature 16. Perito B. & Mastromei G. Molecular basis of bacterial calcium
conditions. It has been shown that biodeposition is carbonate precipitation, Chapter 5, in Molecular
effective in remediation of the porosity of the structures, Biomineralization. Edited by Werner E. G., Springer Berlin
in particular to increase resistance and to reduce the Heidelberg (2011).
permeability of water and aggressive gases, and thereby 17. Achal V., & Pan X. Curr Microbiol, 62(3), 894-902 (2011).
aids in effective bioremediation of materials used by 18. Dupraz C., Reid R. P., Braissant O., Decho A. W., Norman R. S.
construction industry (36, 37, 38). & Visscher P., T. Earth-Sci. Rev, 96(3), 141-162 (2009).
19. Prozorov T., Sem Cell Dev Biol, 46, 36-43 (2015)
20. Li K., Chen C., Chen C., Wang Y., Wei Z., Pan W., & Song T.,
Enzyme Microb Tech, 72, 72-78 (2015).
CONCLUSIONS
21. Frankel R. B. & Bazylinski D. A. Rev. Mineral. Geochem, 54(1),
95-114 (2003)
Urease and carbonic anhydrase play an important role 22. Gray N. D. Berlin Heidelberg (299-309). (2006).
in the production of bioconcrete, biocementation and 23. Khan A., Hashim J., Arshad N., Khan I., Siddiqui N., Wadood A.
biodeposition. All these emerging novel technologies & Choudhary M I. Bioorg Chem, 64, 85-96 (2016).
offer an economical, ecological and suitable alternative 24. Rodriguez-Navarro C., Rodriguez-Gallego M., Chekroun K. B.,
over the traditional methods used by the construction & Gonzalez-Munoz M. T. Appl Environ Microb, 69(4), 2182-2193
industry. Further, since construction materials are vulnerable (2003).
25. Van Tittelboom K., De Muynck W., De Belie N. & Verstraete W.
to several factors leading to fractures and irreversible
In 1st WTA International PhD Symposium: Building materials
damages, these novel technologies apart from promoting
and building technology to preserve the built heritage
self-healing, can offer other benefits such as reducing
International Association for Science and Technology of
the permeability of substances and aggressive gases, Building Maintenance and the Preservation of Monuments
increasing resistance and thereby extending the utility life (WTA), 33, 439-457 (2009).
of construction materials. In addition, application of these 26. Bharti R. K., Srivastava S. & Thakur I. S. Appl Environ Microbiol,
technologies promise to reduce CO2 emissions and mitigate 2(1), 31-39 (2014).
effects associated climate change. 27. Hwang E. T., Gang H. & Gu M. B. J Biotechnol, 168(2), 208-211
(2013).
28. Wong L. S. J Clean Prod, 93, 5-17 (2015)
29. Bachmeier K. L., Williams A. E., Warmington J. R. & Bang S. S. J.
REFERENCES
Biotechnol, 93(2), 171-181 (2002).
30. Yadav R., Wanjari S., Prabhu C., Kumar V., Labhsetwar N.,
1. Khaliq W., & Ehsan M. B. Constr Build Mater, 102, 349-357
Satyanarayanan T., Kotwal S. & Rayalu S. Energy Fuels, 24,
(2016).
6198–6207 (2010).
2. Siddique R., Nanda V., Kadri E. H., Khan M. I., Singh M., &
Rajor, A. Constr Build Mater, 106, 461-469 (2016).
3. Achal V., Pan X., & Özyurt N. Ecol Eng, 37(4), 554-559 (2011).
4. Al-Salloum Y., Abbas H., Sheikh Q. I., Hadi S., Alsayed S., & Readers interested in a full list of references are invited to visit our
Almusallam T. Saudi J Biol Sci, (2016). website at [Link]

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