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Biomineralization for sustainable construction – A review of processes and applications
Biomineralization for Sustainable Construction – A Review of Processes and Applications Varenyam Achal, Abhijit Mukherjee, Deepika Kumari, Qiuzhuo Zhang PII: DOI: Reference:
S0012-8252(15)00091-4 doi: 10.1016/j.earscirev.2015.05.008 EARTH 2120
To appear in:
Earth Science Reviews
Received date: Accepted date:
6 January 2015 11 May 2015
Please cite this article as: Achal, Varenyam, Mukherjee, Abhijit, Kumari, Deepika, Zhang, Qiuzhuo, Biomineralization for Sustainable Construction – A Review of Processes and Applications, Earth Science Reviews (2015), doi: 10.1016/j.earscirev.2015.05.008
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ACCEPTED MANUSCRIPT Biomineralization for Sustainable Construction – A Review of Processes and Applications
School of Ecological and Environmental Sciences, East China Normal University,
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Varenyam Achal1*, Abhijit Mukherjee1,2, Deepika Kumari3, Qiuzhuo Zhang1
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Shanghai 200241, China
Department of Civil Engineering, Curtin University, Bentley, WA 6102, Australia
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Xinjiang Key Laboratory of Environmental Pollution and Bioremediation, Xinjiang
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Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011,
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China
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Running head: Biomineralization for Sustainable Construction
Corresponding author: Varenyam Achal, PhD Associate Professor, School of Ecological and Environmental Sciences, East China Normal University, Shanghai 200241, China Email: [email protected] Phone: +86-21-54341138 Fax: +86-21-54341131 1
ACCEPTED MANUSCRIPT Abstract Modern civilization is facing the dichotomy of rapid development of infrastructure
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that makes concrete as most traded material on the earth other than water. However,
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the production of cement, key ingredient of concrete, releases roughly a tonne of CO2
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into the environment with each tonne of cement production. The environmental concerns and sustainability issues associated with cement and concrete necessitate
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alternative and better approach in the construction. Nature, on the other hand, has a
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plethora of examples of sustainable habitats such as coral reefs, silk webs and ant hills. Recent advances in biotechnology have great potential of emulating nature’s
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way of building in modern days infrastructures at a scale that would sustain increasing
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population. Further, many of the biological materials of nature, be it ceramics or
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polymeric composites formed in the process of biomineralization, provides basis for sustainable construction. This paper elaborates nature’s way of construction based on biomineralization and discusses the progress of different biological pathways for sustainable construction. Main milestones achieved have been identified and the effect of biological intervention on the properties of structural materials has been highlighted. Variety of applications of biomineralization based technology in the construction has been reported. The paper briefly documents the future directions of the technology.
Keywords: Biomineralization; Sustainable construction; Biocement;
Calcium
carbonate; Urease; Building materials 2
ACCEPTED MANUSCRIPT Contents 1. Introduction
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2. Nature’s ways of construction
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3. Alternate pathways for biocementation
3.2. Biologically induced mineralization
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3.2.2. Sulphate reducing bacteria
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3.2.1. Photosynthetic microorganisms
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3.1. Biologically controlled mineralization
3.2.3. Bacteria involving nitrogen cycle in mineralization
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4. Challenges
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3.4. Biocement
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3.3. Microbially induced calcium-carbonate precipitation (MICP)
4.1. Cost of the technology 4.2. Increasing yield
4.3. Alkaline conditions 4.4. Perceived harmful effects- chlorides 5. Structural properties 5.1. Strength 5.2. Water permeability 5.3. Chloride ion permeability 5.4. Reinforcement corrosion 6. Applications 3
ACCEPTED MANUSCRIPT 6.1. As a binder 6.2. As a cement admixture
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6.3. As a barrier layer
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6.4. As a repair system
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7. Economy analysis of biocementation
8.1. Archiving of microbial varieties
8.3. Life cycle assessment
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8.2. Local adaptation
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8. Future directions
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Acknowledgements
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9. Concluding Remarks
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8.4. Expanding application domains
References
1. Introduction
Development of modern infrastructure such as roads, bridges and buildings has proven to bring in economic prosperity. The emerging economies are now emulating the developed ones in rapidly building their infrastructure, resulting in the consumption of construction materials such as cement, brick and steel is at their record highs and it is predicted to grow for the next fifty years (Schnieder et al., 2011). As a result, the construction industry accounts for half of global resource usage, up to 40 percent energy consumption and up to 20 percent of the emission of 4
ACCEPTED MANUSCRIPT greenhouse gasses. Experts have expressed serious doubts about the sustainability of the present construction technologies. Development of an alternative method of
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construction that does not hinder the progress of infrastructure and at the same time
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does not cross the boundaries of sustainability would be a great step to avert the crisis.
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Nature has been building its habitats for millions of years in a sustainable way. It is worthwhile to examine the natural ways of building habitats and to what extent they
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can be emulated in engineering construction.
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Many building materials use huge quantities of energy and produce high volumes of CO2. Steel, brick and concrete are prime among them. However, in the last century
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use of concrete has surpassed all other building materials by a huge margin. Ordinary
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Portland Cement (OPC) is a vital construction material and also a strategic
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commodity (Vlasopoulos, 2010) with a market dominated by China, which is attributed to 57.3% of global consumption (CEMBUREAU, 2012). Despite the incremental improvements in process efficiency that have been adopted by the cement industry in recent years, OPC production is still responsible for around 6% of all anthropogenic global CO2 emissions. In the current global setting, building construction and operation results in 50% of all CO2 emissions worldwide. The push to reduce global CO2 emissions is backed by governments, corporations and citizens who understand that the present rate of release of greenhouse gases into the atmosphere is a serious threat to future life and prosperity on the planet. Cement never appears to become a sustainable material (Gerilla et al., 2007).
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ACCEPTED MANUSCRIPT Further, materials for infrastructure such as concrete, have been developed with a sole focus on strength believing that other functional requirements such as durability,
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thermal or hygral passivity would be automatically achieved. However, in reality,
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infrastructure materials have been found to degrade rather rapidly due to natural as
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well as anthropogenic causes. Often the situation is aggravated due to high CO2 concentration in the atmosphere. The aggressive elements enter the concrete that has
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direct impact on the corrosion of reinforcements (Pacheco-Torgal and Labrincha,
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2013). Corrosion leads to cracking and spalling of concrete that creates pathways for intrusion of deleterious materials. They affect other functional requirements such as
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hygral and thermal insulation. Synthetic agents such as epoxies and polyurethanes,
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water repellents such as silanes or siloxanes, and corrosion inhibitors such as cationic
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surfactants are often not compatible with their substrates and sometimes toxic. On the other hand, Nature has been building habitats such as ant hills, coral reefs and spider webs in a sustainable manner for millions of years. The materials are synthesized at ambient conditions (thus requiring very little energy) and they are optimized to multifunctional requirements. For example, the spider’s web needs to be resilient to be able to tangle the prey and stiff to maintain its shape. Thus, the silk of the web frame and drag lines is more than 1000 times stiffer than the catching spirals (Omenetto and Kaplan, 2010). Although the present technology cannot match the versatility of construction by nature, there are wonderful lessons to be learnt. Moreover, there are early indications of adaptability and scalability of some of the biological processes of nature in the construction industry (Achal et al., 2010a, 6
ACCEPTED MANUSCRIPT 2011a). This paper provides a critical review on nature’s ways of construction. As the
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discussion focuses on concrete, biocementation, its pathways and mechanisms, and
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its application in various building materials have been highlighted. A discussion on
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the economics of this novel biotechnology is provided. Challenges and future work
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2. Nature’s ways of construction
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on biocementation in construction are also outlined.
A large number of high-performance prototypes has been created by nature that
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humans can reverse engineer and in turn use as inspirations for creating synthetic
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products with similar superior performances (Ballarini and Buehler, 2013). There are
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inexhaustible sources of inspiration that come from nature, be it ceramic (tooth enamel, mollusk shell, spicules in sponges, diatoms), polymeric (arthropod exoskeleton, silk, plant cell walls), or fairly balanced composites (feathers, antlers, bones). Biological materials consist virtually of all composites utilizing basic components in different proportions and of a variety of structural architectures with high mechanical strength. Such materials are developed by nature by means of growth or biologically controlled self-assembly, adapting to the environmental conditions and using the most commonly found materials. These materials are composed of only about 10% of about 100 stable elements found in nature. They are notably light, restricted within the first two rows of the periodic table. The principal elements include hydrogen, carbon, oxygen, nitrogen, phosphorous, sulfur, silicon, and calcium. 7
ACCEPTED MANUSCRIPT Common metals such as iron, copper and aluminium are either absent or found in minute quantities, e.g. iron oxide in radular teeth of chiton (Saunders et al., 2009).
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Most likely, reason for absence of metals is that the high temperatures necessary for
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processing of metallic elements are not amenable natural organisms (Chen et al.,
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2012).
Biological organisms produce composites containing both inorganic and organic
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components in complex structures, organized in terms of composition and structure,
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which provide properties for multifunctional performance. Table 1 specifies the major components responsible for mechanical strength in biological materials for natural
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habitats. Coral reefs and bone consists of calcium phosphate and collagen,
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respectively. The main components of these biological materials are calcium
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carbonate, namely aragonite, and hydroxyapatite (Weiner and Addadi, 1997; Sikavitsas et al., 2001; Muller, 2011). Nature grants compressive strength in biological materials by inserting inorganic or mineral elements while the organic component attributes to the ductility. Such combination of strength and ductility leads to high energy absorption prior to failure (Meyers et al., 2008). The most common mineral components include calcium carbonate, calcium phosphate (hydroxyapatite), and amorphous silica. Biological materials are composites of an inorganic mineral phase with a biopolymer, formed in the process called biomineralization (Chen et al., 2012).
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ACCEPTED MANUSCRIPT Table 1: Major inorganic or organic component responsible for mechanical strength in biological materials Inorganic or organic component
Coral reefs
Calcium phosphate/hydroxyapatite
Spicules in sponges, Diatoms
Amorphous silica
Mollusk shells, Reptile eggs
Calcium carbonate/aragonite
Mollusks, Bird eggs
Calcium carbonate/calcite
Bone, Dentine, Tendons, Muscle
Collagen
Arthropod exoskeletons
Chitin
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Biological material
Fibroin
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Silk
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The nature’s way of construction attracts and stimulates scientists and engineers to
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develop building materials and structures with high mechanical strength that are adapted to the environment. Natural load bearing materials can be classified as tension
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elements and compression elements. The tension elements are mostly made of biopolymers. One of the best biological tension elements that inspire construction specialists is silk. The silk produced by orb-web-spinning spiders reveal mechanical properties that are superior to almost all natural and man-made materials. Silks have moduli of 0.003-10 GPa and tensile strengths of 50-1100 MPa (Wegst and Ashby, 2004). Such nature of silk fibre makes it a top choice for springs and outperforms the man-made spring. The superior properties are from two proteins, fibroin and sericin that form a semi-crystalline material with an amorphous region composed of disordered protein chains connected to protein crystals (Gosline et al., 1999; Meyers et al., 2008). This crystal constitutes layers of anti-parallel amino acid sequences, known as b-pleated sheets that provide high tensile strength. Ant hills, on other hand, 9
ACCEPTED MANUSCRIPT are an excellent example of how to achieve multifunctional capability of mechanical strength, thermal insulation and ventilation at a miniscule embodied energy and cost.
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The binder in it is mainly due to a cementing material provided by lignin.
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A large majority of the biological materials originates from biomineralization by
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which living organisms produce minerals, chiefly carbonate products. Mollusk shells are an excellent example of such high-performance biominerals. They are mainly
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consisted of calcium carbonate and evolved to incorporate various design strategies in
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the arrangement of this biomineral. Some of their species showed very high compressive strength due to mineralization. The maximum compressive strengths
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shown by Haliotis rufescens and Araguaia river clam are 540 MPa and 567 MPa,
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respectively (Menig et al., 2000; Chen et al., 2008). Mollusks are made-up of at least
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95% minerals containing calcium carbonate or calcite and aragonite, which exhibit about 3000 times higher toughness than calcite. The natural materials of high mineral contents provide mechanical strength in addition to weaker interfaces with intricate architectures that enhance toughening properties, for example in tooth enamel or nacre (Mirkhalaf et al., 2014). All arthropods that make the largest phylum of animals, are covered by a rigid exoskeleton that not only supports the body and resists mechanical loads, but also provides environmental protection and resistance to desiccation (Neville, 1975; Vincent and Wegst, 2004). The three main components of an exoskeleton are chitin, polysaccharide, structural proteins, and inorganic minerals, typically calcium carbonate, showing a high degree of mineralization (Meyers et al., 2008). The 10
ACCEPTED MANUSCRIPT minerals are mostly deposited within the chitin-protein matrix in the form of crystalline CaCO3.
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Corals provide us a great example of environment-friendly building materials. A
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coral reef is considered as a “carbonate factory” dealing with CaCO3 production and
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loss (Pomar and Hallock, 2008). Corals build reefs by natural interaction between CO2 and water that precipitates as CaCO3 after reaching equilibrium. During reef
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building, corals develop incredible ability to calcify and act as the most prolific
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mineralizer on the planet. Coral reefs give us an excellent example to develop low carbon building materials. They use CO2 as raw material and biomimicking them
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could lead to production of low energy cement, a most widely used building material.
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Therefore, these biological materials serve as a valuable source for research and as an
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inspiration for structural materials development. Calcium is the cation of prime for most organisms. The calcium-bearing minerals cover about 50% of known biominerals (Lowenstam and Weiner, 1989). The dominance of such calcium minerals led to the extensive usage of the term calcification, sometimes, instead of mineralization. The calcium carbonate minerals are the most abundant biogenic minerals, both in terms of the quantities produced and their widespread distribution among many different taxa (Lowenstam and Weiner, 1989). The polymorphs of calcium carbonate present in various biological organisms, which provide mechanical strength has been provided in Table 2.
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ACCEPTED MANUSCRIPT Table 2: Polymorphs of calcium carbonate in biological organisms Calcium carbonate mineral
Formula
Crustaceans cuticle
Calcite
CaCO3
Coral reefs
Calcite
CaCO3
Mollusks shells
Aragonite
Fish head
Aragonite
Echinoderms shells
Mg-calcite
Sea squirt spicules
Vaterite
Gastropods shells
Vaterite
Crustaceans cuticle
Amorphous
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Biological organism
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CaCO3
(Mg/Ca)CO3 CaCO3
CaCO3 CaCO3.nH2O
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CaCO3
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Another amazing phenomenon of natural mineralization is that although they
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consist of brittle materials, they exhibit remarkable fracture toughness. Despite the weakness of constituents present in highly mineralized bio-materials such as teeth,
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nacre, conch shell, glass sponge spicules, and arthropod cuticles; they result in outstanding performance, which led to development of bio-inspired strategies. It is observed that the microstructure of these materials consists of weakly bonded layers that absorb shocks and produce ductility by assembling the brittle constituents. Such precise microstructural assembly has inspired biomimetic designs such as a microstructurally engineered glass that is a few hundred times tougher than monolithic glass (Mirkhalaf et al., 2014). The design solutions available in Nature with finetuned mechanical properties have always inspired engineers in designing structures (Ehrlich, 2010). However, it has been a challenge to emulate them in engineering construction and manufacturing processes. Nature grows the structure through secretion or deposition of minute 12
ACCEPTED MANUSCRIPT quantities of the materials where every step is precisely controlled. Thus, manufacturing the material and the structure are not separate steps, while in
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engineering construction materials are manufactured separately and then used to build
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the structure. The major challenge is to find appropriate methods to combine the two
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processes. One promising route is in-situ synthesis and deposition of carbonate minerals using biological agents. In such a process, precise microstructural control
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should be possible. It also alleviates carbon emission by avoiding the energy intensive
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manufacturing process of materials such as cement and bricks, thereby attaining sustainability. There are several biological pathways based on biomineralization that
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have been explored.
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3. Alternate pathways for biocementation Cementation is a process whereby calcium carbonate either forms or deposits on a surface. In the process, they tend to attach to the substrate creating cementation. When it is the product of a biological process it may be called “biocementation”. In other words, biocementation is the product of ‘biomineralization’. Biomineralization is the process involving organisms in mineral formation as a result of cellular activity that promotes the required physico-chemical conditions for such a formation and growth to take place (Ben Omar et al. 1997). From an evolutionary point of view, this process generated from bacterial activity. The majority of the biominerals contain calcium as their main cation and carbonates in terms of their anions (Perito and Mastromei, 2011).
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ACCEPTED MANUSCRIPT This process has two fundamental types: 1) biologically controlled mineralization, and 2) biologically induced mineralization.
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3.1. Biologically controlled mineralization
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Biologically controlled mineralization (BCM) process can be extra-, inter- or
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intracellular. In extracellular BCM, the cell produces a macromolecular matrix (proteins, polysaccharides or glycoproteins) outside the cell in an area turn out to be
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site of mineralization. Biologically controlled intercellular mineralization process
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typically occurs in single-cell organisms in which the epithelial substrate governs the nucleation and growth of specific biomineral phases over large areas of cell surfaces.
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This type of mineralization is often seen in calcareous algae that nucleate and grow
controlled
intracellular
mineralization
is
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Biologically
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calcite perpendicular to the cell surface (Borowitzka, 1982; Borowitzka et al., 1974).
crystallization process that occurs within specialized vesicles that direct the nucleation of biominerals within the cell (Weiner and Dove, 2003). Many eukaryotes, prevalently tissue-forming multicellular ones, carry out this process (Lowenstam and Weiner, 1989; Mann, 2001), where cellular activity controls the process to a high degree and leads the nucleation, growth, morphology, and final location of the mineral (Decho, 2010). The mineral particles formed are synthesized or deposited intracellularly on or within organic matrices or vesicles in a specific location with respect to the cell. From biomineralization for sustainable construction point of view, biologically induced mineralization is more important and widely reported.
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ACCEPTED MANUSCRIPT 3.2. Biologically induced mineralization In biologically induced mineralization, microorganisms secrete metabolic
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products that react with ions or compounds in the environment resulting in the
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subsequent deposition of mineral particles (Frankel and Bazylinski, 2003). The
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formed bio-minerals, chiefly calcium carbonate, nucleate and grow extracellularly due to metabolic activity of the microorganism and subsequent biochemical reactions
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involving metabolic byproducts. This is a presumably unintended and uncontrolled
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consequence of metabolic activities. With the only exception of Achromatium oxaliferum, calcium carbonate deposition by bacteria has been generally considered to
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be induced, and the type of mineral produced is primarily dependent on environmental
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conditions (Ben Omar et al., 1997; Brennan et al., 2004; Rivadeneyra et al., 1994).
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Calcium carbonate (CaCO3) or calcite precipitation is a common phenomenon found in aquatic environments and soils (Castanier et al., 1999; Ehrlich, 1998), which can be induced by microbes (mainly bacteria). Calcite, aragonite, and vaterite are the major crystalline structural polymorphs of CaCO3 in bacterial systems, as well as in all biogenic systems (Ben Omar et al., 1997), with calcite as most represented isoform (thus, sometimes process is termed as microbially induced calcite precipitation) followed by aragonite and less stable, vaterite. Sulphate reduction, denitrification and urea hydrolysis are the three major metabolic pathways driving these precipitation processes. There are mainly three groups of microorganisms involved in the precipitation of calcium carbonate.
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ACCEPTED MANUSCRIPT 3.2.1. Photosynthetic microorganisms One group involving in calcium carbonate precipitation belongs to photosynthetic
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microorganisms such as cyanobacteria and microalgae, which are most active in
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aquatic environments (Mc Connaughey et al., 2000). In the biologically induced
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process, the mineral precipitates because the organisms change the chemical microenvironment of the water layer adjacent to the cell. In cyanobacterial
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photosynthesis, microorganisms use CO2 in their metabolic process (Eq. 1),
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HCO−3 is transported through the membrane and dissociates within the cell into CO2 and OH− (Eq. 2) (Dittrich and Obst, 2004). CO2 is removed by photosynthesis
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leaving an excess of OH− behind that is pumped out of the cell through the cell
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membrane, causing a rise in pH. The rise in pH causes the carbonic acid equilibrium
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to shift towards an increase in (CO2−3) (Eqs. 2 and 3) resulting in production of CaCO3 in the presence of calcium ion in the system (Eq. 4). CO 2 + H 2O → CH 2O +O 2 − 2
2− 3
2 HCO ↔ CO2 + CO
+ H 2O
CO32 − + H 2O ↔ HCO3− + OH − 2+
− 2
−
Ca + HCO + OH ↔ CaCO3 + 2 H 2O
(1) (2) (3) (4)
3.2.2. Sulphate reducing bacteria The sulphate reducing bacteria (SRB) that are ubiquitous, anaerobic, prokaryotes, morphologically and phylogenetically highly diverse, include species in the Domains Bacteria (δ-subdivision of Proteobacteria and Gram-positive group) and Archaea (Frankel and Bazylinski, 2003). They play a great role in the mineralization of organic matter in anaerobic environments and in the biogeochemical cycling of 16
ACCEPTED MANUSCRIPT sulphur; however, sulphate reduction dominates mineralization in sulphate rich environments.
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Sulphate reducing bacteria can reduce the sulphate generated from abiotic
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dissolution of gypsum (CaSO4.H2O) (Eq. 5) to H2S and HCO3- (Eq. 6) in the
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absence of oxygen. When the hydrogen sulphide degasses, it induces increase in pH (Castanier et al., 1999), resulting in CaCO3 precipitation (Eq. 7).
2− 4
2(CH 2O ) + SO 2+
− 2
−
− 3
(5)
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CaSO4 .H 2O → Ca 2 + + SO42 − + 2 H 2O
→ HS + HCO + CO2 + H 2O −
(6)
Ca + HCO + OH ↔ CaCO3 + 2 H 2O
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(7)
Desulfovibrio, a group of sulphate reducing bacteria, remove sulphates from
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gypsum in a process coupled to calcite production (Atlas and Rude, 1988) by a
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combination of dissolution–precipitation and diffusion processes. In brief, sulphate
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reduction by bacteria from gypsum releases calcium ions that react with carbon dioxide and results calcite formation. The reaction has been summarized in Eq. 8. 6CaSO4 + 4 H 2 O + 6CO2 → 6CaCO3 + 4 H 2 S + 2 S + 11O 2
(8)
In addition, some member of Desulfovibrio such as D. magneticus is known to produce extracellular particles of iron sulphide by mean of biologically induced mineralization while synthesizing intracellular crystals of magnetite via biologically controlled mineralization (Sakaguchi et al., 2002).
3.2.3. Bacteria involving nitrogen cycle in mineralization The third group of bacteria involving in nitrogen cycle is most important with respect to calcium carbonate precipitation in different environments. Such bacteria 17
ACCEPTED MANUSCRIPT produce an enzyme urease (urea amidohydrolase; EC 3.5.1.5), which induce the precipitation of CaCO3 by a series of complex biochemical reactions. This reaction
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requires urea as substrate while calcium source as chief agent for carbonate
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production. During bacterial urease activity, 1 mol of consumed urea contributes to 1
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mol of ammonia and 1 mole of carbamate (Eq. 9) which, on further hydrolysis, contributes to the accumulation of ammonia and carbonic acid (Eq. 10). These
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products subsequently equilibrate in water to form bicarbonate and 2 moles of
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ammonium and hydroxide ions (Eqs. 11 and 12) that results in increase in pH and ultimately shifts the bicarbonate equilibrium, resulting in carbonate ions formation
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(Eq. 13). Due to the increase in pH in the cell, a high extracellular calcium ion
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concentration and a low extracellular proton concentration is required for the
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secretion of carbonate ions. Under this situation, the bacterium undergoes stress due to the electrochemical gradient, which causes an influx of calcium accumulation and excessive expulsion of protons. Survival of bacteria under such condition depends on factors like active export of calcium and compensations of the lost protons. The energy required for these activations can be accomplished by the metabolic CO2, which further contributes to the increase in the level of dissolved inorganic carbon in the microenvironment that ensures the precipitation of the calcium carbonate (Hammes and Verstraete, 2002). High pH condition favours the formation of CO32– from HCO3– (Knoll, 2003). Finally, the carbonate concentration will increase, inducing an increase in supersaturation level leading to CaCO3 precipitation around the cell in the presence of soluble Ca2+ (Eqs. 14 and 15). 18
ACCEPTED MANUSCRIPT CO( NH 2)2 + H 2O → NH 2COOH + NH 3
(9)
NH 2COOH + H 2O → NH 3+ H 2CO3
(10)
H 2CO3↔ HCO3− + H +
(11)
2 NH 3+2H 2O ↔ 2 NH + 2OH − 3
+
+ 4
−
(12)
−
2− 3
+ 4
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+ 4
HCO + H +2 NH + 2OH ↔ CO + 2NH + 2H 2O Ca2+ +Cell → Cell − Ca2+ 2+
2− 3
− 3
(14) (15)
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Cell − Ca +CO → Cell − CaCO
(13)
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3.3. Microbially Induced Calcium-carbonate Precipitation (MICP) Calcium carbonate is the chief cementation material, which can be produced both
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by natural and artificial way by means of microbial metabolic activities. A schematic
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diagram of suggested bacterial calcium metabolism and subsequent CaCO3
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precipitation is presented in Fig. 1.
Fig. 1 Schematic presentation of suggested bacterial calcium metabolism and subsequent CaCO3 precipitation under high-pH and high-Ca2+ extracellular 19
ACCEPTED MANUSCRIPT conditions. Reprinted with permission from Hammes and Verstraete (2002).
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Briefly, MICP is a straightforward biochemical process governed by five key
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factors: (a) the calcium (Ca2+) concentration (to incorporate with carbonate), (b) the
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concentration of dissolved inorganic carbon (to complex with calcium ions, thus reducing calcium carbonate saturation enhancing the calcite precipitation), (c) the pH
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(to favour the formation of CO32– from HCO3–), (d) the availability of nucleation
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sites (to create a strong electrostatic affinity to attract cations and enables the accumulation of calcium ions on the surface of the cell wall), and (e) urease, nickel
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containing metalloenzyme (to enzymatic hydrolysis). Figure 2 shows the crystals of
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calcium carbonate deposited by MICP. Although urease activity is widespread
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among different groups of microorganism, it was mainly bacteria of Bacillus group, such as Bacillus pasteurii (later renamed as Sporosarcina pasteurii), which were shown to proliferate and express the urease genes (Hammes et al., 2003). Due to the presence of several negatively charged groups on the bacterial cell wall positively charged metal ions, here Ca2+, bind on the bacterial surfaces and subsequently reacts with anions (CO32–) and form calcium carbonate. MICP is a natural phenomenon under microbial calcium metabolism in which active calcium metabolism potentially creates unique precipitation conditions, and carbonate precipitation chemically favours bacterial survival and proliferation (Perito and Mastromei, 2011). Besides urease, there is another enzyme, carbonic anhydrase with very important role in MICP. However, the role of this enzyme has not been studied in detail. 20
ACCEPTED MANUSCRIPT Carbonic anhydrase (CA), EC4.2.1.1, is ubiquitously found in organisms, and is essential to many biological processes including photosynthesis, respiration, CO2
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and ion transport, calcification and acid-base balance (Smith and Ferry, 2000). It has
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a wide distribution and participates in all physiological processes that deal with CO2
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and HCO3 handling, such as cellular pH regulation, acid and ion transport (Achal
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and Pan, 2011).
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Fig. 2 Scanning electron micrographs showing calcium carbonate crystals precipitated by rod shaped Bacillus sp. CR2. Reprinted with permission from Achal and Pan (2014).
CA is a zinc-containing enzyme that can catalyse the inter-conversion of CO2 and HCO3−, which would then promote the CaCO3 precipitation with the help of urease. The incorporation of nickel into the active site of urease is dependent on CO2/ HCO3metabolism, which is in turn regulated by CA (Park and Hausinger, 1995). If HCO3− is the source of dissolved inorganic carbon (DIC), CA may catalyse its conversion into CO2, and the overall reactive equations are as follows (Li et al., 2010): 21
ACCEPTED MANUSCRIPT CA HCO3− + H + → H 2O + CO2 2+
− 3
(16)
+
− 3
Ca +2 HCO → CaCO3 + H + HCO → CaCO3 ↓ + H 2O + CO2
(17)
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When CO2 is the source of DIC, CA catalyzes its conversion into HCO3− with overall reactive equations as follows (Li et al., 2010):
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CA CO2 + H 2O → HCO3− + H +
(19)
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Ca 2 + + HCO3− → CaCO3 ↓ + H +
(18)
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Calcium carbonate precipitation is considered as one of the important biogenic
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carbonate precipitations due to their contribution in selective cementation by producing relatively insoluble organic and inorganic compounds intra- or
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extracellularly. Such a compound can serve as a cementitious material giving rise to
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the term “biocement”. Biocement has attracted much attention because it is a real
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‘green material’; relying on Microbially Induced Calcium-carbonate Precipitation (MICP) (Rong and Qian, 2012a). The technique is highly promising as a long term remediation tool for the enhancement of durability of building structures (Achal et al., 2011a, 2011b, 2012a; Bang et al., 2001; De Muynck et al., 2008a,b; Ghosh et al., 2005; Jonkers et al., 2010; Park et al., 2010; Ramachandran et al., 2001). The fundamental understanding of the process of MICP has emerged. The success of MICP has subsequently led to the exploration of this process in a variety of fields. It has been successfully applied in bioremediation of heavy metals and radionuclides (Achal et al., 2012b, 2012c; Fujita et al., 2004), enhancement of oil recovery from oil reservoirs (Nemati and Voordouw, 2003), and strengthening of sand columns (Achal et al., 2009a; De Jong et al., 2006; Qian et al., 2010). Multiple laboratories 22
ACCEPTED MANUSCRIPT have reported similar findings and established repeatability of the process in the laboratory. However, for the MICP to graduate as biocement it has to develop into an
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industrial product.
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3.4. Biocement
Biocement is the product of MICP that, in its commonest form, has three
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constituents, namely, alkalophilic microbes, substrate solution (urea) and calcium ion
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solution (Achal et al., 2013a; Rong and Qian, 2012a). Bacteria use the nutrient to grow cells, urea as a substrate to hydrolyze, and calcium as the energy source to
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form the bio-mineral. These constituents need to coexist with other materials of
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construction and perform their tasks. They can be used as the sole cementing agent
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or in combination with other cementitious materials. The constituents of biocement can be supplied in various forms. They may be converted into a lyophilized powder, tubular capsules or liquid (Achal et al., 2009a; De Belie and De Muynck, 2009; Wang et al., 2010). Existing structures and components can also be treated with the microbial solution in the form of a spray, coating or by dipping (De Belie and De Muynck, 2009; Ghosh et al., 2005). The biocementation process was chiefly experimented with one of the most common construction materials, i.e., sand by most of research groups (Achal et al., 2009a; Qian et al., 2010; Rong et al., 2012). Bio-sandstones were created by filling a tube with sand mixed with bacterial culture or later, by dripping the bacterial solution through the column. The sand in the tube formed a solid column in a few 23
ACCEPTED MANUSCRIPT days (Fig. 3). While estimating the precipitates in the sand column, Achal et al. (2009b) reported that CaCO3 constituted 24% of the total weight of the sand samples
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plugged by S. pasteurii, which was later improved by 33% with a mutant of S.
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pasteurii (Achal et al., 2011b). The strength of bio-sandstones was also measured,
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which could be up to 6.1 MPa (Rong et al., 2012). The encouraging results of biocementation in sand led researchers to think beyond the limited experiment and
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explore its use along with the existing construction materials. However, the existing
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construction materials throw several challenges.
Fig. 3 A typical set-up to prepare bio-sandstone 4. Challenges Although based on the laboratory experiments biocementation is promising, the technology faces various challenges for scaling up to industrial levels of production. 24
ACCEPTED MANUSCRIPT The foremost is cost of the process. Civil infrastructure requires materials in very large quantities. Thus, they are highly cost sensitive. The large quantity requirements
There must be exhaustive
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survive the tough environment of construction processes.
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also mandate that the yield from the technology must be high. The bacteria must also
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life cycle estimates to demonstrate the sustainability credentials of the technology. Above all, biological technologies are almost unknown to structural communities. A
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popular misconception is that all bacteria are harmful. Often the authors face
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questions from the construction community about the risk of endangering lives of the inhabitants because of the bacteria used in the material. This section briefly explains
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such challenges and some progresses in alleviating them.
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4.1. Cost of the technology
The main cost element in the technology is the nutrient. Biocementation process requires nutrients to grow calcium carbonate precipitating bacteria. The cost of the nutrient can be up to 60% of total operating costs (Kristiansen, 2001). The reported nutritional profile for calcium carbonate precipitating bacteria such as S. pasteurii suggested a high preference for protein-based media (Morsdorf and Kaltwasser, 1989). The nutrient media containing yeast extract, nutrient broth (NB), glucose, BactoCasitone, meat extract and peptone are widely used in biocementation (Achal et al., 2009b; De Muynck, 2009; Jonkers et al., 2010; Rodriguez-Navarro et al., 2003; Rong et al., 2012). The cost of such nutrients is estimated to be about US $250 (180 €) per kg, 25
ACCEPTED MANUSCRIPT which could generate around 13L of bacterial culture and produce 5.4×106 unit urease (Achal et al., 2010a). One unit of urease is defined as the amount of enzyme
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hydrolysing one micromole of urea per minute. To reduce this cost while producing
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similar or even higher amount of urease search for alternative inexpensive nutrient
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sources are imperative. Researchers have discovered several replacements of the standard nutrient media with industrial effluents. Many of the industrial effluents that
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have high concentrations of protein can be used as nutrients. Thus dual benefits of
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cost reduction and environmental protection can be achieved. Achal et al. (2009b) successfully replaced the standard nutrients by lactose mother liquor (LML),
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collected from dairy industry, as a medium for biocementation by S. pasteurii. They
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observed that LML is a good source of nutrients supporting bacterial growth, urease
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production and calcite precipitation. The process was further improved by replacing the standard medium with corn steep liquor (CSL), of corn wet milling industry (Achal et al., 2010a). They reported comparable growth rate of S. pasteurii and significantly higher urease production than nutrient broth or yeast extract media. A higher percentage of calcium carbonate was deposited in the sand columns prepared with bacterial cells grown in the medium containing CSL than the standard media. They also measured other performance tests such as strength, permeability and reinforcement corrosion of construction materials. All parameters exhibited significant improvement. It had an additional benefit of recycling a harmful industrial byproduct. This outcome opens up huge opportunities of adapting biomineralization with a host of protein rich wastes ranging from industrial effluents 26
ACCEPTED MANUSCRIPT to domestic sewage available all over the world.
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4.2. Increasing yield
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Biocementation process is highly dependent upon the yield of urease and calcium
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carbonate. However, the process is delicately balanced on the pH levels. It is envisaged that the biological process must coexist with the existing processes of
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cementation. Harsh environment of cement concrete, especially its pH of around 10,
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can make it difficult for the bacteria to survive and reduce the yield. Achal et al. (2009a) developed phenotypic mutants of S. pasteurii by UV
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irradiation to increase the yield. They compared the mutant with the wild-type with
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respect to growth at high pH, urease production and calcite precipitation. Their results
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showed growth ability of at pH range 6-10, while one of mutants Bp M-3 could grow at pH from 5 to 11, suggesting the efficacy of phenotypic mutants in enhancing calcite precipitation. A significant increase in the urease production and higher calcite amount in bio-sandstones were also observed in case of UV induced mutant of S. pasteurii. Further, they found that the mutant had no side effects and it was in fact enhancing the durability of concrete. Improvement of microbial strains for the overproduction of enzymes has been the hallmark of many other commercial processes. In this case too the improved strains can reduce the cost of the biocementation process with increased productivity, survivality and some other special characteristics. However, further research is essential to make a significant progress. 27
ACCEPTED MANUSCRIPT 4.3. Alkaline conditions The alkaline condition of cement due to high pH, which ranges from 10 to 13 (Lea,
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1970), makes hostile environment for the bacterial growth. This is one major
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challenge faced by the biocementation process. However, exposure of cement to
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atmospheric CO2 results in a carbonation reaction, which gradually reduces the surface pH (Ismail et al., 1993). In addition, because of the high pH of the cement,
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bacterial cells might initially be growing slowly and acclimatize to high pH conditions
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during the curing period. Upon cell growth, calcium carbonate would start precipitating on the cell surface and within the cement mortar matrix (Achal et al.,
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2010a). However, if calcium carbonate precipitating alkali-tolerant or alkalophilic
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bacteria can be found, the problem can be alleviated. Some structural components of
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the cell wall of such bacteria, such as teichuronopeptide, contribute to pH homeostasis at alkaline pH and favour bacteria to survive in alkaline environments (Aono et al., 1999).
In order to protect the bacterial cells from high pH of cement, some researchers encapsulated bacterial cells during experimentation. Bang et al. (2001) initiated such research by adopting an innovative immobilization technique for calcium carbonate precipitation in concrete during the biocementation process. They utilized polyurethane (PU) to immobilize the whole cells of B. pasteurii and introduced in the cracks of concrete as PU strips of 10 mm (diameter) × 50 mm (length) with different concentrations of bacterial cells. As a result of biocementation, the compressive strength of the cracked concrete specimens increased by 12% compared with ones 28
ACCEPTED MANUSCRIPT only remediated with PU. The electron micrographs showed calcite crystals throughout the PU matrices, with B. pasteurii embedded in calcite crystals (Fig. 4).
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The physicochemically versatile PU was shown to be an effective enhancement tool
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in MICP in concrete cracks. Such immobilization technique offers several advantages
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for concrete remediation, in which encapsulated cells retain high metabolic activities and are protected from adverse environmental changes in cement. Later, Bachmeier et
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al. (2002) studied the behaviour of the PU-immobilized B. pasteurii urease in calcite
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precipitation at varying temperatures. PU was found to protect the bacterial cells from the high pH of cement and also higher temperature without affecting calcite
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precipitation. The native bacterial isolates from highly alkaline cement should also be
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effective in biocementation in concrete. Achal et al. (2010b) isolated Bacillus sp. CT5
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from commercially available cement, which could sustain calcium carbonate ability at pH 10 within 16 hours and produced 670 U ml-1 of urease.
Fig.
4
Scanning
electron
micrographs
of
calcite
precipitated
by B.
pasteurii immobilized in PU. A. Cells associated with calcite crystals in PU matrices. B. A magnified section boxed in (A). Reprinted with permission from Bang et al. (2001) 29
ACCEPTED MANUSCRIPT Recently, Wang et al. (2012) investigated the possibility of using silica gel as the carrier for protecting the bacteria in cement. Their results showed that silica gel
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immobilized B. sphaericus exhibited a higher urease activity than the polyurethane
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immobilized bacteria, and hence, more CaCO3 precipitated in silica gel (25% by mass)
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than in polyurethane (11% by mass). Thus, there are two routes of ensuring biocementation in concrete- immobilizing the bacteria in a protective environment
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and developing alkalophilic strains. As more results get reported the apprehension of
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bacterial growth in highly alkaline environment is likely to recede.
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4.4. Perceived harmful effects- chlorides
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The most common construction material, concrete is liable to face attacks from
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chlorides. The excessive amounts and penetration of chloride in concrete come from external sources and occurs by various transport mechanisms depending on the exposure conditions. Structures in coastal areas, are widely exposed to airborne chlorides or in direct contact with seawater. A vast majority of concrete structures are reinforced with steel. Steel is susceptible to corrosion in presence of chlorides. Thus, chlorides are avoided in reinforced concrete. In biocementation process calcium chloride is often used as a source of calcium. High solubility of the salt makes it a preferred source. However, the chloride should not cause corrosion in steel. Also, for the technology to be effective in coastal regions the chloride resistivity of the bacteria must be examined. Some researchers have explored alternative sources of calcium such as calcium 30
ACCEPTED MANUSCRIPT nitrate, calcium lactate, calcium acetate and calcium glutamate. Calcium nitrate was used as an efficient calcium source for the deposition of CaCO3 by B. pasteurii in
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order to corrosion protection of cement-based building materials (Qian et al., 2009).
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While using calcium nitrate, in calcium carbonate deposits the main polymorph was
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calcite along with small quantities of vaterite. The CaCO3 precipitated on cement induced by urease greatly improved their surface permeability resistance and resist the
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attack of the acid (Qian et al., 2009). De Muynck et al. (2008a) obtained granular
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grains, presumably of vaterite with calcite crystals on the surface of cement mortars when B. sphaericus utilized calcium acetate as Ca source.
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In order to study biocementation to heal cement stone specimens of dimension 4
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cm3 with B. cohnii, Jonkers et al. (2010) exploited calcium lactate as calcium source
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that resulted into copious amounts of 20-80 µm sized CaCO3 precipitates on crack surfaces. The precipitation based on MICP due to bacterial metabolic conversion of calcium lactate was according to the Eq. 20. CaC6 H10O6 + 6O2 → CaCO3 + 5CO2 + 5 H 2O
(20)
Jonkers et al. (2010) concluded that the process of bacterial mineral formation from calcium lactate represents an alternative calcium source to the more commonly used calcium chloride. In contrast to the calcium chloride, metabolic conversion of calcium lactate does not result in excessive ammonia production what drastically increases the risk of reinforcement corrosion (Neville, 1996) and degradation of concrete matrix, particularly when further oxidized by bacteria to yield nitric acid (Diercks et al., 1991). Recently, Achal and Pan (2014) studied role of various calcium 31
ACCEPTED MANUSCRIPT sources in calcite precipitation and reported urease produced by Bacillus sp. CR2 with
ml-1 in calcium acetate and 389 U ml-1 in calcium oxide.
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calcium chloride was 432 U ml-1 compared to 418 U ml-1 in calcium nitrate, 401 U
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Recently, Xu et al. (2014) reported that the addition of a calcium source led to
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heterogeneous precipitation of CaCO3 crystals both on the biofilm and inside the pores of cement mortars, which played the main role in the coating system. They used
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calcium lactate and calcium glutamate as calcium source to study biocementation by
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B. cohnii. They observed larger thickness of the CaCO3 layer precipitated using calcium glutamate than that from calcium lactate. However, almost the same
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improvement of durability performance on cement mortars was obtained due to a
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calcium glutamate.
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lower porosity of the CaCO3 layer from calcium lactate compared with that from
Most calcium carbonate precipitating bacteria are resistant and active in the environment of high chloride contents. When bacteria were subjected to high salt (NaCl) concentration (0-10%), Bacillus sp. CT-5 was able to grow well at 10% NaCl amended media and other species of Bacillus, such as B. megaterium, B. subtilis and S. pasteurii showed good growth in carbonatogenesis media with 7 or 8% NaCl (Achal et al., 2010b). Most of the researchers reported the ability of bacteria in cementitious materials in the presence of chlorides (Achal et al., 2011a; De Muynck et al., 2008a; Rong and Qian, 2012). Thus, MICP remains largely unaffected in chloride rich environment and one should expect success of biocementation process in coastal and marine conditions. 32
ACCEPTED MANUSCRIPT Other than chlorides (Cl−), concrete structures are also exposed to corrosive substances in the environment such as SO42−, CO2 and atmospheric moisture.
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Maintenance and repair of such structures are important for their continuing function.
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Reddy et al. (2010) reported that at a cell concentration of 105 cells per ml Bacillus
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subtilis increases the concrete resistance to sulphuric acid attack, which was later optimized at 106 cells per ml by Afifudin et al. (2011). Biocementation improves the
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permeation resistance of cementitious materials by forming a protective layer of
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calcium carbonate on the surface of cement-based material with the aid of bacteria (Rong and Qian, 2012). Thus, there is no demonstrated susceptibility of biocement to
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harsh environmental condition.
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The choice of calcium source is an important decision as it ultimately affects cost.
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Calcium chloride seems to be a natural choice. However, there are apprehensions of the presence of chloride ion adversely affecting reinforced concrete due to corrosion. In order to alleviate that concern other calcium sources such as calcium nitrate, calcium lactate and calcium glutamate have been explored. Although the deposition of calcium carbonate was comparable with other salts and found maximum with calcium chloride. Results of investigation of reinforcement corrosion and durability with calcium chloride are reported in the next section.
5. Structural properties To be accepted, biocement must exhibit improvement in structural properties, both as a standalone cementitious agent and as a supplementary cementitious 33
ACCEPTED MANUSCRIPT material with other materials of construction. Thus, structural property tests for a range of functions are imperative. This section reports the structural performance of
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biocement. The quality of cementitious materials is mainly judged by parameters
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such as strength, resistance to moisture and chloride permeability and corrosion of
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reinforcement. To mitigate the risk of unknown consequences new materials are initially used in conjunction with other well known materials. Thus, their
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performance must be judged along with the established structural materials. A large
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number of researchers have reported the structural properties of biocement.
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5.1. Strength
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Biocement has been studied as a deposition on cement mortar and concrete
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(Achal et al., 2010a, 2011a; Ghosh et al., 2005). A cement mortar (or mortar) refers to a building material that is a mixture of cement, sand and water, while concrete contains aggregates in addition to substances that consist of cement mortar. These materials are frequently labelled by their strength. The results of compressive strength tests are generally utilized to establish whether the mortar or concrete mixture as delivered meets the requirement of the specified application. It is measured by subjecting the specimens to compressive forces in a Universal Testing Machine. The strength is calculated from the maximum load withstood by the sample before failure divided by the cross-sectional area resisting the load and expressed in units of psi (Pound-force per square inch) or MPa. Hydration of cement leads to gradual gain in strength. For a standard test, the strength is measured at 34
ACCEPTED MANUSCRIPT different time intervals after casting usually up to an age of 28 days. The strength at 28 days is considered for labelling the grade of the specimen (ASTM C 109-13).
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Biocement was first demonstrated to improve the compressive strength of
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cement mortar cubes by Ramakrishnan et al. (1998, 1999). Later, Ramachandran et
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al. (2001) investigated MICP technology in detail on mortars. They cast mortars of dimension 50.8 mm × 50.8 mm × 50.8 mm that contained 240 g cement, 660 g sand
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and 16.4 ml of B. pasteurii suspended in saline or phosphate buffer. Cement mortar
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cubes were removed from their moulds after 24 hours and placed in urea-CaCl2 medium for 7 and 28 days. When they were tested for compressive strength a
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significant improvement in strength of cement mortar cubes was observed at 28 days.
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Along with bacterial induced carbonate precipitation, they concluded that the overall
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increase of strength followed the presence of an adequate amount of organic substances in the matrix because of microbial biomass, which was later observed by Achal et al. (2011a). The overall trend of an increase in compressive strength might be attributed to the behaviour of microbial cells within the cement matrix, as represented in Fig. 5. Once bacterial cells start precipitation calcite, it plugs the pores on the cement mortar matrix. The impediment of nutrients or oxygen flow to bacterial cells either kill them or turned into endospores and eventually act as organic fibres (Ramachandran et al., 2001). This phenomenon may be associated with the increase of compressive strength of the mortar cubes. Researchers found a monotonic increase in compressive strength as the curing progressed to 28 days. However, the amount of increase may vary with different 35
ACCEPTED MANUSCRIPT microbes and their environment. Ghosh et al. (2005) found 17% increment in the compressive strength after 7 days, which was further increased by 25%, from 23.1
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MPa to 28.9 MPa, at 28 days at 105 cells/ml of Shewanella sp. The performance of
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biocementation to improve compressive strength might be dependent up on media
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used to grow bacterial cells and cure mortar specimens. While replacing nutrient media with LML, Achal et al. (2009b) reported similar compressive strength
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increment of mortars, which was 17% higher at 28 days (26.3 MPa) with S. pasteurii
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compared to control (23.2 MPa). Later, 35% improvement in the compressive strength at 28 d (31.2 MPa) was measured when CSL media was used (Achal et al.
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2010a). Biocementation also showed significant improvement in the split tensile
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strength, as measured by the diametral compression tests in bio-sandstones prepared
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with B. sphaericus and Bacillus sp. (Shirakawa et al., 2011).
Fig. 5 Behavior of bacterial cells inside cement mortar cubes during compressive strength test
5.2. Water permeability Concrete and mortar are porous materials. Understandably, they allow moisture 36
ACCEPTED MANUSCRIPT and other dissolved elements to percolate. Some of these elements may react with the constituents of concrete and alter their properties. Thus, durability of these
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structures can get affected. Permeability governs the rate of flow of fluid into mortar
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or concrete. Water permeability refers to the amount of water migration through
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concrete when it is under a constant water pressure (El-Dieb and Hooton, 1995). As concrete is a porous material, moisture movement can occur by flow, diffusion, or
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absorption. Popular commercially available substances that can be used to hinder
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permeability are synthetic polymers. They have been sometimes found incompatible with the underlying layer due to differences in their thermal expansion coefficient.
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They are also susceptible to environmental exposure such as ultraviolet rays (Reddy
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et al., 2012). Some of these materials are reported as toxic. Through biocementation
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a layer of calcium carbonate can be deposited on the surface of the substrate. Due to the similarity in chemical composition, biocement is compatible with the substrate. It is unaffected by ultraviolet rays and it is non-toxic. These are the main attractions of using biocement to hinder permeability. Biocementation has been proved to reduce the water permeability at a very significant rate. The precipitation of new calcite crystals inside the pores of stone was proposed long back with the application of living cultures of selected calcinogenic bacterial strains (Orial et al., 1992). Such ability of bacteria was later noticed in limestone, when stone specimens were inoculated with Micrococcus sp. and B. subtilis in different sets of experiments, resulted in 60% reduction in water absorption (Tiano et al., 1999). The bacterial strains reduced the stone porosity by 37
ACCEPTED MANUSCRIPT producing high amount of biological matter and calcite crystals, later termed as `biocalcin' (composed mainly of encrusted bacterial bodies mixed with carbonate
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excretates). It ensured the protection of limestone by limiting exchange between the
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interior of the rock and atmosphere, in addition to restricting the penetration of
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degrading agents into the stone (Le Metayer-Levrel et al., 1999). This success led researchers to study the effect biocementation on cementitious materials.
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The effects of MICP on the durability of mortar specimens with different
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porosity were investigated. The surface deposition of calcium carbonate crystals reduced the water absorption by 65 to 90% depending on the porosity of the mortar
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specimens of dimension 40mm×40mm×160mm (De Muynck et al., 2008a). Bacterial
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deposition of a layer of calcite on the surface of the specimens decreased the
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capillary water uptake and permeability (De Muynck et al., 2008b). Concrete cylinders of 80mm diameter and 20mm height were tested for water permeability after treating with B. sphaericus in cementation media and it was observed that biocementation reduced concrete permeability much more than the conventional cement grout repair technique (De Belie and De Muynck, 2009). The presence of Bacillus sp. CT-5, a cement isolate, followed a significant decrease of the water uptake compared to untreated mortars cubes of side dimension 70.6 mm (Achal et al., 2011a). The mortars with bacterial cells absorbed nearly six times less water than the control cubes over a period of 168 hours, while water absorption was five times less in mortars with a mutant of S. pasteurii, Bp M-3 (Achal et al., 2011b). The deposition of a layer of calcium carbonate crystals induced 38
ACCEPTED MANUSCRIPT by bacterial cells on the surface resulted in a decrease in absorption. Achal et al. (2011b) used a mutant of S. pasteurii, Bp M-3, with two different media (containing
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NB and CSL) during concrete preparation and measured both top and side water
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penetration on concrete cubes of dimension 150 mm (M20 grade). Compared to the
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top and side water penetration of 33 mm and 40 mm, respectively in control, the bacterial cells significantly reduced the permeation, measured as 8 mm, 17 mm in
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NB and 11 mm and 25 mm in CSL, on top and side surface, respectively. Scanning
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electron microscope images showed that the calcium carbonate crystals had been deposited into the pores of the interface creating a denser interfacial zone between
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the substrate and the deposit. It was observed that the deposition was effective both
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on horizontal and vertical faces. Thus, both horizontal elements such as slabs and the
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vertical elements such as walls can be remediated. Understandably, the deposition is better on the horizontal surfaces such as slabs due to gravitational assistance. Such results suggest that biocementation is effective in impeding water absorption in the cementitious substrates. It can be used both in new and old structures. Thus, the technology has a potential in repairing deteriorated structures.
5.3. Chloride ion permeability Corrosion of reinforcing steel due to chloride ingress is one of the most common environmental deteriorations that lead to early failure of reinforced concrete structures. The rate of chloride ion ingress into concrete is mainly dependent on the internal pore structure. The transportation of chloride ions into concrete involves 39
ACCEPTED MANUSCRIPT diffusion, capillary suction, permeation and convective flow through the pore system. Biocementation has been successfully demonstrated in reducing chloride ion
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permeation in concrete, thus enhancing its durability. The rapid chloride permeability
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test (RCPT) is used extensively in the concrete industry (Bentz, 2007). ASTM
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C1202-05 specifies the rating of chloride permeability of concrete based on the charge passed through the specimen during a 6-h testing period. The permeability is
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defined in classes such as high, moderate and low.
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Resistance towards chloride penetration of B. sphaericus treated cement mortar cubes of dimension 100 mm3 was measured with the use of an accelerated migration
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test by De Muynck et al. (2008b). Biocementation resulted in significant lower
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chloride migration coefficients compared to untreated specimens. This is due to the
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reduced surface porosity as a result of MICP. The chloride penetration in the cylindrical concrete samples of diameter 100 mm and thickness 50 mm, prepared with and without a mutant of S. pasteurii, Bp M-3, was measured by RCPT (Achal et al., 2011b). The permeability class type was recorded ‘‘moderate’’ for control concrete specimens, while the class changed to ‘‘low’’ type of concrete with bacterial cells as per ASTM C1202-05. For control samples, the average charge passed was 3177 C, whereas for samples prepared with bacterial cells in NB and CSL media it was 1019 C and 1185 C, respectively. Test results indicated 1019 and 1185 Coulombs passed for concretes with bacteria compared to 3177 coulomb for control samples. Thus, biocementation was effective in dramatically reducing the ingress of chlorides into the concrete. 40
ACCEPTED MANUSCRIPT 5.4. Reinforcement corrosion Corrosion of steel reinforcement in concrete is the most common problem
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affecting the durability of such structures. The ingress of moisture, chloride ions, and
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carbon dioxide through the concrete initiates corrosion in the steel. The volume of
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corrosion products is a few multiples higher than the metal. Therefore, surface corrosion of steel creates bursting pressure in concrete leading to cracking and
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spalling. The cracks make access of corrosive substances easier. As a result, corrosion
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is accelerated. Chloride-induced corrosion is one of the main mechanisms of deterioration affecting the long-term performance of such structures. Evidently, high
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permeability can lead to early corrosion. The process is further accelerated in the
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coastal region due to the presence of airborne chlorides.
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There are two ways bio-deposition can help in reducing corrosion, 1) by sealing the pores of concrete, 2) by sealing the cracks formed due to corrosion in deteriorated concrete. The MICP can also use up the oxygen present that would otherwise be involved in the corrosion process of the steel bars. Qian et al. (2009) tested cement mortar cubes biodeposited with S. pasteurii to acid attack by dripping a drop of H2SO4 of pH 0.5 to 5.5 on the mortar surface. The mortar cubes resisted the acid above pH 1.0 and acid resistance value was measured to 1.5. As pH of acid rain in the environment is between 3.5 and 5.6, much higher than pH resistance value of biodeposited mortars, the MICP treatment can resist the corrosion of the acid rain to a certain extent. Later, the detailed investigation leading to positive impact of biocementation in 41
ACCEPTED MANUSCRIPT the reinforced concrete (RC) corrosion prevention was performed by Achal et al. (2012a). They prepared the RC specimens with Bacillus sp. CT-5 of dimension
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200mm×200mm×100mm, cast with one 25 mm diameter twisted deformed steel
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reinforcement bar at the centre of length 300 mm, and induced corrosion by applying
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a constant anodic potential of 40V for 7 days. In the control specimens several cracks appeared due to corrosion. A longitudinal crack of width 0.3 mm developed along the
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reinforcement within 36 hours. The crack continued to grow. At 7 days of accelerated
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corrosion several (at least seven) cracks with width up to 0.2 mm (0.008 in.) were observed on control specimens. In the bacterially treated specimen the appearance of
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cracks was much delayed and their widths were limited. At the end of seven days no
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crack beyond 0.2mm was observed. Thus, biocement deposition had protected the
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reinforcement from corrosion.
The effectiveness of biocementation as a protective agent even after the corrosion has set in was estimated by corrosion rates of specimens (Achal et al., 2012a). In a corroding sample, the rate of corrosion is considered proportional to the corrosion current density Icorr for the specimen. The results indicated that Icorr was limited to 15mA/m2 and 20 mA/m2 for samples with bacteria compared to 61mA/m2 for control samples. A four-fold reduction in corrosion rate by Bacillus sp. CT-5 suggests that the calcite precipitation has the effect of greatly reducing corrosion (Achal et al., 2012a). The results suggest that the formation of calcite might facilitate the protective passive film around the steel and act as a corrosion inhibitor by interrupting the transport process. Further, they also found estimated the residual bond strength between the 42
ACCEPTED MANUSCRIPT corroded reinforcement and the surrounding concrete. The bond strength can be estimated by pulling the reinforcement out of concrete and observing the pullout force.
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The pullout strength was significantly enhanced through biocementation. They
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observed that the reinforcements lost less metal to corrosion when bacterial deposits
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were applied.
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6. Applications
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Clearly, biocementation has shown great promise in the laboratory. The next step is to scale the technology up for field applications. This section envisages the most
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attractive applications of the technology. This technology can be applied as binder,
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cement admixture, barrier or protective layer and also as a repair system in
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construction materials. The various applications of biocementation have been summarized in Table 3. Each application is discussed briefly.
6.1. As a binder
The basic function of a binder of granular materials such as sand is to develop a monolithic structure out of them. Cement performs that function in present day structures. Bacterial deposition can also perform the function in a variety of ways while almost completely avoiding the greenhouse gas emission associate with industrial cement. When the calcium carbonate producing bacterial solution is first introduced into the sand particles, numerous bacteria bind on the surface of sand particles after the solution is fully exudative (Rong and Qian, 2012). In the presence 43
ACCEPTED MANUSCRIPT of substrate and calcium source, bacterial cells precipitate calcite through MICP and the adjacent sand grains are connected to form a whole sand body, known as
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bio-sandstone with a certain degree of strength (Qian et al., 2010). Such
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biocementation process has a huge potential to apply in strengthening the sand crust
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layer in desert or dust fixation in dirt tracks, sealing of the channels and reservoirs in sandy soil, sand dunes, stabilization of sandy soil slopes, construction of aquacultural
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ponds and roads, and to improve the mechanical properties of soil.
6.2. As a cement admixture
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Although cement is known to have high embodied energy it is hard to imagine
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that cement will be completely replaced in foreseeable future. To control the high
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embodied energy of cement several siliceous and calcareous industry byproducts such as fly ash and blast furnace slag, have been used as an admixture to cement. In a similar way, the bacterial product can be used as an admixture to cement or in conjunction with other admixtures. To ensure a positive impact on the embodied energy the admixtures must be locally available and preferably should be an industrial byproduct. Most commonly available admixture is fly ash that is produced due to the combustion of coal for generation of power. There are locally available silicious admixtures that are byproducts of burning crop residues as fuel. Rice husk ash is probably the most common among them. All these residues are recognized as an environmental pollutant (Dhami et al., 2012). Using admixtures such as fly ash and rice husk ash with bacteria for biocementation is an effective way of utilizing such 44
ACCEPTED MANUSCRIPT problematic wastes to combat pollution in the environment. Both these materials have been proved to cause no degradation in the quality of products (Nasly and Yassin,
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2009). While the utilization of fly ash as an admixture for developments of
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biofertilizers has been done by many researchers (Gaind and Gaur, 2003; Kumar and
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Gupta, 2010), fewer studies have been reported on the use of fly ash as an admixture in biocementation studies (Dhami et al., 2012, 2013a). Biocementation was proved to
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enhance the durability of such construction materials containing fly ash and rice husk
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ash.
However, the bacterial admixture needs to be made amenable to mixing with
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cement and it is to be tested for survival in its journey from the cement plant to the
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site of construction. In order to develop efficient formulations using fly ash as a
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cement admixture, Dhami et al. (2013a) optimized the temperature and moisture conditions for maximum survival of bacterial cells. They prepared bacterial formulations by adding bacterial slurry of B. megaterium SS3, B. cereus SS5 and Lysinibacillus fusiformis SS18 to 250 g fly ash, which showed viability of up to 12 months. While studying microbial calcification in sand plugs, Dhami et al. (2013a) reported high amount of calcite precipitates in case of formulated bacterial cells with 28% and 24% reductions in water absorption and porosity, respectively, compared to the control specimens. One of the most high embodied energy materials used by the construction industry is brick. It uses agricultural soil as its raw material and bakes it at a high temperature. Thus, energy consumption of baked bricks is around 3 MJ/Kg. To reduce the energy 45
ACCEPTED MANUSCRIPT consumption concrete blocks that do not need baking is used. Bricks made of soil and sand with a small quantity of cement that do not require baking are the most energy
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efficient alternative (Reddy and Jagadish, 2003). However, due to the presence of high
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proportion of clay such blocks are known to absorb moisture and swell. Use of
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biocementation in the clay can reduce water absorption to a great degree (Dhami et al., 2013b). Soil–cement blocks with density 1.75 Kg/m3, soil: sand 1:1 along with 7%
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cement content were mixed with 10% B. megaterium culture medium. Cylinder
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specimens of diameter 3.8 cm and height 7.6 cm were tested. Biocementation resulted in the formation of a whitish layer on the surface of blocks that was attributed to the
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formation of CaCO3 precipitates with 32% content and resulted into 40% reduction in
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the water absorption. Mercury intrusion porosimetry data of bacterial and control
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soil-cement specimens reflected that total porosity of control specimens was 25.3 % upon calcification, which was reduced by 31%. These are a few initial applications of bacteria as an admixture. The authors believe that there are many other applications waiting to be explored.
6.3. As a barrier layer
Structures are often protected by a barrier layer to impede intrusion of moisture and other deleterious substances. A common barrier layer is synthetic polymeric coatings such as polyurethane. The barrier layer must be durable, non-toxic, and compatible with the substrate. Another important aspect is breathability of the layer i.e. moisture inside the substrate should be able to escape. Although polymeric coatings are very effective in the short term, they are known to degrade with exposure 46
ACCEPTED MANUSCRIPT to ultraviolet rays. They are sometimes toxic. When they are not breathable due to the vapour pressure, they leave the substrate and blisters appear. An ideal barrier layer is
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the one that is closest in composition (and hence in properties) with the substrate.
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cement. Thus, it should be a good barrier material.
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Biocementation produces a deposit that is very close in composition with that of
Early applications of biocementation as a protective layer on limestone
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monuments have shown that the protective effect of the bacterially deposited calcite
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layer lasted for several years (Castanier et al., 1999). More recently, biocementation has been investigated by several researchers for the deposition of a protective surface
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layer as barrier, with consolidating and waterproofing properties, on the surfaces of
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building materials as biodeposition (De Muynck et al., 2010). In the patented ‘Calcite
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Bioconcept’process, a layer of selected bacteria is sprayed on the surface together with specific nutrients and calcium ions (Adolphe et al., 1990). Over time, this layer is supplied repeatedly with the nutrient medium and eventually a layer of calcite is precipitated.
The common way of testing bacterial cells as a barrier layer is explained by De Muynck et al. (2008a). They immersed the mortar cubes and concrete cylinders for 24 hours in 0.3 and 0.6L, respectively, of a 1-day-old stock culture of B. sphaericus prior to submersion in the nutrient solution, and tested for different parameters such as capillary water suction and permeability. They concluded that deposition of a layer of bacterial calcite on the surface of the specimens resulted in a decrease of capillary suction and a decrease in gas permeability. Biodeposition was also reported to better 47
ACCEPTED MANUSCRIPT resist the migration of chlorides in concrete cubes than that of the acrylic coating and the water repellent silanes and silicones or silanes/siloxanes mixture (De Muynck et
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al., 2008b).
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The bio-treatment was applied on the plaster as a barrier in order to protect
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degradation of historical buildings (Anne et al., 2010). The freeze-dried B. cereus was re-hydrated with the nutritive solution and the culture medium was sprayed onto the
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building facade (about 1L m-2). The bacteria were fed with a nutritive solution for 24,
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32, 48 and 72 hours after spraying. Upon bio-treatment, a calcite coating was visualized on the plaster surface. It was noted that water penetration and surface
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deposition.
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porosity was considerably reduced in the treated samples due to calcium carbonate
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A ureolytic biodeposition treatment was applied to conserve limestone in order to investigate the effect of pore structure on the protective performance of a biogenic carbonate surface treatment (De Muynck et al., 2011). The presence of biogenic carbonate as a barrier layer resulted in a 20-fold decrease in the rate of water absorption, which resulted in increased resistance to sodium sulphate attack and freezing and thawing. Rong and Qian (2012) introduced the brushing method to apply bacterial cells as a barrier layer on the surface of cementitious materials. Large numbers of bacteria were absorbed on the surface of cement mortar of dimension 30mm3 due to multiple brushing. The result showed a layer of white precipitation substances on the surface of mortar after 3 days. After curing for 7 days, there was a dense white precipitation on the surface, identified as calcite by EDS and XRD 48
ACCEPTED MANUSCRIPT analyses. The water absorption of mortar surface was decreased by 85% due to biocementation. The deposits from B. megaterium were found to be very effective to
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treat fly ash bricks that showed significant reduction in water absorption, better frost
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resistance and increased compressive strength due to calcite deposition on the surface
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and voids of bricks (Dhami et al., 2012). The water absorption was reduced by 46%, while the compressive strength was enhanced by 24.2% compared to the control
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specimens.
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Another potential application of the technology is in curing of concrete. Cement hydration in concrete is a slow process that continues for several years after casting.
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However, most of the hardening and strength gain happens in the first seven days.
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During this time, water is regularly supplied to concrete to ensure proper hydration of
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cement. This process is called curing of concrete. It is possible to supply the bacterial culture instead of water at the time of curing. With bacterial culture one hopes to deposit a layer of calcium carbonate on the surface of the structure automatically creating a barrier layer. Such structures should be more durable than water cured ones. Although the initial results are promising there are several steps before one can hope to scale the technology up to industrial level. The technology must be packaged in a manner that is convenient to industrial application; the field personnel must be trained; and above all, its durability must be studied with long term tests.
6.4. As a repair system Cracking is a common phenomenon in concrete due to its relatively low tensile 49
ACCEPTED MANUSCRIPT strength. In addition, natural processes such as weathering, faults, land subsidence, earthquakes, and human activities create fractures in concrete structures that affects
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the service life of the structure. Though a variety of techniques is available for crack
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repair, those repair systems often have different thermal expansion coefficient
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compared to concrete and are environmental and health hazards (Jonkers et al., 2010). The basic concept in applying biocementation in repair systems is to embed calcite
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producing bacteria, which grow inside the cracks and precipitate calcium carbonate
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thereby closing the gaps.
Gollapudi et al. (1995) investigated such crack repair system where they indicated
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complete plugging in damaged rock within a day of nutrient injection in a sand
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packed column containing S. pasteurii. This biocementation process was highly
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effective in the presence of fractures as they appear to create nucleation sites for bacteria clusters, leading to improved selective plugging. Later, such technique was effectively shown in repairing cracks in cementitious materials. A novel approach of microbiologically-enhanced crack remediation (MECR) was successfully initiated in concrete (Ramachandran et al., 2001). Cement mortar cubes of dimension 50.8 mm3 were provided with a cut of constant width 3.175mm and different depths of 12.7, 19.05 and 25.4 mm, to simulate cracks. The cracks in the specimens were filled with natural sand and S. pasteurii, while with only sand in control specimens to evaluate MECR efficiency. The specimens were cured in urea-CaCl2 broth for 28 days and tested for their compressive strength. Upon physical examination it was noticed that the sand particles were held together due to MICP in 50
ACCEPTED MANUSCRIPT the crack remediated areas. In the mortar specimens with the deepest crack of 25.4 mm, biocementation increased the compressive strength by 60% than that of the
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control. Distinct, sharp-edged calcite crystals with rod-shaped holes, occupied by the
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in crack remediation (Ramachandran et al., 2001).
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S. pasteurii were seen under SEM examination, confirmed the role of biocementation
MECR experiments were also conducted on larger concrete specimens. They used
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ultrasonic waves for verifying crack sealing. Ultrasonic wave transmission is a novel
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way to study effectiveness of crack healing in concrete. Ultrasonic waves travel easily through hardened concrete. When they find an interface of water or air, as in a crack,
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they get reflected from the crack face. Thus, the existence of the crack can be
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determined. When the crack is sealed the interface of concrete with air or water is
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eliminated. Therefore, the waves are able to travel through the sealant resulting in reduced reflection (Van Tittelboom et al., 2010). De Belie and De Muynck (2009) made standardized cracks in concrete samples of 160mm × 160mm × 70mm by introducing copper plates of 0.3 mm thickness up to a depth of 10 or 20mm into the fresh concrete. They also obtained realistic cracks by splitting fibre reinforced polymer concrete cylinders. Biodeposition treatment was conducted by immersing the concrete samples for 24 hours in a B. sphaericus culture grown overnight in 20g/L yeast extract and 20 g/L urea, followed by 3 days in equimolar solution of urea and CaCl2. The MECR was evaluated by determining the transmission time of ultrasound waves and the water permeability coefficient. Like other researchers they also reported that a deposition of calcium carbonate was visible at the crack edges and on 51
ACCEPTED MANUSCRIPT concrete surface (Achal et al., 2010a; De Muynck et al., 2010; Ramachandran et al., 2001). The ultrasound transmission testing proved that complete sealing of artificial
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cracks of 0.3 mm wide and 10 mm deep due to biodeposition and also such samples
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executed improved water permeation resistance. Similar experiments were carried out
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by Van Tittelboom et al. (2010). They immobilized the B. sphaericus cells prior to crack repair in order to protect against high pH in concrete. Biocementation resulted
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in an increase in ultrasonic pulse velocity, which indicated the formation of crack
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bridging due to MICP. Further, Visual examination of the cracks proved that this technique resulted in complete healing of the cracks. Microbial remediation of
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concrete cracks was observed to be more efficient in shallow cracks than in deeper
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Muynck et al., 2010).
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cracks primarily due to aerobic nature of the bacteria used (Achal et al., 2010a; De
Further, self-healing is an ultimate dream of construction engineers. As the world’s population stabilises the necessity of new infrastructure would alleviate. With present technologies, often it is more expensive to maintain a structure than to build a new one. In this scenario, the idea of self-healing inspired from natural phenomena such as skin of animals looks promising (Van Breugel, 2007). Self-healing is an automatically initiated response to damage or failure and in order to perform the healing, self-healing system must be capable of identifying and healing failures (Fischer, 2010). One of the most promising routes to self-healing structures can be achieved through biocementation. The most common self-healing system is based on microencapsulation. 52
ACCEPTED MANUSCRIPT Recently, the concept of using microcapsules as carriers for self-healing agents in concrete was adopted by Wang et al. (2014), which provides an additional advantage
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of getting protected of the harsh environment inside the concrete. They encapsulated
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bacterial spores from B. sphaericus with concentration 109 spores/g in melamine
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based microcapsules and incorporated them into long reinforced prisms of dimension 30mm × 30mm × 360mm. After curing for 28 days the specimens were subjected to a
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tensile force to create multiple cracks. The enhanced self-healing efficiency in cracked
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specimens contributed by microencapsulated bacterial spores was demonstrated based on the experimental results from light microscopy and the water permeability test. The
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specimens with bacteria containing microcapsules had much higher crack healing
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ratio (48% to 80%) compared to the ones without bacteria, where only 18% to 50% of
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healing was observed. The maximum crack width healed in the specimens with bacterial microcapsules was 970 µm, which was about 4 times wider compared to the specimens without bacteria (max 250 µm). When the capsules are ruptured, for example, by damage, the self-healing mechanism is triggered through the release of the healing agent in the region of damage (Jonkers and Schlangen, 2008; Van Tittelboom and De Belie, 2013; Zemskov et al., 2013). Copious production of bacteria-mediated minerals results in sealing off the crack; hence, the permeability of concrete and leakage is reduced to enhance the durability of structures.
7. Economy analysis of biocementation Biocementation clearly shows the promise of field application. However, its 53
ACCEPTED MANUSCRIPT economic viability must be examined to evaluate time scale of its industrial acceptance. Economic analysis must be carried out in terms of life cycle estimates
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considering its sustainability incentives. Moreover, economies of scale would also
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apply. Detailed economic analysis awaits data from pilot field evaluations.
experiments and results would be of interest.
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Meanwhile, an analysis of the cost of biocementation based on laboratory
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The cost of biocementation depends largely on the price of nutrients, and to some
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extent on the price of the bacterial strain. In this example prices at currency of the United States is used. Price of bacteria varies from country to country; however, one
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standard bacterial strain, if bought from ATCC costs $500, and from MTCC costs
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$10, while CGMCC sells at $200 (Achal, 2015). Such bacterial strain can be used in
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construction work for many years by sub-culturing it at regular time interval. De Muynck et al. (2010) provided a detailed report on the cost analysis of biocement based
on
personal
communication
with
FTB
Remmers,
2008;
http://www.ftbremmers.com/). The biodeposition cost was calculated about $4 per m2, providing the price of 1 kg lyophilized bacteria about $1,500. The cost of nutrients is estimated to be about US $250 per kg. Finally, it was calculated that for microbial concrete application in the area of 0.04–0.08 kg per m2, keeping the cost of nutrients to US $7–15 per m2, the total product cost would be around US $31–39 per m2. The additional cost during the preparation of biological mortar or microbial concrete will be that of the nutrient. However, the cost of nutrients can be reduced 54
ACCEPTED MANUSCRIPT significantly by replacing standard or commercially available nutrients with such industrial byproducts, rich in carbohydrate, protein and energy sources. Achal et al.
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(2009b, 2010a) successfully reduced the cost of microbial concrete production by
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replacing standard nutrient with lactose mother liquor (LML) and corn steep liquor
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(CSL) where LML showed effectiveness equal to standard nutrient, while CSL was significantly better in terms of microbial concrete production. LML and CSL are
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available locally with a price of around $1 and $2 per litre, respectively, which is far
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cheaper compared to the standard nutrient medium and this brings the biodeposition cost to $0.25–1.0. On many occasions such industrial effluents can even be obtained
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free of cost. While the performance of LML was up to the level of standard
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commercially available nutrients, CSL was significantly better in terms of
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biocementation. Hence, CSL offers an economic advantage over the standard nutrient medium and the overall process cost reduces dramatically. One can find local sources of such economic nutrients. Thus, the technology can be significantly cost effective provided it is adapted to the local bacterial strains and locally available nutrient rich byproducts.
8. Future directions This section attempts to map some of the future directions of the technology. It is evident that there are several opportunities and challenges. This section discusses some of the opportunities that, in the opinion of the authors, future research should explore. 55
ACCEPTED MANUSCRIPT 8.1. Archiving of microbial varieties Biomineralization in construction is a new field of research that combines two
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communities- biotechnologists and civil engineers who are relatively unknown to
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each other. Therefore, both communities must document the existing knowledge in a
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manner that is comprehensible to the other. In order to make this novel process of biotechnology popular in construction engineering, it is warranted to catalogue and
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archive the microbial varieties that are amenable to biocementation. The
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microorganisms including bacteria and fungi produce various enzymes. However, for biocementation urease is an important enzyme. Same bacterium has been
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reported to produce different amounts of urease by researchers from different
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countries. Therefore, it is important to catalogue them in various geo-climatic
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domains. It is always preferable to use the local varieties that are acclimatised to local conditions. It is also more cost-effective to harvest local micro-organisms than buying them.
The census will help researchers and engineers differentiate between various microorganisms and to choose the perfect one for usage. It is envisaged that for adaptation of the technology in construction civil engineers and microbiologists must work closely. The catalogue will help engineers to contact microbiologists for assistance. The civil engineers, on the other hand, should contribute in documenting past results with a goal to develop standard testing protocols for construction materials with biocementation. A publicly-accessible, easy-to-search database of carbonate 56
ACCEPTED MANUSCRIPT producing microbes and their role in biocementation with available past results and standard testing and application methodologies will be imperative for the industrial
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success of the technology.
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8.2. Local adaptation
The biocementation ability based on MICP depends on several factors including
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geographical environment. Thus, the performance of the microorganisms varies
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depending on the geo-climatic conditions. While applying this technology the geographical domain of a product must be identified. This is contrary to the style of
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production and trade of conventional building products that are marginally affected
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by the geo-climatic conditions. The locally available microorganisms are likely to be
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acclimatised to the local conditions and thus, have higher chance of success. Thus, the technology must be locally adapted. As a result, experiments must be repeated several times to develop local varieties. The regulatory limitations of transporting microorganisms from another country also mandate use of local microorganism. Further, the locally available nutrients in the form of industrial byproducts should be used. That would not only reduce the product cost but also allow recycling of wastes and alleviate pollution.
8.3. Life cycle assessment An assessment of the biocementation technology in terms of its overall economic and environmental impact is imperative. In recent years, a cradle to grave approach 57
ACCEPTED MANUSCRIPT of assessment has developed. These methods attempt to evaluate a project for its entire life time both in terms of economy and environmental sustainability. A new
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protocol for Life Cycle Assessment (LCA) is slowly emerging (Horne et al., 2009).
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It is being argued that sustainability is a way of achieving economy (Parish and
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Chester, 2009). These methods are based on algorithms that need considerable field data. For conventional materials the methodologies are at an emerging stage (Bribián
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et al., 2011). A challenge for LCA for a new technology such as biocementation
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would be availability of reliable data. Although field data are scant there is adequate information to start the life cycle evaluation of the material. It is important to initiate
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LCA of biocementation.
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8.5. Expanding application domains Biocementation has been applied in sand, rocks and stones, cement and concrete. However, it can benefit several areas of construction. It can be utilized in rehabilitation of heritage stone and lime mortar structures. The process can also be carefully employed in remediation of structures that contain hazardous materials. Encapsulation of hazardous chemicals in the bio-deposition is a promising application. It has been reported that building materials from old demolished structures are found unsuitable for recycling due to the presence of heavy metals (Kawai et al., 2014). The MICP technology has been widely used in the remediation of various heavy metals such as arsenic, copper, lead and chromium (Achal et al., 2011c, 2012b, 2012d, 2013). Biocementation was successful even in presence of 58
ACCEPTED MANUSCRIPT radioactive chemicals such as strontium (Sr). Warren et al. (2001) observed significant strontium uptake into calcite generated by S. pasteurii induced calcium
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carbonate precipitation. They together with Fujita et al. (2004) concluded that
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biocementation was a promising approach for 90Sr remediation in aquifers rich in
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calcium carbonate. Recently, Achal et al. (2012c) demonstrated Halomonas sp. SR4 induced calcite precipitation in sequestering soluble strontium as biominerals (Fig. 6)
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that could play an important role in strontium bioremediation as well repairing
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nuclear structures containing this harmful substance. Such results open new areas of
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application of biocementation technology.
Fig. 6 A schematic representation of strontium sequestering based on MICP, also showing distinct calcite crystals as depicted by SEM. Reprint with permission from Achal et al. (2012c)
9. Concluding remarks Present day world faces the dichotomy of infrastructural growth without compromising sustainability. While present building technologies are struggling to 59
ACCEPTED MANUSCRIPT move towards sustainability, natural habitats are being built sustainably for millions of years. Recent advances in the understanding of natural systems and biotechnology
Concrete is the world’s most widely consumed construction material.
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construction.
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let us attempt to emulate some of nature’s sustainable ways in infrastructural
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Cement used in concrete is the most energy consuming component. Biocementation is a means of creating a cement like binder using microbes that has the potential to
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be a significant step towards sustainability. There are several pathways to
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biocementation. Some of them such as MICP has been more widely studied than the others. Laboratory experiments have demonstrated that biocementation is able to
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improve compressive strength of cement based materials, reduce porosity, and thus
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diminish diffusion of moisture and other deleterious materials. By reducing
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intrusions biocementation should improve the durability of structures, another step towards sustainability. The technology can be used in a variety of ways both in new infrastructure and maintenance of existing ones. There are immense opportunities in improving the technology in structural performance, economy and environmental sustainability. However, there are daunting challenges for industrialisation of this emerging technology. Biosafety could be a major concern especially for researchers or engineers of non-microbiology background working in the area of biocement due to their limited knowledge on bacteria. Working with pathogenic bacterial strains for biocementation could be a dangerous step that can be avoided either by checking pathogenicity of isolated bacteria or by procuring urease producing bacteria from established culture collection bank. Moreover, it is very important to create 60
ACCEPTED MANUSCRIPT collaboration between two seemingly disparate groups- microbiologists and civil engineers. Creating dialogues between the two groups can be seminal in future
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success of the technology. The present paper is an attempt towards that direction.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China
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(No. 41450110458) and the Research Innovation Fund from East China Normal
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University (No. 78210267). We are grateful to editor and anonymous journal referees for commenting earlier versions of the manuscript.
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Table 3: Various applications of biocementation on construction materials and their benefits Media
Limestone
Micrococcus sp., B. subtilis,
B4 media, M-3, M-3P media
S. pasteurii
NB, urea, CaCl2
Sand
S. pasteurii, Mutant of S.
Reference
Protection from decaying
Tiano
et
al.
(1999),
Jimenez-Lopez et al. (2007) Crack remediation
Zhong and Islam (1995)
NB/LML/CSL, urea, CaCl2
Sand plugging of high strength
Achal et al. (2009b, 2010a)
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Granite
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Myxococcus xanthus
Benefit
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Bacteria
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Construction material
pasteurii Alkalophilic bacteria
Yeast extract, peptone, urea, CaCl2
Sand plugging of high strength
Rong and Qian (2012)
Cement mortar
S. pasteurii, B. pseudofirmus,
Phosphate buffer/NB/CSL/alkaline
Crack remediation/Self healing
Ramakrishnan et al. (2001);
B. cohnii, B. alkalinitrilicus
media/yeast extract, urea, CaCl2/ca
CE
PT ED
Sand
Achal et al. (2010b); Jonkers
lactate
Shewanella sp., S. pasteurii, Bacillus sp. CT5
NB/LML/CSL, urea, CaCl2
Improved compressive strength
Ghosh et al. (2005); Achal et
AC
Cement mortar
et al. (2010)
al. (2009a, 2009b, 2010a, 2011a)
Cement mortar
B. sphaericus
NB, NaHCO3, NH4Cl, urea
Improved reduced
compressive water
strength,
absorption,
gas
De Muynck et al. (2008a, 2008b)
permeability and carbonation
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Concrete
Bacillus sp. CT5
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NB/CSL, urea, CaCl2
Reduced
water
and
chloride
Achal et al. (2011b)
NB/CSL, urea, CaCl2
Crack remediation/Self healing
De Belie and De Muynck (2009); Van Tittelboom et al. (2010)
Corrosion protection
Achal et al. (2012a)
PT ED
MA
Bacillus sp. CT5
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Yeast extract, urea, ca nitrate
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Concrete
B. sphaericus
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Concrete
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permeability
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S0012-8252(15)00091-4 doi: 10.1016/j.earscirev.2015.05.008 EARTH 2120
To appear in:
Earth Science Reviews
Received date: Accepted date:
6 January 2015 11 May 2015
Please cite this article as: Achal, Varenyam, Mukherjee, Abhijit, Kumari, Deepika, Zhang, Qiuzhuo, Biomineralization for Sustainable Construction – A Review of Processes and Applications, Earth Science Reviews (2015), doi: 10.1016/j.earscirev.2015.05.008
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ACCEPTED MANUSCRIPT Biomineralization for Sustainable Construction – A Review of Processes and Applications
School of Ecological and Environmental Sciences, East China Normal University,
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1
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Varenyam Achal1*, Abhijit Mukherjee1,2, Deepika Kumari3, Qiuzhuo Zhang1
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Shanghai 200241, China
Department of Civil Engineering, Curtin University, Bentley, WA 6102, Australia
3
Xinjiang Key Laboratory of Environmental Pollution and Bioremediation, Xinjiang
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2
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Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011,
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China
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Running head: Biomineralization for Sustainable Construction
Corresponding author: Varenyam Achal, PhD Associate Professor, School of Ecological and Environmental Sciences, East China Normal University, Shanghai 200241, China Email: [email protected] Phone: +86-21-54341138 Fax: +86-21-54341131 1
ACCEPTED MANUSCRIPT Abstract Modern civilization is facing the dichotomy of rapid development of infrastructure
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that makes concrete as most traded material on the earth other than water. However,
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the production of cement, key ingredient of concrete, releases roughly a tonne of CO2
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into the environment with each tonne of cement production. The environmental concerns and sustainability issues associated with cement and concrete necessitate
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alternative and better approach in the construction. Nature, on the other hand, has a
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plethora of examples of sustainable habitats such as coral reefs, silk webs and ant hills. Recent advances in biotechnology have great potential of emulating nature’s
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way of building in modern days infrastructures at a scale that would sustain increasing
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population. Further, many of the biological materials of nature, be it ceramics or
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polymeric composites formed in the process of biomineralization, provides basis for sustainable construction. This paper elaborates nature’s way of construction based on biomineralization and discusses the progress of different biological pathways for sustainable construction. Main milestones achieved have been identified and the effect of biological intervention on the properties of structural materials has been highlighted. Variety of applications of biomineralization based technology in the construction has been reported. The paper briefly documents the future directions of the technology.
Keywords: Biomineralization; Sustainable construction; Biocement;
Calcium
carbonate; Urease; Building materials 2
ACCEPTED MANUSCRIPT Contents 1. Introduction
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2. Nature’s ways of construction
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3. Alternate pathways for biocementation
3.2. Biologically induced mineralization
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3.2.2. Sulphate reducing bacteria
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3.2.1. Photosynthetic microorganisms
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3.1. Biologically controlled mineralization
3.2.3. Bacteria involving nitrogen cycle in mineralization
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4. Challenges
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3.4. Biocement
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3.3. Microbially induced calcium-carbonate precipitation (MICP)
4.1. Cost of the technology 4.2. Increasing yield
4.3. Alkaline conditions 4.4. Perceived harmful effects- chlorides 5. Structural properties 5.1. Strength 5.2. Water permeability 5.3. Chloride ion permeability 5.4. Reinforcement corrosion 6. Applications 3
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6.3. As a barrier layer
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6.4. As a repair system
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7. Economy analysis of biocementation
8.1. Archiving of microbial varieties
8.3. Life cycle assessment
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8.2. Local adaptation
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8. Future directions
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Acknowledgements
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9. Concluding Remarks
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8.4. Expanding application domains
References
1. Introduction
Development of modern infrastructure such as roads, bridges and buildings has proven to bring in economic prosperity. The emerging economies are now emulating the developed ones in rapidly building their infrastructure, resulting in the consumption of construction materials such as cement, brick and steel is at their record highs and it is predicted to grow for the next fifty years (Schnieder et al., 2011). As a result, the construction industry accounts for half of global resource usage, up to 40 percent energy consumption and up to 20 percent of the emission of 4
ACCEPTED MANUSCRIPT greenhouse gasses. Experts have expressed serious doubts about the sustainability of the present construction technologies. Development of an alternative method of
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construction that does not hinder the progress of infrastructure and at the same time
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does not cross the boundaries of sustainability would be a great step to avert the crisis.
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Nature has been building its habitats for millions of years in a sustainable way. It is worthwhile to examine the natural ways of building habitats and to what extent they
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can be emulated in engineering construction.
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Many building materials use huge quantities of energy and produce high volumes of CO2. Steel, brick and concrete are prime among them. However, in the last century
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use of concrete has surpassed all other building materials by a huge margin. Ordinary
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Portland Cement (OPC) is a vital construction material and also a strategic
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commodity (Vlasopoulos, 2010) with a market dominated by China, which is attributed to 57.3% of global consumption (CEMBUREAU, 2012). Despite the incremental improvements in process efficiency that have been adopted by the cement industry in recent years, OPC production is still responsible for around 6% of all anthropogenic global CO2 emissions. In the current global setting, building construction and operation results in 50% of all CO2 emissions worldwide. The push to reduce global CO2 emissions is backed by governments, corporations and citizens who understand that the present rate of release of greenhouse gases into the atmosphere is a serious threat to future life and prosperity on the planet. Cement never appears to become a sustainable material (Gerilla et al., 2007).
5
ACCEPTED MANUSCRIPT Further, materials for infrastructure such as concrete, have been developed with a sole focus on strength believing that other functional requirements such as durability,
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thermal or hygral passivity would be automatically achieved. However, in reality,
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infrastructure materials have been found to degrade rather rapidly due to natural as
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well as anthropogenic causes. Often the situation is aggravated due to high CO2 concentration in the atmosphere. The aggressive elements enter the concrete that has
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direct impact on the corrosion of reinforcements (Pacheco-Torgal and Labrincha,
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2013). Corrosion leads to cracking and spalling of concrete that creates pathways for intrusion of deleterious materials. They affect other functional requirements such as
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hygral and thermal insulation. Synthetic agents such as epoxies and polyurethanes,
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water repellents such as silanes or siloxanes, and corrosion inhibitors such as cationic
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surfactants are often not compatible with their substrates and sometimes toxic. On the other hand, Nature has been building habitats such as ant hills, coral reefs and spider webs in a sustainable manner for millions of years. The materials are synthesized at ambient conditions (thus requiring very little energy) and they are optimized to multifunctional requirements. For example, the spider’s web needs to be resilient to be able to tangle the prey and stiff to maintain its shape. Thus, the silk of the web frame and drag lines is more than 1000 times stiffer than the catching spirals (Omenetto and Kaplan, 2010). Although the present technology cannot match the versatility of construction by nature, there are wonderful lessons to be learnt. Moreover, there are early indications of adaptability and scalability of some of the biological processes of nature in the construction industry (Achal et al., 2010a, 6
ACCEPTED MANUSCRIPT 2011a). This paper provides a critical review on nature’s ways of construction. As the
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discussion focuses on concrete, biocementation, its pathways and mechanisms, and
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its application in various building materials have been highlighted. A discussion on
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the economics of this novel biotechnology is provided. Challenges and future work
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2. Nature’s ways of construction
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on biocementation in construction are also outlined.
A large number of high-performance prototypes has been created by nature that
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humans can reverse engineer and in turn use as inspirations for creating synthetic
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products with similar superior performances (Ballarini and Buehler, 2013). There are
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inexhaustible sources of inspiration that come from nature, be it ceramic (tooth enamel, mollusk shell, spicules in sponges, diatoms), polymeric (arthropod exoskeleton, silk, plant cell walls), or fairly balanced composites (feathers, antlers, bones). Biological materials consist virtually of all composites utilizing basic components in different proportions and of a variety of structural architectures with high mechanical strength. Such materials are developed by nature by means of growth or biologically controlled self-assembly, adapting to the environmental conditions and using the most commonly found materials. These materials are composed of only about 10% of about 100 stable elements found in nature. They are notably light, restricted within the first two rows of the periodic table. The principal elements include hydrogen, carbon, oxygen, nitrogen, phosphorous, sulfur, silicon, and calcium. 7
ACCEPTED MANUSCRIPT Common metals such as iron, copper and aluminium are either absent or found in minute quantities, e.g. iron oxide in radular teeth of chiton (Saunders et al., 2009).
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Most likely, reason for absence of metals is that the high temperatures necessary for
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processing of metallic elements are not amenable natural organisms (Chen et al.,
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2012).
Biological organisms produce composites containing both inorganic and organic
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components in complex structures, organized in terms of composition and structure,
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which provide properties for multifunctional performance. Table 1 specifies the major components responsible for mechanical strength in biological materials for natural
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habitats. Coral reefs and bone consists of calcium phosphate and collagen,
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respectively. The main components of these biological materials are calcium
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carbonate, namely aragonite, and hydroxyapatite (Weiner and Addadi, 1997; Sikavitsas et al., 2001; Muller, 2011). Nature grants compressive strength in biological materials by inserting inorganic or mineral elements while the organic component attributes to the ductility. Such combination of strength and ductility leads to high energy absorption prior to failure (Meyers et al., 2008). The most common mineral components include calcium carbonate, calcium phosphate (hydroxyapatite), and amorphous silica. Biological materials are composites of an inorganic mineral phase with a biopolymer, formed in the process called biomineralization (Chen et al., 2012).
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ACCEPTED MANUSCRIPT Table 1: Major inorganic or organic component responsible for mechanical strength in biological materials Inorganic or organic component
Coral reefs
Calcium phosphate/hydroxyapatite
Spicules in sponges, Diatoms
Amorphous silica
Mollusk shells, Reptile eggs
Calcium carbonate/aragonite
Mollusks, Bird eggs
Calcium carbonate/calcite
Bone, Dentine, Tendons, Muscle
Collagen
Arthropod exoskeletons
Chitin
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Biological material
Fibroin
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Silk
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The nature’s way of construction attracts and stimulates scientists and engineers to
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develop building materials and structures with high mechanical strength that are adapted to the environment. Natural load bearing materials can be classified as tension
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elements and compression elements. The tension elements are mostly made of biopolymers. One of the best biological tension elements that inspire construction specialists is silk. The silk produced by orb-web-spinning spiders reveal mechanical properties that are superior to almost all natural and man-made materials. Silks have moduli of 0.003-10 GPa and tensile strengths of 50-1100 MPa (Wegst and Ashby, 2004). Such nature of silk fibre makes it a top choice for springs and outperforms the man-made spring. The superior properties are from two proteins, fibroin and sericin that form a semi-crystalline material with an amorphous region composed of disordered protein chains connected to protein crystals (Gosline et al., 1999; Meyers et al., 2008). This crystal constitutes layers of anti-parallel amino acid sequences, known as b-pleated sheets that provide high tensile strength. Ant hills, on other hand, 9
ACCEPTED MANUSCRIPT are an excellent example of how to achieve multifunctional capability of mechanical strength, thermal insulation and ventilation at a miniscule embodied energy and cost.
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The binder in it is mainly due to a cementing material provided by lignin.
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A large majority of the biological materials originates from biomineralization by
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which living organisms produce minerals, chiefly carbonate products. Mollusk shells are an excellent example of such high-performance biominerals. They are mainly
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consisted of calcium carbonate and evolved to incorporate various design strategies in
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the arrangement of this biomineral. Some of their species showed very high compressive strength due to mineralization. The maximum compressive strengths
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shown by Haliotis rufescens and Araguaia river clam are 540 MPa and 567 MPa,
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respectively (Menig et al., 2000; Chen et al., 2008). Mollusks are made-up of at least
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95% minerals containing calcium carbonate or calcite and aragonite, which exhibit about 3000 times higher toughness than calcite. The natural materials of high mineral contents provide mechanical strength in addition to weaker interfaces with intricate architectures that enhance toughening properties, for example in tooth enamel or nacre (Mirkhalaf et al., 2014). All arthropods that make the largest phylum of animals, are covered by a rigid exoskeleton that not only supports the body and resists mechanical loads, but also provides environmental protection and resistance to desiccation (Neville, 1975; Vincent and Wegst, 2004). The three main components of an exoskeleton are chitin, polysaccharide, structural proteins, and inorganic minerals, typically calcium carbonate, showing a high degree of mineralization (Meyers et al., 2008). The 10
ACCEPTED MANUSCRIPT minerals are mostly deposited within the chitin-protein matrix in the form of crystalline CaCO3.
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Corals provide us a great example of environment-friendly building materials. A
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coral reef is considered as a “carbonate factory” dealing with CaCO3 production and
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loss (Pomar and Hallock, 2008). Corals build reefs by natural interaction between CO2 and water that precipitates as CaCO3 after reaching equilibrium. During reef
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building, corals develop incredible ability to calcify and act as the most prolific
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mineralizer on the planet. Coral reefs give us an excellent example to develop low carbon building materials. They use CO2 as raw material and biomimicking them
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could lead to production of low energy cement, a most widely used building material.
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Therefore, these biological materials serve as a valuable source for research and as an
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inspiration for structural materials development. Calcium is the cation of prime for most organisms. The calcium-bearing minerals cover about 50% of known biominerals (Lowenstam and Weiner, 1989). The dominance of such calcium minerals led to the extensive usage of the term calcification, sometimes, instead of mineralization. The calcium carbonate minerals are the most abundant biogenic minerals, both in terms of the quantities produced and their widespread distribution among many different taxa (Lowenstam and Weiner, 1989). The polymorphs of calcium carbonate present in various biological organisms, which provide mechanical strength has been provided in Table 2.
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ACCEPTED MANUSCRIPT Table 2: Polymorphs of calcium carbonate in biological organisms Calcium carbonate mineral
Formula
Crustaceans cuticle
Calcite
CaCO3
Coral reefs
Calcite
CaCO3
Mollusks shells
Aragonite
Fish head
Aragonite
Echinoderms shells
Mg-calcite
Sea squirt spicules
Vaterite
Gastropods shells
Vaterite
Crustaceans cuticle
Amorphous
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Biological organism
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CaCO3
(Mg/Ca)CO3 CaCO3
CaCO3 CaCO3.nH2O
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CaCO3
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Another amazing phenomenon of natural mineralization is that although they
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consist of brittle materials, they exhibit remarkable fracture toughness. Despite the weakness of constituents present in highly mineralized bio-materials such as teeth,
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nacre, conch shell, glass sponge spicules, and arthropod cuticles; they result in outstanding performance, which led to development of bio-inspired strategies. It is observed that the microstructure of these materials consists of weakly bonded layers that absorb shocks and produce ductility by assembling the brittle constituents. Such precise microstructural assembly has inspired biomimetic designs such as a microstructurally engineered glass that is a few hundred times tougher than monolithic glass (Mirkhalaf et al., 2014). The design solutions available in Nature with finetuned mechanical properties have always inspired engineers in designing structures (Ehrlich, 2010). However, it has been a challenge to emulate them in engineering construction and manufacturing processes. Nature grows the structure through secretion or deposition of minute 12
ACCEPTED MANUSCRIPT quantities of the materials where every step is precisely controlled. Thus, manufacturing the material and the structure are not separate steps, while in
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engineering construction materials are manufactured separately and then used to build
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the structure. The major challenge is to find appropriate methods to combine the two
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processes. One promising route is in-situ synthesis and deposition of carbonate minerals using biological agents. In such a process, precise microstructural control
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should be possible. It also alleviates carbon emission by avoiding the energy intensive
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manufacturing process of materials such as cement and bricks, thereby attaining sustainability. There are several biological pathways based on biomineralization that
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have been explored.
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3. Alternate pathways for biocementation Cementation is a process whereby calcium carbonate either forms or deposits on a surface. In the process, they tend to attach to the substrate creating cementation. When it is the product of a biological process it may be called “biocementation”. In other words, biocementation is the product of ‘biomineralization’. Biomineralization is the process involving organisms in mineral formation as a result of cellular activity that promotes the required physico-chemical conditions for such a formation and growth to take place (Ben Omar et al. 1997). From an evolutionary point of view, this process generated from bacterial activity. The majority of the biominerals contain calcium as their main cation and carbonates in terms of their anions (Perito and Mastromei, 2011).
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ACCEPTED MANUSCRIPT This process has two fundamental types: 1) biologically controlled mineralization, and 2) biologically induced mineralization.
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3.1. Biologically controlled mineralization
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Biologically controlled mineralization (BCM) process can be extra-, inter- or
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intracellular. In extracellular BCM, the cell produces a macromolecular matrix (proteins, polysaccharides or glycoproteins) outside the cell in an area turn out to be
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site of mineralization. Biologically controlled intercellular mineralization process
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typically occurs in single-cell organisms in which the epithelial substrate governs the nucleation and growth of specific biomineral phases over large areas of cell surfaces.
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This type of mineralization is often seen in calcareous algae that nucleate and grow
controlled
intracellular
mineralization
is
a
compartmentalized
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Biologically
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calcite perpendicular to the cell surface (Borowitzka, 1982; Borowitzka et al., 1974).
crystallization process that occurs within specialized vesicles that direct the nucleation of biominerals within the cell (Weiner and Dove, 2003). Many eukaryotes, prevalently tissue-forming multicellular ones, carry out this process (Lowenstam and Weiner, 1989; Mann, 2001), where cellular activity controls the process to a high degree and leads the nucleation, growth, morphology, and final location of the mineral (Decho, 2010). The mineral particles formed are synthesized or deposited intracellularly on or within organic matrices or vesicles in a specific location with respect to the cell. From biomineralization for sustainable construction point of view, biologically induced mineralization is more important and widely reported.
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ACCEPTED MANUSCRIPT 3.2. Biologically induced mineralization In biologically induced mineralization, microorganisms secrete metabolic
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products that react with ions or compounds in the environment resulting in the
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subsequent deposition of mineral particles (Frankel and Bazylinski, 2003). The
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formed bio-minerals, chiefly calcium carbonate, nucleate and grow extracellularly due to metabolic activity of the microorganism and subsequent biochemical reactions
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involving metabolic byproducts. This is a presumably unintended and uncontrolled
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consequence of metabolic activities. With the only exception of Achromatium oxaliferum, calcium carbonate deposition by bacteria has been generally considered to
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be induced, and the type of mineral produced is primarily dependent on environmental
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conditions (Ben Omar et al., 1997; Brennan et al., 2004; Rivadeneyra et al., 1994).
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Calcium carbonate (CaCO3) or calcite precipitation is a common phenomenon found in aquatic environments and soils (Castanier et al., 1999; Ehrlich, 1998), which can be induced by microbes (mainly bacteria). Calcite, aragonite, and vaterite are the major crystalline structural polymorphs of CaCO3 in bacterial systems, as well as in all biogenic systems (Ben Omar et al., 1997), with calcite as most represented isoform (thus, sometimes process is termed as microbially induced calcite precipitation) followed by aragonite and less stable, vaterite. Sulphate reduction, denitrification and urea hydrolysis are the three major metabolic pathways driving these precipitation processes. There are mainly three groups of microorganisms involved in the precipitation of calcium carbonate.
15
ACCEPTED MANUSCRIPT 3.2.1. Photosynthetic microorganisms One group involving in calcium carbonate precipitation belongs to photosynthetic
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microorganisms such as cyanobacteria and microalgae, which are most active in
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aquatic environments (Mc Connaughey et al., 2000). In the biologically induced
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process, the mineral precipitates because the organisms change the chemical microenvironment of the water layer adjacent to the cell. In cyanobacterial
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photosynthesis, microorganisms use CO2 in their metabolic process (Eq. 1),
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HCO−3 is transported through the membrane and dissociates within the cell into CO2 and OH− (Eq. 2) (Dittrich and Obst, 2004). CO2 is removed by photosynthesis
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leaving an excess of OH− behind that is pumped out of the cell through the cell
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membrane, causing a rise in pH. The rise in pH causes the carbonic acid equilibrium
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to shift towards an increase in (CO2−3) (Eqs. 2 and 3) resulting in production of CaCO3 in the presence of calcium ion in the system (Eq. 4). CO 2 + H 2O → CH 2O +O 2 − 2
2− 3
2 HCO ↔ CO2 + CO
+ H 2O
CO32 − + H 2O ↔ HCO3− + OH − 2+
− 2
−
Ca + HCO + OH ↔ CaCO3 + 2 H 2O
(1) (2) (3) (4)
3.2.2. Sulphate reducing bacteria The sulphate reducing bacteria (SRB) that are ubiquitous, anaerobic, prokaryotes, morphologically and phylogenetically highly diverse, include species in the Domains Bacteria (δ-subdivision of Proteobacteria and Gram-positive group) and Archaea (Frankel and Bazylinski, 2003). They play a great role in the mineralization of organic matter in anaerobic environments and in the biogeochemical cycling of 16
ACCEPTED MANUSCRIPT sulphur; however, sulphate reduction dominates mineralization in sulphate rich environments.
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Sulphate reducing bacteria can reduce the sulphate generated from abiotic
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dissolution of gypsum (CaSO4.H2O) (Eq. 5) to H2S and HCO3- (Eq. 6) in the
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absence of oxygen. When the hydrogen sulphide degasses, it induces increase in pH (Castanier et al., 1999), resulting in CaCO3 precipitation (Eq. 7).
2− 4
2(CH 2O ) + SO 2+
− 2
−
− 3
(5)
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CaSO4 .H 2O → Ca 2 + + SO42 − + 2 H 2O
→ HS + HCO + CO2 + H 2O −
(6)
Ca + HCO + OH ↔ CaCO3 + 2 H 2O
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(7)
Desulfovibrio, a group of sulphate reducing bacteria, remove sulphates from
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gypsum in a process coupled to calcite production (Atlas and Rude, 1988) by a
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combination of dissolution–precipitation and diffusion processes. In brief, sulphate
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reduction by bacteria from gypsum releases calcium ions that react with carbon dioxide and results calcite formation. The reaction has been summarized in Eq. 8. 6CaSO4 + 4 H 2 O + 6CO2 → 6CaCO3 + 4 H 2 S + 2 S + 11O 2
(8)
In addition, some member of Desulfovibrio such as D. magneticus is known to produce extracellular particles of iron sulphide by mean of biologically induced mineralization while synthesizing intracellular crystals of magnetite via biologically controlled mineralization (Sakaguchi et al., 2002).
3.2.3. Bacteria involving nitrogen cycle in mineralization The third group of bacteria involving in nitrogen cycle is most important with respect to calcium carbonate precipitation in different environments. Such bacteria 17
ACCEPTED MANUSCRIPT produce an enzyme urease (urea amidohydrolase; EC 3.5.1.5), which induce the precipitation of CaCO3 by a series of complex biochemical reactions. This reaction
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requires urea as substrate while calcium source as chief agent for carbonate
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production. During bacterial urease activity, 1 mol of consumed urea contributes to 1
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mol of ammonia and 1 mole of carbamate (Eq. 9) which, on further hydrolysis, contributes to the accumulation of ammonia and carbonic acid (Eq. 10). These
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products subsequently equilibrate in water to form bicarbonate and 2 moles of
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ammonium and hydroxide ions (Eqs. 11 and 12) that results in increase in pH and ultimately shifts the bicarbonate equilibrium, resulting in carbonate ions formation
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(Eq. 13). Due to the increase in pH in the cell, a high extracellular calcium ion
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concentration and a low extracellular proton concentration is required for the
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secretion of carbonate ions. Under this situation, the bacterium undergoes stress due to the electrochemical gradient, which causes an influx of calcium accumulation and excessive expulsion of protons. Survival of bacteria under such condition depends on factors like active export of calcium and compensations of the lost protons. The energy required for these activations can be accomplished by the metabolic CO2, which further contributes to the increase in the level of dissolved inorganic carbon in the microenvironment that ensures the precipitation of the calcium carbonate (Hammes and Verstraete, 2002). High pH condition favours the formation of CO32– from HCO3– (Knoll, 2003). Finally, the carbonate concentration will increase, inducing an increase in supersaturation level leading to CaCO3 precipitation around the cell in the presence of soluble Ca2+ (Eqs. 14 and 15). 18
ACCEPTED MANUSCRIPT CO( NH 2)2 + H 2O → NH 2COOH + NH 3
(9)
NH 2COOH + H 2O → NH 3+ H 2CO3
(10)
H 2CO3↔ HCO3− + H +
(11)
2 NH 3+2H 2O ↔ 2 NH + 2OH − 3
+
+ 4
−
(12)
−
2− 3
+ 4
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+ 4
HCO + H +2 NH + 2OH ↔ CO + 2NH + 2H 2O Ca2+ +Cell → Cell − Ca2+ 2+
2− 3
− 3
(14) (15)
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Cell − Ca +CO → Cell − CaCO
(13)
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3.3. Microbially Induced Calcium-carbonate Precipitation (MICP) Calcium carbonate is the chief cementation material, which can be produced both
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by natural and artificial way by means of microbial metabolic activities. A schematic
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diagram of suggested bacterial calcium metabolism and subsequent CaCO3
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precipitation is presented in Fig. 1.
Fig. 1 Schematic presentation of suggested bacterial calcium metabolism and subsequent CaCO3 precipitation under high-pH and high-Ca2+ extracellular 19
ACCEPTED MANUSCRIPT conditions. Reprinted with permission from Hammes and Verstraete (2002).
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Briefly, MICP is a straightforward biochemical process governed by five key
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factors: (a) the calcium (Ca2+) concentration (to incorporate with carbonate), (b) the
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concentration of dissolved inorganic carbon (to complex with calcium ions, thus reducing calcium carbonate saturation enhancing the calcite precipitation), (c) the pH
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(to favour the formation of CO32– from HCO3–), (d) the availability of nucleation
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sites (to create a strong electrostatic affinity to attract cations and enables the accumulation of calcium ions on the surface of the cell wall), and (e) urease, nickel
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containing metalloenzyme (to enzymatic hydrolysis). Figure 2 shows the crystals of
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calcium carbonate deposited by MICP. Although urease activity is widespread
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among different groups of microorganism, it was mainly bacteria of Bacillus group, such as Bacillus pasteurii (later renamed as Sporosarcina pasteurii), which were shown to proliferate and express the urease genes (Hammes et al., 2003). Due to the presence of several negatively charged groups on the bacterial cell wall positively charged metal ions, here Ca2+, bind on the bacterial surfaces and subsequently reacts with anions (CO32–) and form calcium carbonate. MICP is a natural phenomenon under microbial calcium metabolism in which active calcium metabolism potentially creates unique precipitation conditions, and carbonate precipitation chemically favours bacterial survival and proliferation (Perito and Mastromei, 2011). Besides urease, there is another enzyme, carbonic anhydrase with very important role in MICP. However, the role of this enzyme has not been studied in detail. 20
ACCEPTED MANUSCRIPT Carbonic anhydrase (CA), EC4.2.1.1, is ubiquitously found in organisms, and is essential to many biological processes including photosynthesis, respiration, CO2
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and ion transport, calcification and acid-base balance (Smith and Ferry, 2000). It has
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a wide distribution and participates in all physiological processes that deal with CO2
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and HCO3 handling, such as cellular pH regulation, acid and ion transport (Achal
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and Pan, 2011).
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Fig. 2 Scanning electron micrographs showing calcium carbonate crystals precipitated by rod shaped Bacillus sp. CR2. Reprinted with permission from Achal and Pan (2014).
CA is a zinc-containing enzyme that can catalyse the inter-conversion of CO2 and HCO3−, which would then promote the CaCO3 precipitation with the help of urease. The incorporation of nickel into the active site of urease is dependent on CO2/ HCO3metabolism, which is in turn regulated by CA (Park and Hausinger, 1995). If HCO3− is the source of dissolved inorganic carbon (DIC), CA may catalyse its conversion into CO2, and the overall reactive equations are as follows (Li et al., 2010): 21
ACCEPTED MANUSCRIPT CA HCO3− + H + → H 2O + CO2 2+
− 3
(16)
+
− 3
Ca +2 HCO → CaCO3 + H + HCO → CaCO3 ↓ + H 2O + CO2
(17)
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When CO2 is the source of DIC, CA catalyzes its conversion into HCO3− with overall reactive equations as follows (Li et al., 2010):
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CA CO2 + H 2O → HCO3− + H +
(19)
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Ca 2 + + HCO3− → CaCO3 ↓ + H +
(18)
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Calcium carbonate precipitation is considered as one of the important biogenic
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carbonate precipitations due to their contribution in selective cementation by producing relatively insoluble organic and inorganic compounds intra- or
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extracellularly. Such a compound can serve as a cementitious material giving rise to
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the term “biocement”. Biocement has attracted much attention because it is a real
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‘green material’; relying on Microbially Induced Calcium-carbonate Precipitation (MICP) (Rong and Qian, 2012a). The technique is highly promising as a long term remediation tool for the enhancement of durability of building structures (Achal et al., 2011a, 2011b, 2012a; Bang et al., 2001; De Muynck et al., 2008a,b; Ghosh et al., 2005; Jonkers et al., 2010; Park et al., 2010; Ramachandran et al., 2001). The fundamental understanding of the process of MICP has emerged. The success of MICP has subsequently led to the exploration of this process in a variety of fields. It has been successfully applied in bioremediation of heavy metals and radionuclides (Achal et al., 2012b, 2012c; Fujita et al., 2004), enhancement of oil recovery from oil reservoirs (Nemati and Voordouw, 2003), and strengthening of sand columns (Achal et al., 2009a; De Jong et al., 2006; Qian et al., 2010). Multiple laboratories 22
ACCEPTED MANUSCRIPT have reported similar findings and established repeatability of the process in the laboratory. However, for the MICP to graduate as biocement it has to develop into an
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industrial product.
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3.4. Biocement
Biocement is the product of MICP that, in its commonest form, has three
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constituents, namely, alkalophilic microbes, substrate solution (urea) and calcium ion
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solution (Achal et al., 2013a; Rong and Qian, 2012a). Bacteria use the nutrient to grow cells, urea as a substrate to hydrolyze, and calcium as the energy source to
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form the bio-mineral. These constituents need to coexist with other materials of
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construction and perform their tasks. They can be used as the sole cementing agent
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or in combination with other cementitious materials. The constituents of biocement can be supplied in various forms. They may be converted into a lyophilized powder, tubular capsules or liquid (Achal et al., 2009a; De Belie and De Muynck, 2009; Wang et al., 2010). Existing structures and components can also be treated with the microbial solution in the form of a spray, coating or by dipping (De Belie and De Muynck, 2009; Ghosh et al., 2005). The biocementation process was chiefly experimented with one of the most common construction materials, i.e., sand by most of research groups (Achal et al., 2009a; Qian et al., 2010; Rong et al., 2012). Bio-sandstones were created by filling a tube with sand mixed with bacterial culture or later, by dripping the bacterial solution through the column. The sand in the tube formed a solid column in a few 23
ACCEPTED MANUSCRIPT days (Fig. 3). While estimating the precipitates in the sand column, Achal et al. (2009b) reported that CaCO3 constituted 24% of the total weight of the sand samples
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plugged by S. pasteurii, which was later improved by 33% with a mutant of S.
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pasteurii (Achal et al., 2011b). The strength of bio-sandstones was also measured,
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which could be up to 6.1 MPa (Rong et al., 2012). The encouraging results of biocementation in sand led researchers to think beyond the limited experiment and
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explore its use along with the existing construction materials. However, the existing
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construction materials throw several challenges.
Fig. 3 A typical set-up to prepare bio-sandstone 4. Challenges Although based on the laboratory experiments biocementation is promising, the technology faces various challenges for scaling up to industrial levels of production. 24
ACCEPTED MANUSCRIPT The foremost is cost of the process. Civil infrastructure requires materials in very large quantities. Thus, they are highly cost sensitive. The large quantity requirements
There must be exhaustive
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survive the tough environment of construction processes.
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also mandate that the yield from the technology must be high. The bacteria must also
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life cycle estimates to demonstrate the sustainability credentials of the technology. Above all, biological technologies are almost unknown to structural communities. A
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popular misconception is that all bacteria are harmful. Often the authors face
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questions from the construction community about the risk of endangering lives of the inhabitants because of the bacteria used in the material. This section briefly explains
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such challenges and some progresses in alleviating them.
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4.1. Cost of the technology
The main cost element in the technology is the nutrient. Biocementation process requires nutrients to grow calcium carbonate precipitating bacteria. The cost of the nutrient can be up to 60% of total operating costs (Kristiansen, 2001). The reported nutritional profile for calcium carbonate precipitating bacteria such as S. pasteurii suggested a high preference for protein-based media (Morsdorf and Kaltwasser, 1989). The nutrient media containing yeast extract, nutrient broth (NB), glucose, BactoCasitone, meat extract and peptone are widely used in biocementation (Achal et al., 2009b; De Muynck, 2009; Jonkers et al., 2010; Rodriguez-Navarro et al., 2003; Rong et al., 2012). The cost of such nutrients is estimated to be about US $250 (180 €) per kg, 25
ACCEPTED MANUSCRIPT which could generate around 13L of bacterial culture and produce 5.4×106 unit urease (Achal et al., 2010a). One unit of urease is defined as the amount of enzyme
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hydrolysing one micromole of urea per minute. To reduce this cost while producing
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similar or even higher amount of urease search for alternative inexpensive nutrient
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sources are imperative. Researchers have discovered several replacements of the standard nutrient media with industrial effluents. Many of the industrial effluents that
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have high concentrations of protein can be used as nutrients. Thus dual benefits of
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cost reduction and environmental protection can be achieved. Achal et al. (2009b) successfully replaced the standard nutrients by lactose mother liquor (LML),
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collected from dairy industry, as a medium for biocementation by S. pasteurii. They
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observed that LML is a good source of nutrients supporting bacterial growth, urease
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production and calcite precipitation. The process was further improved by replacing the standard medium with corn steep liquor (CSL), of corn wet milling industry (Achal et al., 2010a). They reported comparable growth rate of S. pasteurii and significantly higher urease production than nutrient broth or yeast extract media. A higher percentage of calcium carbonate was deposited in the sand columns prepared with bacterial cells grown in the medium containing CSL than the standard media. They also measured other performance tests such as strength, permeability and reinforcement corrosion of construction materials. All parameters exhibited significant improvement. It had an additional benefit of recycling a harmful industrial byproduct. This outcome opens up huge opportunities of adapting biomineralization with a host of protein rich wastes ranging from industrial effluents 26
ACCEPTED MANUSCRIPT to domestic sewage available all over the world.
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4.2. Increasing yield
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Biocementation process is highly dependent upon the yield of urease and calcium
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carbonate. However, the process is delicately balanced on the pH levels. It is envisaged that the biological process must coexist with the existing processes of
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cementation. Harsh environment of cement concrete, especially its pH of around 10,
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can make it difficult for the bacteria to survive and reduce the yield. Achal et al. (2009a) developed phenotypic mutants of S. pasteurii by UV
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irradiation to increase the yield. They compared the mutant with the wild-type with
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respect to growth at high pH, urease production and calcite precipitation. Their results
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showed growth ability of at pH range 6-10, while one of mutants Bp M-3 could grow at pH from 5 to 11, suggesting the efficacy of phenotypic mutants in enhancing calcite precipitation. A significant increase in the urease production and higher calcite amount in bio-sandstones were also observed in case of UV induced mutant of S. pasteurii. Further, they found that the mutant had no side effects and it was in fact enhancing the durability of concrete. Improvement of microbial strains for the overproduction of enzymes has been the hallmark of many other commercial processes. In this case too the improved strains can reduce the cost of the biocementation process with increased productivity, survivality and some other special characteristics. However, further research is essential to make a significant progress. 27
ACCEPTED MANUSCRIPT 4.3. Alkaline conditions The alkaline condition of cement due to high pH, which ranges from 10 to 13 (Lea,
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1970), makes hostile environment for the bacterial growth. This is one major
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challenge faced by the biocementation process. However, exposure of cement to
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atmospheric CO2 results in a carbonation reaction, which gradually reduces the surface pH (Ismail et al., 1993). In addition, because of the high pH of the cement,
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bacterial cells might initially be growing slowly and acclimatize to high pH conditions
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during the curing period. Upon cell growth, calcium carbonate would start precipitating on the cell surface and within the cement mortar matrix (Achal et al.,
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2010a). However, if calcium carbonate precipitating alkali-tolerant or alkalophilic
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bacteria can be found, the problem can be alleviated. Some structural components of
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the cell wall of such bacteria, such as teichuronopeptide, contribute to pH homeostasis at alkaline pH and favour bacteria to survive in alkaline environments (Aono et al., 1999).
In order to protect the bacterial cells from high pH of cement, some researchers encapsulated bacterial cells during experimentation. Bang et al. (2001) initiated such research by adopting an innovative immobilization technique for calcium carbonate precipitation in concrete during the biocementation process. They utilized polyurethane (PU) to immobilize the whole cells of B. pasteurii and introduced in the cracks of concrete as PU strips of 10 mm (diameter) × 50 mm (length) with different concentrations of bacterial cells. As a result of biocementation, the compressive strength of the cracked concrete specimens increased by 12% compared with ones 28
ACCEPTED MANUSCRIPT only remediated with PU. The electron micrographs showed calcite crystals throughout the PU matrices, with B. pasteurii embedded in calcite crystals (Fig. 4).
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The physicochemically versatile PU was shown to be an effective enhancement tool
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in MICP in concrete cracks. Such immobilization technique offers several advantages
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for concrete remediation, in which encapsulated cells retain high metabolic activities and are protected from adverse environmental changes in cement. Later, Bachmeier et
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al. (2002) studied the behaviour of the PU-immobilized B. pasteurii urease in calcite
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precipitation at varying temperatures. PU was found to protect the bacterial cells from the high pH of cement and also higher temperature without affecting calcite
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precipitation. The native bacterial isolates from highly alkaline cement should also be
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effective in biocementation in concrete. Achal et al. (2010b) isolated Bacillus sp. CT5
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from commercially available cement, which could sustain calcium carbonate ability at pH 10 within 16 hours and produced 670 U ml-1 of urease.
Fig.
4
Scanning
electron
micrographs
of
calcite
precipitated
by B.
pasteurii immobilized in PU. A. Cells associated with calcite crystals in PU matrices. B. A magnified section boxed in (A). Reprinted with permission from Bang et al. (2001) 29
ACCEPTED MANUSCRIPT Recently, Wang et al. (2012) investigated the possibility of using silica gel as the carrier for protecting the bacteria in cement. Their results showed that silica gel
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immobilized B. sphaericus exhibited a higher urease activity than the polyurethane
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immobilized bacteria, and hence, more CaCO3 precipitated in silica gel (25% by mass)
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than in polyurethane (11% by mass). Thus, there are two routes of ensuring biocementation in concrete- immobilizing the bacteria in a protective environment
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and developing alkalophilic strains. As more results get reported the apprehension of
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bacterial growth in highly alkaline environment is likely to recede.
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4.4. Perceived harmful effects- chlorides
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The most common construction material, concrete is liable to face attacks from
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chlorides. The excessive amounts and penetration of chloride in concrete come from external sources and occurs by various transport mechanisms depending on the exposure conditions. Structures in coastal areas, are widely exposed to airborne chlorides or in direct contact with seawater. A vast majority of concrete structures are reinforced with steel. Steel is susceptible to corrosion in presence of chlorides. Thus, chlorides are avoided in reinforced concrete. In biocementation process calcium chloride is often used as a source of calcium. High solubility of the salt makes it a preferred source. However, the chloride should not cause corrosion in steel. Also, for the technology to be effective in coastal regions the chloride resistivity of the bacteria must be examined. Some researchers have explored alternative sources of calcium such as calcium 30
ACCEPTED MANUSCRIPT nitrate, calcium lactate, calcium acetate and calcium glutamate. Calcium nitrate was used as an efficient calcium source for the deposition of CaCO3 by B. pasteurii in
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order to corrosion protection of cement-based building materials (Qian et al., 2009).
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While using calcium nitrate, in calcium carbonate deposits the main polymorph was
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calcite along with small quantities of vaterite. The CaCO3 precipitated on cement induced by urease greatly improved their surface permeability resistance and resist the
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attack of the acid (Qian et al., 2009). De Muynck et al. (2008a) obtained granular
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grains, presumably of vaterite with calcite crystals on the surface of cement mortars when B. sphaericus utilized calcium acetate as Ca source.
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In order to study biocementation to heal cement stone specimens of dimension 4
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cm3 with B. cohnii, Jonkers et al. (2010) exploited calcium lactate as calcium source
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that resulted into copious amounts of 20-80 µm sized CaCO3 precipitates on crack surfaces. The precipitation based on MICP due to bacterial metabolic conversion of calcium lactate was according to the Eq. 20. CaC6 H10O6 + 6O2 → CaCO3 + 5CO2 + 5 H 2O
(20)
Jonkers et al. (2010) concluded that the process of bacterial mineral formation from calcium lactate represents an alternative calcium source to the more commonly used calcium chloride. In contrast to the calcium chloride, metabolic conversion of calcium lactate does not result in excessive ammonia production what drastically increases the risk of reinforcement corrosion (Neville, 1996) and degradation of concrete matrix, particularly when further oxidized by bacteria to yield nitric acid (Diercks et al., 1991). Recently, Achal and Pan (2014) studied role of various calcium 31
ACCEPTED MANUSCRIPT sources in calcite precipitation and reported urease produced by Bacillus sp. CR2 with
ml-1 in calcium acetate and 389 U ml-1 in calcium oxide.
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calcium chloride was 432 U ml-1 compared to 418 U ml-1 in calcium nitrate, 401 U
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Recently, Xu et al. (2014) reported that the addition of a calcium source led to
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heterogeneous precipitation of CaCO3 crystals both on the biofilm and inside the pores of cement mortars, which played the main role in the coating system. They used
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calcium lactate and calcium glutamate as calcium source to study biocementation by
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B. cohnii. They observed larger thickness of the CaCO3 layer precipitated using calcium glutamate than that from calcium lactate. However, almost the same
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improvement of durability performance on cement mortars was obtained due to a
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calcium glutamate.
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lower porosity of the CaCO3 layer from calcium lactate compared with that from
Most calcium carbonate precipitating bacteria are resistant and active in the environment of high chloride contents. When bacteria were subjected to high salt (NaCl) concentration (0-10%), Bacillus sp. CT-5 was able to grow well at 10% NaCl amended media and other species of Bacillus, such as B. megaterium, B. subtilis and S. pasteurii showed good growth in carbonatogenesis media with 7 or 8% NaCl (Achal et al., 2010b). Most of the researchers reported the ability of bacteria in cementitious materials in the presence of chlorides (Achal et al., 2011a; De Muynck et al., 2008a; Rong and Qian, 2012). Thus, MICP remains largely unaffected in chloride rich environment and one should expect success of biocementation process in coastal and marine conditions. 32
ACCEPTED MANUSCRIPT Other than chlorides (Cl−), concrete structures are also exposed to corrosive substances in the environment such as SO42−, CO2 and atmospheric moisture.
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Maintenance and repair of such structures are important for their continuing function.
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Reddy et al. (2010) reported that at a cell concentration of 105 cells per ml Bacillus
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subtilis increases the concrete resistance to sulphuric acid attack, which was later optimized at 106 cells per ml by Afifudin et al. (2011). Biocementation improves the
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permeation resistance of cementitious materials by forming a protective layer of
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calcium carbonate on the surface of cement-based material with the aid of bacteria (Rong and Qian, 2012). Thus, there is no demonstrated susceptibility of biocement to
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harsh environmental condition.
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The choice of calcium source is an important decision as it ultimately affects cost.
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Calcium chloride seems to be a natural choice. However, there are apprehensions of the presence of chloride ion adversely affecting reinforced concrete due to corrosion. In order to alleviate that concern other calcium sources such as calcium nitrate, calcium lactate and calcium glutamate have been explored. Although the deposition of calcium carbonate was comparable with other salts and found maximum with calcium chloride. Results of investigation of reinforcement corrosion and durability with calcium chloride are reported in the next section.
5. Structural properties To be accepted, biocement must exhibit improvement in structural properties, both as a standalone cementitious agent and as a supplementary cementitious 33
ACCEPTED MANUSCRIPT material with other materials of construction. Thus, structural property tests for a range of functions are imperative. This section reports the structural performance of
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biocement. The quality of cementitious materials is mainly judged by parameters
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such as strength, resistance to moisture and chloride permeability and corrosion of
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reinforcement. To mitigate the risk of unknown consequences new materials are initially used in conjunction with other well known materials. Thus, their
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performance must be judged along with the established structural materials. A large
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number of researchers have reported the structural properties of biocement.
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5.1. Strength
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Biocement has been studied as a deposition on cement mortar and concrete
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(Achal et al., 2010a, 2011a; Ghosh et al., 2005). A cement mortar (or mortar) refers to a building material that is a mixture of cement, sand and water, while concrete contains aggregates in addition to substances that consist of cement mortar. These materials are frequently labelled by their strength. The results of compressive strength tests are generally utilized to establish whether the mortar or concrete mixture as delivered meets the requirement of the specified application. It is measured by subjecting the specimens to compressive forces in a Universal Testing Machine. The strength is calculated from the maximum load withstood by the sample before failure divided by the cross-sectional area resisting the load and expressed in units of psi (Pound-force per square inch) or MPa. Hydration of cement leads to gradual gain in strength. For a standard test, the strength is measured at 34
ACCEPTED MANUSCRIPT different time intervals after casting usually up to an age of 28 days. The strength at 28 days is considered for labelling the grade of the specimen (ASTM C 109-13).
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Biocement was first demonstrated to improve the compressive strength of
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cement mortar cubes by Ramakrishnan et al. (1998, 1999). Later, Ramachandran et
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al. (2001) investigated MICP technology in detail on mortars. They cast mortars of dimension 50.8 mm × 50.8 mm × 50.8 mm that contained 240 g cement, 660 g sand
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and 16.4 ml of B. pasteurii suspended in saline or phosphate buffer. Cement mortar
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cubes were removed from their moulds after 24 hours and placed in urea-CaCl2 medium for 7 and 28 days. When they were tested for compressive strength a
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significant improvement in strength of cement mortar cubes was observed at 28 days.
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Along with bacterial induced carbonate precipitation, they concluded that the overall
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increase of strength followed the presence of an adequate amount of organic substances in the matrix because of microbial biomass, which was later observed by Achal et al. (2011a). The overall trend of an increase in compressive strength might be attributed to the behaviour of microbial cells within the cement matrix, as represented in Fig. 5. Once bacterial cells start precipitation calcite, it plugs the pores on the cement mortar matrix. The impediment of nutrients or oxygen flow to bacterial cells either kill them or turned into endospores and eventually act as organic fibres (Ramachandran et al., 2001). This phenomenon may be associated with the increase of compressive strength of the mortar cubes. Researchers found a monotonic increase in compressive strength as the curing progressed to 28 days. However, the amount of increase may vary with different 35
ACCEPTED MANUSCRIPT microbes and their environment. Ghosh et al. (2005) found 17% increment in the compressive strength after 7 days, which was further increased by 25%, from 23.1
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MPa to 28.9 MPa, at 28 days at 105 cells/ml of Shewanella sp. The performance of
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biocementation to improve compressive strength might be dependent up on media
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used to grow bacterial cells and cure mortar specimens. While replacing nutrient media with LML, Achal et al. (2009b) reported similar compressive strength
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increment of mortars, which was 17% higher at 28 days (26.3 MPa) with S. pasteurii
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compared to control (23.2 MPa). Later, 35% improvement in the compressive strength at 28 d (31.2 MPa) was measured when CSL media was used (Achal et al.
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2010a). Biocementation also showed significant improvement in the split tensile
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strength, as measured by the diametral compression tests in bio-sandstones prepared
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with B. sphaericus and Bacillus sp. (Shirakawa et al., 2011).
Fig. 5 Behavior of bacterial cells inside cement mortar cubes during compressive strength test
5.2. Water permeability Concrete and mortar are porous materials. Understandably, they allow moisture 36
ACCEPTED MANUSCRIPT and other dissolved elements to percolate. Some of these elements may react with the constituents of concrete and alter their properties. Thus, durability of these
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structures can get affected. Permeability governs the rate of flow of fluid into mortar
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or concrete. Water permeability refers to the amount of water migration through
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concrete when it is under a constant water pressure (El-Dieb and Hooton, 1995). As concrete is a porous material, moisture movement can occur by flow, diffusion, or
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absorption. Popular commercially available substances that can be used to hinder
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permeability are synthetic polymers. They have been sometimes found incompatible with the underlying layer due to differences in their thermal expansion coefficient.
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They are also susceptible to environmental exposure such as ultraviolet rays (Reddy
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et al., 2012). Some of these materials are reported as toxic. Through biocementation
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a layer of calcium carbonate can be deposited on the surface of the substrate. Due to the similarity in chemical composition, biocement is compatible with the substrate. It is unaffected by ultraviolet rays and it is non-toxic. These are the main attractions of using biocement to hinder permeability. Biocementation has been proved to reduce the water permeability at a very significant rate. The precipitation of new calcite crystals inside the pores of stone was proposed long back with the application of living cultures of selected calcinogenic bacterial strains (Orial et al., 1992). Such ability of bacteria was later noticed in limestone, when stone specimens were inoculated with Micrococcus sp. and B. subtilis in different sets of experiments, resulted in 60% reduction in water absorption (Tiano et al., 1999). The bacterial strains reduced the stone porosity by 37
ACCEPTED MANUSCRIPT producing high amount of biological matter and calcite crystals, later termed as `biocalcin' (composed mainly of encrusted bacterial bodies mixed with carbonate
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excretates). It ensured the protection of limestone by limiting exchange between the
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interior of the rock and atmosphere, in addition to restricting the penetration of
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degrading agents into the stone (Le Metayer-Levrel et al., 1999). This success led researchers to study the effect biocementation on cementitious materials.
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The effects of MICP on the durability of mortar specimens with different
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porosity were investigated. The surface deposition of calcium carbonate crystals reduced the water absorption by 65 to 90% depending on the porosity of the mortar
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specimens of dimension 40mm×40mm×160mm (De Muynck et al., 2008a). Bacterial
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deposition of a layer of calcite on the surface of the specimens decreased the
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capillary water uptake and permeability (De Muynck et al., 2008b). Concrete cylinders of 80mm diameter and 20mm height were tested for water permeability after treating with B. sphaericus in cementation media and it was observed that biocementation reduced concrete permeability much more than the conventional cement grout repair technique (De Belie and De Muynck, 2009). The presence of Bacillus sp. CT-5, a cement isolate, followed a significant decrease of the water uptake compared to untreated mortars cubes of side dimension 70.6 mm (Achal et al., 2011a). The mortars with bacterial cells absorbed nearly six times less water than the control cubes over a period of 168 hours, while water absorption was five times less in mortars with a mutant of S. pasteurii, Bp M-3 (Achal et al., 2011b). The deposition of a layer of calcium carbonate crystals induced 38
ACCEPTED MANUSCRIPT by bacterial cells on the surface resulted in a decrease in absorption. Achal et al. (2011b) used a mutant of S. pasteurii, Bp M-3, with two different media (containing
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NB and CSL) during concrete preparation and measured both top and side water
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penetration on concrete cubes of dimension 150 mm (M20 grade). Compared to the
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top and side water penetration of 33 mm and 40 mm, respectively in control, the bacterial cells significantly reduced the permeation, measured as 8 mm, 17 mm in
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NB and 11 mm and 25 mm in CSL, on top and side surface, respectively. Scanning
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electron microscope images showed that the calcium carbonate crystals had been deposited into the pores of the interface creating a denser interfacial zone between
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the substrate and the deposit. It was observed that the deposition was effective both
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on horizontal and vertical faces. Thus, both horizontal elements such as slabs and the
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vertical elements such as walls can be remediated. Understandably, the deposition is better on the horizontal surfaces such as slabs due to gravitational assistance. Such results suggest that biocementation is effective in impeding water absorption in the cementitious substrates. It can be used both in new and old structures. Thus, the technology has a potential in repairing deteriorated structures.
5.3. Chloride ion permeability Corrosion of reinforcing steel due to chloride ingress is one of the most common environmental deteriorations that lead to early failure of reinforced concrete structures. The rate of chloride ion ingress into concrete is mainly dependent on the internal pore structure. The transportation of chloride ions into concrete involves 39
ACCEPTED MANUSCRIPT diffusion, capillary suction, permeation and convective flow through the pore system. Biocementation has been successfully demonstrated in reducing chloride ion
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permeation in concrete, thus enhancing its durability. The rapid chloride permeability
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test (RCPT) is used extensively in the concrete industry (Bentz, 2007). ASTM
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C1202-05 specifies the rating of chloride permeability of concrete based on the charge passed through the specimen during a 6-h testing period. The permeability is
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defined in classes such as high, moderate and low.
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Resistance towards chloride penetration of B. sphaericus treated cement mortar cubes of dimension 100 mm3 was measured with the use of an accelerated migration
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test by De Muynck et al. (2008b). Biocementation resulted in significant lower
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chloride migration coefficients compared to untreated specimens. This is due to the
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reduced surface porosity as a result of MICP. The chloride penetration in the cylindrical concrete samples of diameter 100 mm and thickness 50 mm, prepared with and without a mutant of S. pasteurii, Bp M-3, was measured by RCPT (Achal et al., 2011b). The permeability class type was recorded ‘‘moderate’’ for control concrete specimens, while the class changed to ‘‘low’’ type of concrete with bacterial cells as per ASTM C1202-05. For control samples, the average charge passed was 3177 C, whereas for samples prepared with bacterial cells in NB and CSL media it was 1019 C and 1185 C, respectively. Test results indicated 1019 and 1185 Coulombs passed for concretes with bacteria compared to 3177 coulomb for control samples. Thus, biocementation was effective in dramatically reducing the ingress of chlorides into the concrete. 40
ACCEPTED MANUSCRIPT 5.4. Reinforcement corrosion Corrosion of steel reinforcement in concrete is the most common problem
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affecting the durability of such structures. The ingress of moisture, chloride ions, and
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carbon dioxide through the concrete initiates corrosion in the steel. The volume of
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corrosion products is a few multiples higher than the metal. Therefore, surface corrosion of steel creates bursting pressure in concrete leading to cracking and
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spalling. The cracks make access of corrosive substances easier. As a result, corrosion
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is accelerated. Chloride-induced corrosion is one of the main mechanisms of deterioration affecting the long-term performance of such structures. Evidently, high
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permeability can lead to early corrosion. The process is further accelerated in the
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coastal region due to the presence of airborne chlorides.
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There are two ways bio-deposition can help in reducing corrosion, 1) by sealing the pores of concrete, 2) by sealing the cracks formed due to corrosion in deteriorated concrete. The MICP can also use up the oxygen present that would otherwise be involved in the corrosion process of the steel bars. Qian et al. (2009) tested cement mortar cubes biodeposited with S. pasteurii to acid attack by dripping a drop of H2SO4 of pH 0.5 to 5.5 on the mortar surface. The mortar cubes resisted the acid above pH 1.0 and acid resistance value was measured to 1.5. As pH of acid rain in the environment is between 3.5 and 5.6, much higher than pH resistance value of biodeposited mortars, the MICP treatment can resist the corrosion of the acid rain to a certain extent. Later, the detailed investigation leading to positive impact of biocementation in 41
ACCEPTED MANUSCRIPT the reinforced concrete (RC) corrosion prevention was performed by Achal et al. (2012a). They prepared the RC specimens with Bacillus sp. CT-5 of dimension
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200mm×200mm×100mm, cast with one 25 mm diameter twisted deformed steel
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reinforcement bar at the centre of length 300 mm, and induced corrosion by applying
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a constant anodic potential of 40V for 7 days. In the control specimens several cracks appeared due to corrosion. A longitudinal crack of width 0.3 mm developed along the
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reinforcement within 36 hours. The crack continued to grow. At 7 days of accelerated
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corrosion several (at least seven) cracks with width up to 0.2 mm (0.008 in.) were observed on control specimens. In the bacterially treated specimen the appearance of
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cracks was much delayed and their widths were limited. At the end of seven days no
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crack beyond 0.2mm was observed. Thus, biocement deposition had protected the
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reinforcement from corrosion.
The effectiveness of biocementation as a protective agent even after the corrosion has set in was estimated by corrosion rates of specimens (Achal et al., 2012a). In a corroding sample, the rate of corrosion is considered proportional to the corrosion current density Icorr for the specimen. The results indicated that Icorr was limited to 15mA/m2 and 20 mA/m2 for samples with bacteria compared to 61mA/m2 for control samples. A four-fold reduction in corrosion rate by Bacillus sp. CT-5 suggests that the calcite precipitation has the effect of greatly reducing corrosion (Achal et al., 2012a). The results suggest that the formation of calcite might facilitate the protective passive film around the steel and act as a corrosion inhibitor by interrupting the transport process. Further, they also found estimated the residual bond strength between the 42
ACCEPTED MANUSCRIPT corroded reinforcement and the surrounding concrete. The bond strength can be estimated by pulling the reinforcement out of concrete and observing the pullout force.
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The pullout strength was significantly enhanced through biocementation. They
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observed that the reinforcements lost less metal to corrosion when bacterial deposits
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were applied.
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6. Applications
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Clearly, biocementation has shown great promise in the laboratory. The next step is to scale the technology up for field applications. This section envisages the most
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attractive applications of the technology. This technology can be applied as binder,
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cement admixture, barrier or protective layer and also as a repair system in
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construction materials. The various applications of biocementation have been summarized in Table 3. Each application is discussed briefly.
6.1. As a binder
The basic function of a binder of granular materials such as sand is to develop a monolithic structure out of them. Cement performs that function in present day structures. Bacterial deposition can also perform the function in a variety of ways while almost completely avoiding the greenhouse gas emission associate with industrial cement. When the calcium carbonate producing bacterial solution is first introduced into the sand particles, numerous bacteria bind on the surface of sand particles after the solution is fully exudative (Rong and Qian, 2012). In the presence 43
ACCEPTED MANUSCRIPT of substrate and calcium source, bacterial cells precipitate calcite through MICP and the adjacent sand grains are connected to form a whole sand body, known as
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bio-sandstone with a certain degree of strength (Qian et al., 2010). Such
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biocementation process has a huge potential to apply in strengthening the sand crust
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layer in desert or dust fixation in dirt tracks, sealing of the channels and reservoirs in sandy soil, sand dunes, stabilization of sandy soil slopes, construction of aquacultural
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ponds and roads, and to improve the mechanical properties of soil.
6.2. As a cement admixture
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Although cement is known to have high embodied energy it is hard to imagine
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that cement will be completely replaced in foreseeable future. To control the high
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embodied energy of cement several siliceous and calcareous industry byproducts such as fly ash and blast furnace slag, have been used as an admixture to cement. In a similar way, the bacterial product can be used as an admixture to cement or in conjunction with other admixtures. To ensure a positive impact on the embodied energy the admixtures must be locally available and preferably should be an industrial byproduct. Most commonly available admixture is fly ash that is produced due to the combustion of coal for generation of power. There are locally available silicious admixtures that are byproducts of burning crop residues as fuel. Rice husk ash is probably the most common among them. All these residues are recognized as an environmental pollutant (Dhami et al., 2012). Using admixtures such as fly ash and rice husk ash with bacteria for biocementation is an effective way of utilizing such 44
ACCEPTED MANUSCRIPT problematic wastes to combat pollution in the environment. Both these materials have been proved to cause no degradation in the quality of products (Nasly and Yassin,
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2009). While the utilization of fly ash as an admixture for developments of
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biofertilizers has been done by many researchers (Gaind and Gaur, 2003; Kumar and
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Gupta, 2010), fewer studies have been reported on the use of fly ash as an admixture in biocementation studies (Dhami et al., 2012, 2013a). Biocementation was proved to
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enhance the durability of such construction materials containing fly ash and rice husk
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ash.
However, the bacterial admixture needs to be made amenable to mixing with
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cement and it is to be tested for survival in its journey from the cement plant to the
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site of construction. In order to develop efficient formulations using fly ash as a
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cement admixture, Dhami et al. (2013a) optimized the temperature and moisture conditions for maximum survival of bacterial cells. They prepared bacterial formulations by adding bacterial slurry of B. megaterium SS3, B. cereus SS5 and Lysinibacillus fusiformis SS18 to 250 g fly ash, which showed viability of up to 12 months. While studying microbial calcification in sand plugs, Dhami et al. (2013a) reported high amount of calcite precipitates in case of formulated bacterial cells with 28% and 24% reductions in water absorption and porosity, respectively, compared to the control specimens. One of the most high embodied energy materials used by the construction industry is brick. It uses agricultural soil as its raw material and bakes it at a high temperature. Thus, energy consumption of baked bricks is around 3 MJ/Kg. To reduce the energy 45
ACCEPTED MANUSCRIPT consumption concrete blocks that do not need baking is used. Bricks made of soil and sand with a small quantity of cement that do not require baking are the most energy
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efficient alternative (Reddy and Jagadish, 2003). However, due to the presence of high
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proportion of clay such blocks are known to absorb moisture and swell. Use of
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biocementation in the clay can reduce water absorption to a great degree (Dhami et al., 2013b). Soil–cement blocks with density 1.75 Kg/m3, soil: sand 1:1 along with 7%
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cement content were mixed with 10% B. megaterium culture medium. Cylinder
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specimens of diameter 3.8 cm and height 7.6 cm were tested. Biocementation resulted in the formation of a whitish layer on the surface of blocks that was attributed to the
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formation of CaCO3 precipitates with 32% content and resulted into 40% reduction in
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the water absorption. Mercury intrusion porosimetry data of bacterial and control
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soil-cement specimens reflected that total porosity of control specimens was 25.3 % upon calcification, which was reduced by 31%. These are a few initial applications of bacteria as an admixture. The authors believe that there are many other applications waiting to be explored.
6.3. As a barrier layer
Structures are often protected by a barrier layer to impede intrusion of moisture and other deleterious substances. A common barrier layer is synthetic polymeric coatings such as polyurethane. The barrier layer must be durable, non-toxic, and compatible with the substrate. Another important aspect is breathability of the layer i.e. moisture inside the substrate should be able to escape. Although polymeric coatings are very effective in the short term, they are known to degrade with exposure 46
ACCEPTED MANUSCRIPT to ultraviolet rays. They are sometimes toxic. When they are not breathable due to the vapour pressure, they leave the substrate and blisters appear. An ideal barrier layer is
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the one that is closest in composition (and hence in properties) with the substrate.
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cement. Thus, it should be a good barrier material.
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Biocementation produces a deposit that is very close in composition with that of
Early applications of biocementation as a protective layer on limestone
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monuments have shown that the protective effect of the bacterially deposited calcite
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layer lasted for several years (Castanier et al., 1999). More recently, biocementation has been investigated by several researchers for the deposition of a protective surface
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layer as barrier, with consolidating and waterproofing properties, on the surfaces of
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building materials as biodeposition (De Muynck et al., 2010). In the patented ‘Calcite
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Bioconcept’process, a layer of selected bacteria is sprayed on the surface together with specific nutrients and calcium ions (Adolphe et al., 1990). Over time, this layer is supplied repeatedly with the nutrient medium and eventually a layer of calcite is precipitated.
The common way of testing bacterial cells as a barrier layer is explained by De Muynck et al. (2008a). They immersed the mortar cubes and concrete cylinders for 24 hours in 0.3 and 0.6L, respectively, of a 1-day-old stock culture of B. sphaericus prior to submersion in the nutrient solution, and tested for different parameters such as capillary water suction and permeability. They concluded that deposition of a layer of bacterial calcite on the surface of the specimens resulted in a decrease of capillary suction and a decrease in gas permeability. Biodeposition was also reported to better 47
ACCEPTED MANUSCRIPT resist the migration of chlorides in concrete cubes than that of the acrylic coating and the water repellent silanes and silicones or silanes/siloxanes mixture (De Muynck et
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al., 2008b).
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The bio-treatment was applied on the plaster as a barrier in order to protect
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degradation of historical buildings (Anne et al., 2010). The freeze-dried B. cereus was re-hydrated with the nutritive solution and the culture medium was sprayed onto the
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building facade (about 1L m-2). The bacteria were fed with a nutritive solution for 24,
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32, 48 and 72 hours after spraying. Upon bio-treatment, a calcite coating was visualized on the plaster surface. It was noted that water penetration and surface
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deposition.
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porosity was considerably reduced in the treated samples due to calcium carbonate
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A ureolytic biodeposition treatment was applied to conserve limestone in order to investigate the effect of pore structure on the protective performance of a biogenic carbonate surface treatment (De Muynck et al., 2011). The presence of biogenic carbonate as a barrier layer resulted in a 20-fold decrease in the rate of water absorption, which resulted in increased resistance to sodium sulphate attack and freezing and thawing. Rong and Qian (2012) introduced the brushing method to apply bacterial cells as a barrier layer on the surface of cementitious materials. Large numbers of bacteria were absorbed on the surface of cement mortar of dimension 30mm3 due to multiple brushing. The result showed a layer of white precipitation substances on the surface of mortar after 3 days. After curing for 7 days, there was a dense white precipitation on the surface, identified as calcite by EDS and XRD 48
ACCEPTED MANUSCRIPT analyses. The water absorption of mortar surface was decreased by 85% due to biocementation. The deposits from B. megaterium were found to be very effective to
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treat fly ash bricks that showed significant reduction in water absorption, better frost
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resistance and increased compressive strength due to calcite deposition on the surface
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and voids of bricks (Dhami et al., 2012). The water absorption was reduced by 46%, while the compressive strength was enhanced by 24.2% compared to the control
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specimens.
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Another potential application of the technology is in curing of concrete. Cement hydration in concrete is a slow process that continues for several years after casting.
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However, most of the hardening and strength gain happens in the first seven days.
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During this time, water is regularly supplied to concrete to ensure proper hydration of
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cement. This process is called curing of concrete. It is possible to supply the bacterial culture instead of water at the time of curing. With bacterial culture one hopes to deposit a layer of calcium carbonate on the surface of the structure automatically creating a barrier layer. Such structures should be more durable than water cured ones. Although the initial results are promising there are several steps before one can hope to scale the technology up to industrial level. The technology must be packaged in a manner that is convenient to industrial application; the field personnel must be trained; and above all, its durability must be studied with long term tests.
6.4. As a repair system Cracking is a common phenomenon in concrete due to its relatively low tensile 49
ACCEPTED MANUSCRIPT strength. In addition, natural processes such as weathering, faults, land subsidence, earthquakes, and human activities create fractures in concrete structures that affects
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the service life of the structure. Though a variety of techniques is available for crack
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repair, those repair systems often have different thermal expansion coefficient
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compared to concrete and are environmental and health hazards (Jonkers et al., 2010). The basic concept in applying biocementation in repair systems is to embed calcite
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producing bacteria, which grow inside the cracks and precipitate calcium carbonate
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thereby closing the gaps.
Gollapudi et al. (1995) investigated such crack repair system where they indicated
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complete plugging in damaged rock within a day of nutrient injection in a sand
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packed column containing S. pasteurii. This biocementation process was highly
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effective in the presence of fractures as they appear to create nucleation sites for bacteria clusters, leading to improved selective plugging. Later, such technique was effectively shown in repairing cracks in cementitious materials. A novel approach of microbiologically-enhanced crack remediation (MECR) was successfully initiated in concrete (Ramachandran et al., 2001). Cement mortar cubes of dimension 50.8 mm3 were provided with a cut of constant width 3.175mm and different depths of 12.7, 19.05 and 25.4 mm, to simulate cracks. The cracks in the specimens were filled with natural sand and S. pasteurii, while with only sand in control specimens to evaluate MECR efficiency. The specimens were cured in urea-CaCl2 broth for 28 days and tested for their compressive strength. Upon physical examination it was noticed that the sand particles were held together due to MICP in 50
ACCEPTED MANUSCRIPT the crack remediated areas. In the mortar specimens with the deepest crack of 25.4 mm, biocementation increased the compressive strength by 60% than that of the
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control. Distinct, sharp-edged calcite crystals with rod-shaped holes, occupied by the
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in crack remediation (Ramachandran et al., 2001).
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S. pasteurii were seen under SEM examination, confirmed the role of biocementation
MECR experiments were also conducted on larger concrete specimens. They used
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ultrasonic waves for verifying crack sealing. Ultrasonic wave transmission is a novel
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way to study effectiveness of crack healing in concrete. Ultrasonic waves travel easily through hardened concrete. When they find an interface of water or air, as in a crack,
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they get reflected from the crack face. Thus, the existence of the crack can be
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determined. When the crack is sealed the interface of concrete with air or water is
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eliminated. Therefore, the waves are able to travel through the sealant resulting in reduced reflection (Van Tittelboom et al., 2010). De Belie and De Muynck (2009) made standardized cracks in concrete samples of 160mm × 160mm × 70mm by introducing copper plates of 0.3 mm thickness up to a depth of 10 or 20mm into the fresh concrete. They also obtained realistic cracks by splitting fibre reinforced polymer concrete cylinders. Biodeposition treatment was conducted by immersing the concrete samples for 24 hours in a B. sphaericus culture grown overnight in 20g/L yeast extract and 20 g/L urea, followed by 3 days in equimolar solution of urea and CaCl2. The MECR was evaluated by determining the transmission time of ultrasound waves and the water permeability coefficient. Like other researchers they also reported that a deposition of calcium carbonate was visible at the crack edges and on 51
ACCEPTED MANUSCRIPT concrete surface (Achal et al., 2010a; De Muynck et al., 2010; Ramachandran et al., 2001). The ultrasound transmission testing proved that complete sealing of artificial
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cracks of 0.3 mm wide and 10 mm deep due to biodeposition and also such samples
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executed improved water permeation resistance. Similar experiments were carried out
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by Van Tittelboom et al. (2010). They immobilized the B. sphaericus cells prior to crack repair in order to protect against high pH in concrete. Biocementation resulted
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in an increase in ultrasonic pulse velocity, which indicated the formation of crack
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bridging due to MICP. Further, Visual examination of the cracks proved that this technique resulted in complete healing of the cracks. Microbial remediation of
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concrete cracks was observed to be more efficient in shallow cracks than in deeper
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Muynck et al., 2010).
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cracks primarily due to aerobic nature of the bacteria used (Achal et al., 2010a; De
Further, self-healing is an ultimate dream of construction engineers. As the world’s population stabilises the necessity of new infrastructure would alleviate. With present technologies, often it is more expensive to maintain a structure than to build a new one. In this scenario, the idea of self-healing inspired from natural phenomena such as skin of animals looks promising (Van Breugel, 2007). Self-healing is an automatically initiated response to damage or failure and in order to perform the healing, self-healing system must be capable of identifying and healing failures (Fischer, 2010). One of the most promising routes to self-healing structures can be achieved through biocementation. The most common self-healing system is based on microencapsulation. 52
ACCEPTED MANUSCRIPT Recently, the concept of using microcapsules as carriers for self-healing agents in concrete was adopted by Wang et al. (2014), which provides an additional advantage
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of getting protected of the harsh environment inside the concrete. They encapsulated
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bacterial spores from B. sphaericus with concentration 109 spores/g in melamine
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based microcapsules and incorporated them into long reinforced prisms of dimension 30mm × 30mm × 360mm. After curing for 28 days the specimens were subjected to a
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tensile force to create multiple cracks. The enhanced self-healing efficiency in cracked
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specimens contributed by microencapsulated bacterial spores was demonstrated based on the experimental results from light microscopy and the water permeability test. The
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specimens with bacteria containing microcapsules had much higher crack healing
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ratio (48% to 80%) compared to the ones without bacteria, where only 18% to 50% of
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healing was observed. The maximum crack width healed in the specimens with bacterial microcapsules was 970 µm, which was about 4 times wider compared to the specimens without bacteria (max 250 µm). When the capsules are ruptured, for example, by damage, the self-healing mechanism is triggered through the release of the healing agent in the region of damage (Jonkers and Schlangen, 2008; Van Tittelboom and De Belie, 2013; Zemskov et al., 2013). Copious production of bacteria-mediated minerals results in sealing off the crack; hence, the permeability of concrete and leakage is reduced to enhance the durability of structures.
7. Economy analysis of biocementation Biocementation clearly shows the promise of field application. However, its 53
ACCEPTED MANUSCRIPT economic viability must be examined to evaluate time scale of its industrial acceptance. Economic analysis must be carried out in terms of life cycle estimates
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considering its sustainability incentives. Moreover, economies of scale would also
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apply. Detailed economic analysis awaits data from pilot field evaluations.
experiments and results would be of interest.
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Meanwhile, an analysis of the cost of biocementation based on laboratory
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The cost of biocementation depends largely on the price of nutrients, and to some
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extent on the price of the bacterial strain. In this example prices at currency of the United States is used. Price of bacteria varies from country to country; however, one
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standard bacterial strain, if bought from ATCC costs $500, and from MTCC costs
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$10, while CGMCC sells at $200 (Achal, 2015). Such bacterial strain can be used in
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construction work for many years by sub-culturing it at regular time interval. De Muynck et al. (2010) provided a detailed report on the cost analysis of biocement based
on
personal
communication
with
FTB
Remmers,
2008;
http://www.ftbremmers.com/). The biodeposition cost was calculated about $4 per m2, providing the price of 1 kg lyophilized bacteria about $1,500. The cost of nutrients is estimated to be about US $250 per kg. Finally, it was calculated that for microbial concrete application in the area of 0.04–0.08 kg per m2, keeping the cost of nutrients to US $7–15 per m2, the total product cost would be around US $31–39 per m2. The additional cost during the preparation of biological mortar or microbial concrete will be that of the nutrient. However, the cost of nutrients can be reduced 54
ACCEPTED MANUSCRIPT significantly by replacing standard or commercially available nutrients with such industrial byproducts, rich in carbohydrate, protein and energy sources. Achal et al.
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(2009b, 2010a) successfully reduced the cost of microbial concrete production by
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replacing standard nutrient with lactose mother liquor (LML) and corn steep liquor
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(CSL) where LML showed effectiveness equal to standard nutrient, while CSL was significantly better in terms of microbial concrete production. LML and CSL are
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available locally with a price of around $1 and $2 per litre, respectively, which is far
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cheaper compared to the standard nutrient medium and this brings the biodeposition cost to $0.25–1.0. On many occasions such industrial effluents can even be obtained
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free of cost. While the performance of LML was up to the level of standard
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commercially available nutrients, CSL was significantly better in terms of
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biocementation. Hence, CSL offers an economic advantage over the standard nutrient medium and the overall process cost reduces dramatically. One can find local sources of such economic nutrients. Thus, the technology can be significantly cost effective provided it is adapted to the local bacterial strains and locally available nutrient rich byproducts.
8. Future directions This section attempts to map some of the future directions of the technology. It is evident that there are several opportunities and challenges. This section discusses some of the opportunities that, in the opinion of the authors, future research should explore. 55
ACCEPTED MANUSCRIPT 8.1. Archiving of microbial varieties Biomineralization in construction is a new field of research that combines two
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communities- biotechnologists and civil engineers who are relatively unknown to
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each other. Therefore, both communities must document the existing knowledge in a
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manner that is comprehensible to the other. In order to make this novel process of biotechnology popular in construction engineering, it is warranted to catalogue and
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archive the microbial varieties that are amenable to biocementation. The
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microorganisms including bacteria and fungi produce various enzymes. However, for biocementation urease is an important enzyme. Same bacterium has been
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reported to produce different amounts of urease by researchers from different
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countries. Therefore, it is important to catalogue them in various geo-climatic
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domains. It is always preferable to use the local varieties that are acclimatised to local conditions. It is also more cost-effective to harvest local micro-organisms than buying them.
The census will help researchers and engineers differentiate between various microorganisms and to choose the perfect one for usage. It is envisaged that for adaptation of the technology in construction civil engineers and microbiologists must work closely. The catalogue will help engineers to contact microbiologists for assistance. The civil engineers, on the other hand, should contribute in documenting past results with a goal to develop standard testing protocols for construction materials with biocementation. A publicly-accessible, easy-to-search database of carbonate 56
ACCEPTED MANUSCRIPT producing microbes and their role in biocementation with available past results and standard testing and application methodologies will be imperative for the industrial
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success of the technology.
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8.2. Local adaptation
The biocementation ability based on MICP depends on several factors including
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geographical environment. Thus, the performance of the microorganisms varies
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depending on the geo-climatic conditions. While applying this technology the geographical domain of a product must be identified. This is contrary to the style of
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production and trade of conventional building products that are marginally affected
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by the geo-climatic conditions. The locally available microorganisms are likely to be
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acclimatised to the local conditions and thus, have higher chance of success. Thus, the technology must be locally adapted. As a result, experiments must be repeated several times to develop local varieties. The regulatory limitations of transporting microorganisms from another country also mandate use of local microorganism. Further, the locally available nutrients in the form of industrial byproducts should be used. That would not only reduce the product cost but also allow recycling of wastes and alleviate pollution.
8.3. Life cycle assessment An assessment of the biocementation technology in terms of its overall economic and environmental impact is imperative. In recent years, a cradle to grave approach 57
ACCEPTED MANUSCRIPT of assessment has developed. These methods attempt to evaluate a project for its entire life time both in terms of economy and environmental sustainability. A new
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protocol for Life Cycle Assessment (LCA) is slowly emerging (Horne et al., 2009).
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It is being argued that sustainability is a way of achieving economy (Parish and
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Chester, 2009). These methods are based on algorithms that need considerable field data. For conventional materials the methodologies are at an emerging stage (Bribián
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et al., 2011). A challenge for LCA for a new technology such as biocementation
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would be availability of reliable data. Although field data are scant there is adequate information to start the life cycle evaluation of the material. It is important to initiate
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LCA of biocementation.
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8.5. Expanding application domains Biocementation has been applied in sand, rocks and stones, cement and concrete. However, it can benefit several areas of construction. It can be utilized in rehabilitation of heritage stone and lime mortar structures. The process can also be carefully employed in remediation of structures that contain hazardous materials. Encapsulation of hazardous chemicals in the bio-deposition is a promising application. It has been reported that building materials from old demolished structures are found unsuitable for recycling due to the presence of heavy metals (Kawai et al., 2014). The MICP technology has been widely used in the remediation of various heavy metals such as arsenic, copper, lead and chromium (Achal et al., 2011c, 2012b, 2012d, 2013). Biocementation was successful even in presence of 58
ACCEPTED MANUSCRIPT radioactive chemicals such as strontium (Sr). Warren et al. (2001) observed significant strontium uptake into calcite generated by S. pasteurii induced calcium
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carbonate precipitation. They together with Fujita et al. (2004) concluded that
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biocementation was a promising approach for 90Sr remediation in aquifers rich in
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calcium carbonate. Recently, Achal et al. (2012c) demonstrated Halomonas sp. SR4 induced calcite precipitation in sequestering soluble strontium as biominerals (Fig. 6)
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that could play an important role in strontium bioremediation as well repairing
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nuclear structures containing this harmful substance. Such results open new areas of
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application of biocementation technology.
Fig. 6 A schematic representation of strontium sequestering based on MICP, also showing distinct calcite crystals as depicted by SEM. Reprint with permission from Achal et al. (2012c)
9. Concluding remarks Present day world faces the dichotomy of infrastructural growth without compromising sustainability. While present building technologies are struggling to 59
ACCEPTED MANUSCRIPT move towards sustainability, natural habitats are being built sustainably for millions of years. Recent advances in the understanding of natural systems and biotechnology
Concrete is the world’s most widely consumed construction material.
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construction.
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let us attempt to emulate some of nature’s sustainable ways in infrastructural
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Cement used in concrete is the most energy consuming component. Biocementation is a means of creating a cement like binder using microbes that has the potential to
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be a significant step towards sustainability. There are several pathways to
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biocementation. Some of them such as MICP has been more widely studied than the others. Laboratory experiments have demonstrated that biocementation is able to
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improve compressive strength of cement based materials, reduce porosity, and thus
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diminish diffusion of moisture and other deleterious materials. By reducing
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intrusions biocementation should improve the durability of structures, another step towards sustainability. The technology can be used in a variety of ways both in new infrastructure and maintenance of existing ones. There are immense opportunities in improving the technology in structural performance, economy and environmental sustainability. However, there are daunting challenges for industrialisation of this emerging technology. Biosafety could be a major concern especially for researchers or engineers of non-microbiology background working in the area of biocement due to their limited knowledge on bacteria. Working with pathogenic bacterial strains for biocementation could be a dangerous step that can be avoided either by checking pathogenicity of isolated bacteria or by procuring urease producing bacteria from established culture collection bank. Moreover, it is very important to create 60
ACCEPTED MANUSCRIPT collaboration between two seemingly disparate groups- microbiologists and civil engineers. Creating dialogues between the two groups can be seminal in future
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success of the technology. The present paper is an attempt towards that direction.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China
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(No. 41450110458) and the Research Innovation Fund from East China Normal
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University (No. 78210267). We are grateful to editor and anonymous journal referees for commenting earlier versions of the manuscript.
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Table 3: Various applications of biocementation on construction materials and their benefits Media
Limestone
Micrococcus sp., B. subtilis,
B4 media, M-3, M-3P media
S. pasteurii
NB, urea, CaCl2
Sand
S. pasteurii, Mutant of S.
Reference
Protection from decaying
Tiano
et
al.
(1999),
Jimenez-Lopez et al. (2007) Crack remediation
Zhong and Islam (1995)
NB/LML/CSL, urea, CaCl2
Sand plugging of high strength
Achal et al. (2009b, 2010a)
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Granite
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Myxococcus xanthus
Benefit
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Bacteria
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Construction material
pasteurii Alkalophilic bacteria
Yeast extract, peptone, urea, CaCl2
Sand plugging of high strength
Rong and Qian (2012)
Cement mortar
S. pasteurii, B. pseudofirmus,
Phosphate buffer/NB/CSL/alkaline
Crack remediation/Self healing
Ramakrishnan et al. (2001);
B. cohnii, B. alkalinitrilicus
media/yeast extract, urea, CaCl2/ca
CE
PT ED
Sand
Achal et al. (2010b); Jonkers
lactate
Shewanella sp., S. pasteurii, Bacillus sp. CT5
NB/LML/CSL, urea, CaCl2
Improved compressive strength
Ghosh et al. (2005); Achal et
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Cement mortar
et al. (2010)
al. (2009a, 2009b, 2010a, 2011a)
Cement mortar
B. sphaericus
NB, NaHCO3, NH4Cl, urea
Improved reduced
compressive water
strength,
absorption,
gas
De Muynck et al. (2008a, 2008b)
permeability and carbonation
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Concrete
Bacillus sp. CT5
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NB/CSL, urea, CaCl2
Reduced
water
and
chloride
Achal et al. (2011b)
NB/CSL, urea, CaCl2
Crack remediation/Self healing
De Belie and De Muynck (2009); Van Tittelboom et al. (2010)
Corrosion protection
Achal et al. (2012a)
PT ED
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Bacillus sp. CT5
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Yeast extract, urea, ca nitrate
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Concrete
B. sphaericus
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Concrete
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permeability
78