BIOCALCIFICATION OF CONCRETE USING CARBONIC ANYHDRASE PRODUCED BY SOIL BACTERIAL ISOLATES

ABSTRACT

Two (2) bacterial isolates obtained from Garima construction sites were named GARIMA (A) and (B), and code-named GA (A) and GA (B) respectively. They were screened for their carbonic anhydrase (CA) producing ability. Isolates GA (A) and GA (B) showed positive reaction to para-nitrophenylacetate (pNPA), producing yellow and peach/orange coloured colonies respectively. The isolates were used to produce the crude CA and the mean enzyme activity with standard error of mean for the CA from isolates GA (A) and GA (B) were 0.0321±0.0012 and 0.0351±0.0002 mmoles/mL-1/sec-1 respectively, the GA (B) was subsequently used for the large scale production of the crude CA. Isolate GA (B) was identified as Alcaligenes faecalis subsp. parafaecalis strain G, using cultural, biochemical and molecular characterizations. Optimum substrate concentration for the CA from isolate GA (A) was 5 mM, with 50oC optimum temperature and an optimum pH of 8.5. While CA from A. faecalis subsp. parafaecalis Strain G had an optimum substrate concentration of 7 mM, optimum temperature of 50oC, and optimum pH of 9.5. The crude CA extract was used to reinforce concrete; the results showed an increase in crushing strength on days 7, 14 and 28 with mean crushing strength values of 11.54, 15.52 and 22.28 N/mm2 respectively, and with a percentage strength gain of 48.29%. The scanning electron micrographs revealed distinctly visible precipitates of calcium carbonate crystals on the surfaces of the concrete treated with the crude CA. These results indicate that CA could have huge potential applications in the bio-calcification and healing of concrete.

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the Study

A very important material composition for building infrastructure and civil construction that is relatively inexpensive, within reach and easy to cast is concrete; a term used to describe any materials that have its components of cement, fine aggregates of sand, coarse aggregates of pebbles or stones, and water (Khattra, et al., 2016; Patil et al., 2016; Viduthalai et al., 2018). The materials compositions of concrete are such that are able to withstand compressive weight to some extent, until the applied weight is greater that the resistance limit, thus causing a reduction in strength by forming cracks (Viduthalai et al., 2018). The lack of durability in concrete is oftentimes connected to increased permeability, often as a result of the increasing permeability matrix or availability of cracks  (De  Belie and  Wang,  2016).  Concrete  is  sensitive to  crack  formation,  thus endangering its durability, and often requiring repairs and of course attracting more cost. The formation of crack results from its lack of enough tensile strength fortification, reason for the addition of steel to concrete in order to accommodate the stretched weight (Van Tittelboom et al., 2010).

When the tensile stresses are high on concrete, it causes deformation as a result of amongst others, silica reaction, gradients in the temperature and corrosion of the reinforcements. The development of small concrete cracks create an avenue for the passage of liquids and gases through them, damaging the concrete and corroding also the reinforcements, therefore leading to structural failure. Implying that the formation of cracks in concrete is a fundamental cause of structural failure, and a delay and inaccurate response to  the cracks  could  cause  further  enlargement  and  additional  repair  costs (Bhavana, 2017; Neha et al., 2018).

The occurrence of cracks in concrete is as a result of several processes which include tensile and mechanical compressive forces, shrinkages (Gandhimathi et al., 2015). Micro- cracks in concrete, over time accelerate its deterioration as it allows permeation of deteriorating elements such as carbon (IV) oxide (CO2), sulphates and chloride ions into the concrete body. And once the permeation of these elements is repetitive, it causes an expansion in the cracks width, leading to acceleration in the concrete deterioration (Choi et al., 2016). Adequate attention must be focused on preventing the aforementioned process affecting concrete durability (Jokyani and Chouhan, 2018).

The conventional repair mechanisms for compromised concrete relies on the process of matching materials of dissimilar characteristics such as a composition of inorganic and organic calcium hydrosilicate and epoxies of petroleum origin respectively. A viable alternative to this approach is the application of bacteria and/or their metabolic products in concrete strengthening and repairs (Rahbar et al., 2021). The method of concrete strengthening to heal these cracks has been employed in a quest to overcome these challenges associated with concrete cracking, in a technique called Bio-calcification or Microbially Induced Calcium Carbonate Precipitation (MICCP), involving a selective microbiologically mediated filling process whereby calcium carbonate (CaCO3) is precipitated, and acting as a microbial sealant is encouraged by metabolic activities of the microorganisms, resulting in the reduction of the concentration of the openings and an improvement of the concrete’s serviceability, longevity and compressive strength (Parnnika and Das, 2013; Gandhimathi et al., 2015). An array of complex biochemical processes is involved in MICCP, which includes the precipitation of calcium carbonate (CaCO3) by the action of the enzyme carbonic anhydrase (Varenyam et al., 2010).

Also, these greenhouse gases’ (GHGs) concentration in the atmosphere has assume an alarming dimension caused primarily from the activities of humans, which has led the global campaign geared towards restoring the integrity of the climate. The greenhouse gases include but not limited to carbon iv) oxide (CO2), methane and chlorofluorocarbons with CO2 being the most prominent GHG. CO2 arises from the combustion of fossil fuels and also from the processes of industrial activities such as the production of cement (Christopher et al. 2013).

The ubiquitous distribution of microorganisms makes them huge reservoirs of biochemically important enzymes and important candidates in enzyme production. Microbial enzymes are naturally robust and highly thermo- and pH- stable, and also highly multi-functional, making them ideal candidates for a wide range processes of biotechnology (Thapa et al., 2019). Carbonic anhydrase (CA) is a metalloenzyme containing zinc, and that is able to catalyse the reversible hydration reaction of CO2 (CO2 + H2O ⇔ HCO3–  + H+). Therefore, CA has the potential to speed up the process of precipitation of calcite under favorable conditions (Zhang et al., 2011). Eventually, this acts as a sealant to the formed fissures and cracks in concrete by self-assembling on concrete surfaces and inside the cracks thus producing crystal substances that are stable and have these cracks filled with their solid precipitates. CA in the presence of calcium can be utilized to form calcium carbonate rapidly, and in addition the calcium carbonate produced has materials similarities with the concrete mechanical characteristics, thus creating a final product that is almost non-differentiable. Also atmospheric carbon (iv) oxide is sequestrated by the catalytic reaction of CA precipitation of calcium carbonate, and also its decomposition is not offensive or threatening to the lives of human. Although most of the CA are dependent on or contain zinc (Zn2+), there have been reports of some cadmium (Cd2+) and Iron/Ferrous (Fe2+) containing CAs (Tomazett et al., 2016; Rahbar et al., 2021).

This biological technique can be employed to enhance the compressive strength and stiffness of concretes with cracks. MICCP or MECR can take place within or out of the cell of the bacteria, more so a shift away in concrete, with the metabolic activities causing the precipitation of minerals as a result of the changes in solution chemistry (Patil et al., 2016). Some bacteria that can carry out such calcification function include Bacillus sphaericus, B. megaterium, which generally are regarded to as safe bacteria as they are non-pathogenic, and which apart from their calcification ability, thrive in a pH of 9.0 optimum  (surviving in  harsh environmental  conditions  as  the high  pH support  the activities of the bacteria), are spore formers, affording them stay for a longer time in cement and concrete as capsules and also the secretion of exoploysaccharide substances that aids concrete adhesion (Acuna et al., 2018; Whitaker et al., 2018; Agereh et al., 2019).

Carbonic anhydrase produced by most calcifying bacteria have been reported by Bansal et al. (2016) to have greater potential as a biocatalyst for CaCO3  precipitation and formation from carbon (iv) oxide hydration, in the presence of a calcium source. Application of MICCP by exploring the potentials of carbonic anhydrase is an environmentally friendly approach as it does not cause the depletion of the resources of nature and also does not result in the release of harmful substances to the environment, prompting the proposal for its use in the construction industry as it helps in achieving a denser and stronger concrete (Satinder et al., 2017). This research therefore aims at biocalcifying concrete using the carbonic anhydrase produced by isolated soil bacterial isolates.

1.2 Statement of Research Problem

Concrete is a major player in the civil construction industry, and it is being bedeviled by cracks formation that actually threatens concrete durability as the cracks allows for the permeation of liquids and gaseous substances into the spaces created by the cracks. This exposes the concrete reinforcements to environmental stress, often leading to corrosion of these reinforcements and structural failure subsequently which could come in the form of building, bridge collapse, pavements caving in (Van Tittelboom and De Belie, 2010; Patil et al., 2016). The aforementioned can be tackled by constant maintenance and repairs. However, some cracks called micro-cracks are not easily noticeable, and could grow into bigger cracks that will eventually lead to structure collapse.

Odeyemi et al. (2019) while studying the trends of building collapses from 2009-2019, reported an alarming rate of cases of collapsed buildings in Nigeria. These spates of building collapse can be likened to the cracks on concrete infrastructure. Initially, when micro cracks are formed in concrete, they create huge damage to the ability of the concrete to be serviceable resulting in maintenance cost in the long run.

In addition, the cement industry is a critical player in the consumption of fossil fuels, contributing considerably to global carbon emissions. The production of cement releases carbon (IV) oxide (CO2), a greenhouse gas into the atmosphere that causes the depletion of the ozone layer and ultimately global warming (Ali et al., 2015).

The  methods  that  are  conventionally employed  for  concretes  crack  repairs  involve treatment with chemical adhesives or sealants. Conventional concrete cracks repairing methods such as the treatments with chemical sealants or adhesives for the prevention of the widening of the cracks are generally environmentally unfriendly and of high cost implications, and in addition there is also the issue of incompatibility with concrete in terms of the materials.

1.3 Justification for the Study

Considering the alarming rate of concrete structural failure arising from cracks, and the attendant monetary and human costs, a successful use of bacteria and/or their products for remediation of this menace is a great step towards having a revolutionary technology of using microbial products in civil construction and concrete strengthening. In addition, the race for the adoption of green technologies that are environmentally friendly is fast dominating global center stage, and the application of carbonic anhydrase (CA) which itself is a bacterial product will relatively help reduce green-house gas emission as it will sequestrate CO2 and in the presence of calcium produces CaCO3, which therefore calcify the concrete. Carbonic anhydrase is of great importance in this regard, as it is able to sequester CO2 in a series of biochemical reactions to produce CaCO3 (Bond et al., 2001; Kanbar, 2008).

The use of microbial approach to concrete crack repair and healing has gained prominence and growing acceptance recently and has been reported to offer a better alternative, as it involves the application of microorganisms and/or their products for the treatment of concrete. This method is not only environmentally friendly; it also confers on the concrete the ability to self-heal on its own (Joshi et al., 2017). This is a process known as bio- calcification or microbially induced calcium carbonate precipitation (MICCP); a highly effective technological process that has been applied in repairing or remediating concrete cracks, improving the strength of concretes, soil consolidation and even in restoring monuments made of stones. This process is eco-friendly, and very cost effective. Microorganisms can actually produce metabolites that can precipitate CaCO3  to fill in these cracks and sealing them, thus affording self-healing of concretes and reducing the permeation of liquid and gases into the crevices when formed and of course, therefore saving repair  and maintenance cost  while improving the durability of the concrete (Tambunan et al., 2019; Rahbar et al., 2021). This research could be revolutionary, as it could help in strengthening concrete by way of bio-calcification, which could be applied in roads, bridges and pavements construction, and also housing infrastructure.

1.4 Aim and Objectives of Study

This research was aimed at biocalcifying concrete using carbonic anhydrase produced from soil bacteria isolates. The objectives of this study were to:

i.          isolate and screen bacteria from soil of selected construction sites for carbonic anhydrase (CA) production.

ii.         identify the bacteria with the higher potential for the production of carbonic anhydrase.

iii.        determine  carbonic  anhydrase  activity  and  optimize  the  parameters  for carbonic anhydrase activity.

iv.        determine the effect of the carbonic anhydrase on the bio-calcification of concrete.

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