CHARACTERIZATION OF UREASE OF BACTERIAL ISOLATES FROM CEMENT FOR CONCRETE ENHANCEMENT

ABSTRACT

Ureases are a group of enzymes that hydrolyse urea producing carbon dioxide and ammonia. Urease producing bacteria have shown their importance in microbially induced calcium carbonate precipitation in the construction industry. This study focused on the characterization of bacterial isolates from cement for concrete enhancement. Two (2) bacterial isolates assigned codes CA (B) and CA (F) were isolated from cement samples using enrichment culture technique. The isolates were screened for urease production and both tested positive. Enzyme activities of the two isolates were determined. Isolate CA (F) had the mean enzyme activity of 0.0005 mg/mM/s while isolate CA (B) had 0.0002 mg/mM/s. Optimum substrate concentration for urease activity of isolate CA (F) was 1mM while that of isolate CA (B) was at 3mM. The optimum temperature of urease activity for isolate CA (B) was 70℃, while  isolate CA (F) had the optimum activity at 50℃. Optimum pH of both isolates was 9.5. Isolate CA (F) was identified as Lysinibacillus fusiformis strain 5B, using cultural, biochemical and molecular characterizations.   It was used for large scale production of urease due to its higher enzyme activity. The urease produced was used in the production of bio-concretes. A control without urease was also cast. The cubes were cured for 7, 14 and 28 days and compressive strength of the cubes was determined. The compressive strength values of concrete calcified with urease on day 7, 14 and 28 were high with mean values of 24.71, 27.55 and 28.14 N/mm2 respectively. The control also had a high compressive strength of 20.46, 23.11 and 22.53 N/mm2 when compared to the International Standard comprehensive strengths for grade 25 concrete which are 16.25, 22.50 and 24.75 N/mm2. Scanning electron micrographs revealed visible precipitates of calcium carbonate crystals on the surface of concrete treated with urease, while the control had no visible crystals. The results of this study showed that urease produced by Lysinibacillus fusiformis strain 5B was able to enhance concrete strength.

CHAPTER ONE

1.0       INTRODUCTION

1.1       Background to the Study

Concrete is an important material meant for buildings, civil engineering, and general construction in modern society (Abudoleh et al., 2019). It is primarily a mixture of coarse and fine aggregate, cement and water. The most important part of concrete material is cement. It binds the Segregates; it also fills the space between fine and coarse particles (Seifan et al., 2016). In the construction industry, concrete is one of the most consumed materials but is prone to micro-crack formation due to the presence of pores (Chu, 2016). Although micro-cracks do not affect the firmness of the structure, but it can spread to form a network of micro and large cracks that may consequently affect the permeability of concrete. Plastic settlement, formwork movement and plastic shrinkage due to rapid loss of water from concrete surface result in formation of crack in plastic state, while cracks formation in the hardened state area is a result of thermal stress, weathering, error in design and detailing, chemical reaction, constant overload external load and drying shrinkage (Seifan et al., 2016).

These cracks in concrete are greatly unwanted. This is because they provide a corridor for the entry of water and other harmful substances that may lead to corrosion of reinforcement and reduce the durability and strength of concrete. The repairs of concrete cracks all over the world are highly expensive (Chu, 2016). A variety of methods to repair concrete is available, but the majority of these methods are chemically based, expensive and often lead to environmental and health safety hazards (Chaparro-Acuña et al., 2017). There are two methods by which concretes can be repaired: these are via (i) autogenous and (ii) autonomous healing. In autogenous healing, the self-healing process occurs with the use of products formed when water and carbon monoxide dehydrate are present.

Hydration products such as C-S-H or calcium carbonate are formed so as to cause crack healing. The introduction of chemicals such as bentonite and magnesium oxide has the ability to achieve high sealing of cracks with an initial width of about 0.18 mm. On the other hand, autonomous healing is the use of bacteria or its products, organic compounds and encapsulated materials with pozzolan to seal cracks. The use of chemicals such as calcium lactate and microorganism, notably the use of bacteria are distinguished in this treatment (Stanaszek, 2020).

A stronger and more durable concrete has been developed by incorporating a biological approach,  namely bacteria,  in  a  technique  called  microbiologically enhanced  crack remediation (MECR) or microbial induced carbonate precipitation (MICP) (Cheng et al., 2019). This approach utilizes bacteria mineral precipitation to increase the strength and durability of concrete, thereby leading to the production of a more durable concrete. Thus, reducing the maintenance cost. In bio-concrete development, microorganisms are used for sealing cracks, increasing compressive strength and durability and sometimes used as water purifiers (Cheng et al., 2019).

Microbiologically induced calcite precipitation has been projected as a more effective repair technique for the plugging of pores and micro-cracks in concrete. This remediation technique is preferred to other techniques because it is bio-based, cost-effective, eco- friendly and durable (Omoregie et al., 2017). Microorganisms play a crucial role in preventing weakening in porous materials, improve properties of sand, repair of limestone monuments and sealing of concrete cracks to more durable material and enhance the durability of building materials (Rao et al., 2017). The initial sets of microorganisms that can induce the carbonate precipitation are photosynthetic microorganisms (e.g., cyanobacteria and microalgae), sulphate-reducing bacteria and some other microorganisms  that  are  involved  in  nitrogen  cycle.  Bacteria  can  induce  calcium carbonate precipitation using mechanisms such as denitrification, sulphate reduction, urea hydrolysis and iron reduction (Abudoleh et al., 2019). Microorganisms able to synthesize the urease are usually called urea-hydrolyzing bacteria, ureolytic bacteria, or urease- positive bacteria. During microbial induced calcite precipitation, microorganisms secret one or more metabolic products such as CO32- that react with ions such as Ca2+  in the environment leading to the formation of CaCO3 precipitate (Dhami et al., 2014). Urease positive bacteria have been found to influence the precipitation of calcium carbonate (calcite) by the production of urease. This particular enzyme catalyzes the hydrolysis of urea to CO2 and ammonia, resulting in an increase in the pH and calcite precipitation in the bacterial environment. The calcium carbonate (CaCO3) precipitation by urea hydrolysis is a straightforward and a controllable mechanism of MICP that can produce high concentrations of CaCO3 within a short period of time. The MICP can occur inside or outside the bacterial cell or even a distance away from the concrete and often bacterial activities simply trigger a change in solution chemistry, which leads to over saturation and mineral precipitation (Vekariya  and Pitroda, 2013). Microorganisms have been employed to repair cracks in channels so as to prevent leaching and remediation of granite, mortar, limestone, and concrete (Pheng et al., 2018).

1.2        Statement of the Research Problem

Concrete is a strong and fairly cheap construction material mostly used worldwide. One drawback in the use of concrete for construction work is cracking, a phenomenon that hampers the structural integrity and durability of the concrete materials. Concrete cracking is a major concern, as it leads to a loss of structural strength over time, and constructions often suffer from cracking that leads to deterioration and shortening of their service life. The sum of money spent on maintenance and repair of concrete structures is enormous and greatly affect national economy. Building collapse can lead to injury or death of individuals (Ghasemi et al., 2019). It is estimated that damage due to corrosion of concrete in the US alone is $276 billion, with an annual cost in repairs to be $18 – 21 billion per year (Mondal et al., 2019). The indirect costs due to traffic jams and loss of productivity due to reparation are greater than the direct costs of maintenance and repair. Also a study by Oseghale (2015), on the cases and effect of building collapse in Lagos state, stated that on two cases studied total direct loss to the building owners was thirty eight million three hundred and eight five thousand, seven hundred and twenty one naira (38,385,721) which is about One hundred and ninety four thousand, eighty hundred and fifty one dollars ($194,851) at one hundred and ninety seven naira to one US dollars, central bank of Nigeria exchange rate as at 14th March, 2015. One of the causes of this building collapse was linked to corrosion of concrete.

There are many disadvantages involved in traditional repair systems. Key among them are huge cost, thermal expansion, weak bonding, environmental and health safety hazards. According to Lee et al. (2018) durability problems can be tackled by manual inspection and repair; this involves impregnation of cracks with cement or epoxy-based or other synthetic fillers. Concrete with increasingly high compressive strength have been applied to civil engineering in the last decade (Mondal et al., 2019). Even though the addition of powders and steel fiber has the ability to improve several properties of concrete, most of these materials are brittle. In most cases, the brittleness increases as does the compressive strength. This poses possible dangers or fracture failures of the concrete.

1.3       Justification for the Study

The increasing rate of cases of concrete structural failure often arising from defective concrete which has led to the collapse of buildings, bridges, roads and huge financial costs has necessitated the need to constantly improving the strength of concrete using different approaches such as the use of use of fibers, glasses, petroleum epoxies and other chemical enhancers. All these approaches are not without any shortcomings (Mondal et al., 2019). For example the use chemical adhesives pose serious environmental threat. Researchers have also employed the use of microorganisms for bio cementation and concrete enhancement, an approach that has gained prominence and growing global traction, and has been reported to be a viable 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 strength on the concrete and in addition, the ability of self-healing on the concrete (Joshi et al., 2017). This process is known as microbially induced calcium carbonate precipitation (MICCP); a highly effective technological process that has been applied in strengthening, repairing, remediating concrete cracks, consolidation and stabilization of soil.

However, studies have further gone into the use of microbial products for concrete enhancement. Microorganisms are able to produce metabolic products that can aid the precipitation of calcite, thereby calcifying concretes and also improving their serviceability and durability. These metabolic products such as Urease have not only proven to be effective and environmentally friendly, it reduces the risk of hazardous implication in humans. It is cost effective as it utilizes microorganisms or their products (Cheng et al., 2019).This study could be revolutionary, as it could help in strengthening concrete by way of calcite precipitation, which could be used in roads, bridges and construction of houses.

1.4       Aim and Objectives of the Study

The aim of the study was to characterize urease of bacterial isolates from cement for concrete enhancement. The objectives of this study were to:

i.    isolate by enrichment, characterize and identify urease producing bacteria from cement.

ii.  determine urease activity and  identify the organism  with the highest  urease activity.

iii. optimize the parameters for urease activity.

iv. produce crude urease for concrete production.

v.   determine the strength characteristics of concrete produced.

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