Effect of Bacillus cohnii on Some Physicomechanical and Microstructural Properties of Ordinary Portland Cement

Cement-made materials face durability and sustainability challenges. (is is majorly caused by the presence of cracks. Cracking affects the mechanical strength of cement-basedmaterials. Microbiologically induced calcite precipitation (MICP) has been found to enhance compressive strength, thus enhancing on the mechanical and durability properties of these materials. (is paper presents the findings of a study conducted to investigate the effect of Bacillus cohnii on compressive strength development of OPC mortar prisms and the effect of Bacillus cohnii on cement setting time and soundness. Microbial concentration of 1.0×10 cells·ml was used. Compressive strength tests analyses were carried out for each category of mortar prisms. Compressive strength tests were carried out on the 2, 7, 14, 28, 56, and 90 day of curing in distilled water and microbial solutions. All microbial mortars exhibited a greater compressive strength compared to the control with the highest observed at 90 days. Highest percentage gain in compressive strength was observed at 90 days which is 28.3%. Microstructural analysis was carried out using a scanning electron microscope (SEM) after 28 days of curing. (e results indicated the presence of calcium carbonate and more calcium silicate hydrate (CSH) deposits on the bacterial mortars. (e bacteria did not have an effect on cement soundness. Setting time was significantly accelerated.


Introduction
ere is an increasing need to enhance the durability and sustainability of cement-made materials. ese materials are expected to have a long service life of more than fifty years [1]. However, due to degradation, this is sometimes impossible [2]. Due to this, there is a need for continuous monitoring and repair of cement-made materials which is expensive in the long run [3].
To a great extent, durability aspects of mortar/concrete are possibly connected with its pore structure characteristics [4]. Deleterious processes, such as rebar corrosion, caused by ingress of chloride or carbonation, are aided by connectivity within the pore system of the cement matrix. e vulnerability of these cement-based materials to the deleterious processes is more if the pore structure is more open and interconnected [5,6]. Densification of the cement matrix microstructure is one method of sealing the pores. is increases the resistance of the material for ingress of the deleterious materials such as CO 2 , chlorides, and sulphates [7].
Ordinary Portland cement (OPC) exhibits shorter setting times and achieves high compressive strength earlier (at 28 days of curing) compared to blended cements [8]. Due to these properties, OPC is preferred in the construction of dams and bridges in Kenya [8,9]. OPC is, however, prone to attack by deleterious substances such as chlorides and sulphates because of high amount of Ca(OH) 2 released as a hydration product [8]. e pore structure of OPC is more open compared to that of blended cement structures [9,10]. Hence, there is a need for densification and refinement of its microstructure. Cement-made structures using OPC have a higher permeability due to their higher tendency to form cracks. ese cracks tend to form a continuous pathway for ingress of aggressive media/deleterious substances. eir presence is thus undesirable in these cement-based materials since they are a major cause of deterioration and which compromises durability [9].
Measures aimed at crack remediation in cement-based materials are therefore being undertaken to ensure serviceability of the structures [11]. In this regard, a novel technique that uses bacterial sealing in which bacterial metabolism aids in calcite precipitation has been utilized [12]. Urea hydrolyzing bacteria has been explored, hence the name bacterial concrete. Bacteria in cement give it a special ability to repair itself autonomously [13]. Bacteria incorporation in cement also aids in improving the inherent characteristics of cement-made materials in most environments [14].
Long-term durability and sustainability of cement-based materials is important in order to avert economic losses and safety considerations [1]. Deterioration of cement-made materials is mainly triggered by ingress of aggressive substances [2]. e downgrading of cement-based materials/ structures durability and mechanical aspects is influenced majorly by its pore structure [15]. Application of MICP has been found to be an environmentally innocuous process in enhancing the properties of cement [3,15]. Researchers [16,17] have established that MICP enhances strength of bricks and consolidating sand. ey [16,17] further noted that permeability aspects of concrete were also improved [15]. is is by decreasing water and chloride ion permeability by sealing/plugging the small holes and cracks in concrete [15,18]. Incorporation of microorganisms in mortar/concrete enables self-healing and enhances other durability characteristics of mortar/concrete that would save the environment [19]. is is possible since other materials, for example, chloride salts, used in strength enhancement and improvement of durability are not environmental friendly [11]. e materials are expensive compared to microbial concrete; require regular maintenance; as well as limited availability [1,11].
MICP mainly depends upon pH as well as presence of growth site among other factors [20,21]. e MICP process is also influenced by Ca 2+ ion concentration as well as dissolved inorganic carbon [22]. Studies by Balazas [21] and Chahal et al. [23] using concrete demonstrated that the optimum cell concentration of Bacillus subtilis was 10 6 cells per ml. Leena and Vishakha [24] used Bacillus cohnii and observed a 15% gain in compressive strength in relation to the control.  [25]. Analysis was performed in accordance with KS EAS 18-1:2017 [26]. 100.0 g was pulverized and ground to pass through a 76 μm sieve. XRF was used to analyze the oxides in the cement.

Loss on Ignition (LOI).
Triplicate samples of test cement were analyzed for loss on ignition (LOI). Gravimetric method described in KS EAS 18:1-2017 [26] was used to determine LOI. 1.000 g of the test cement was accurately weighed and put into a crucible whose mass had been predetermined. e crucible and its content were placed in a furnace preset at 975°C. is was done for one hour after which the crucible and its content were removed from the furnace and cooled in a desiccator. LOI was expressed as a percentage of the difference of the mass before and after ignition.
Chemical composition of test cement is given in Table 1.
From the results, the test cement met the stipulated requirement by Kenya Bureau of Standards [26].

Nutrients for Bacterial
Culturing. Analytical grade (AR) chemicals were used in this work. ey were acquired from Highway Laboratory Chemical Equipment, Nairobi, Kenya. ese included peptone from casein, calcium lactate, sodium hydrogen carbonate, distilled water, meat extract, and anhydrous sodium carbonate. Bacillus cohnii was sourced from Germany, Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH.

Microbial Cultivation of Bacillus cohnii.
Microbial solution of Bacillus cohnii was cultured as stipulated in the manual from the supplier (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). e media (liquid) considered for the culturing the bacteria was constituted by adding 5.00 g of peptone to 3.95 g of calcium acetate and 3.00 g of meat extract in 1 litre of distilled water. e resultant mixture constituted the liquid medium per stock culture. Sterilization of the mixture was carried out by autoclaving at a temperature of 121°C. is was done for 20 minutes. is mixture was then cooled to room temperature. 5.30 g of anhydrous Na 2 CO 3 and 4.20 g of NaHCO 3 were mixed and a solution of one litre prepared which was 1 M Na-sesquicarbonate (1.0 ml in 10.0 ml) and then added to the stock culture, and this adjusted the pH of the stock solution to 9.7. At this pH, bacterium spore powder was added to the mixture. Using a shaker incubator and at 130 rotations per minute, these cultures were incubated for 72 hours at a temperature of 30°C. Using an atomic absorbance spectrophotometer (G10S UV-Vis, USA), test for optical density (OD) was performed to determine the amount of culture solution required to mix so as to achieve the desired concentration. Bacterium growing media was considered as the blank sample for checking the microbial solutions. 0.5 ml of both the blank and microbial solution was separately put in the spectrophotometer set at a wavelength of 600 nanometres, and the machine was set to read. 1.0 × 10 7 cells·ml −1 was recorded as the concentration and used in this investigation.

Preparation of Mortar Prisms.
Test prisms were prepared using 40 mm × 40 mm × 160 mm moulds according to KS EAS 18-1:2017 [26]. To prepare mortar with w/c of 0.5, model number 1616-6-1358 at East African Portland Cement Company Limited, Athi River, Kenya. 225.0 ml of distilled water was added to the cement in the basin. e mix basin was then clamped onto the automatic mixer, and 1350 ± 1 g of standard sand was added little by little till all was added from an automatic pour trough. e resultant mortar was used to prepare three prisms. e cast prisms were stored in a humidity cabinet for 24 hours at 22°C ± 1°C and then demolded. e demolded mortars were labeled and cured in their respective curing media.

Soundness Test.
e test was performed according to KS EAS 148-3:2017 [27]. A lightly oiled mould was put on a lightly oiled glass sheet and filled with mortar paste formed by gauging cement with 0.78 times the distilled water to prepare the control cement paste labeled as OPC (H 2 O). e same steps were followed but with 0.78 times of bacterial solution and denoted as OPC (BC). is gave a cement paste of standard consistency for each mortar category. is was done in triplicates.

Setting Time
(1) Initial Setting Time and Final Setting Time. Fresh cement pastes for the control and microbial were prepared according to KS EAS 148-3:2017 [27].
is was done in triplicates for both initial and final setting time, and their average was reported.

Compressive Strength Analysis.
Compressive strength analysis was conducted according to KS EAS 148-3:2017 [27]. is was done at 2 nd , 7 th , 14 th , 28 th , 56 th , and 90 th day of curing the control and microbial mortar prisms. Analysis was performed using a compressive strength machine number CH 8224lohningent. ree mortar prisms for each mortar category were picked from the curing tub and wiped off any deposits. ey were placed on the testing machine after noting their identities and load applied vertically at a rate of 50 N/s until failure to obtain prism halves. e obtained halves were smoothly crushed by load application at 2400 N/s. e average strength of the three prisms was considered as the final compressive strength.

Scanning Electron Microscopy (SEM)
. SEM was performed on control and microbial samples after 28 th day of curing. is was done using the SEM model Zeiss Ultra Plug FEG-SEM according to Scrivener et al. [28]. Table 2 gives the results for setting time and soundness for the control and microbial cement samples.

Setting Time and Soundness.
From the results, the initial and final setting times of the control were observed as 78 and 165 minutes, respectively. e initial setting time was accelerated by 15 minutes when compared with the control. e findings indicated that both initial and final setting times were significantly accelerated by Bacillus cohnii. e soundness was not affected by the bacteria. Figures 1-3 presents the compressive strength results for the control and microbial mortar prisms at varied curing period and curing regimes as outlined in Section 2.1.3. Table 3 gives the results of comparison of compressive strength development of the mortars with curing age. e control mortars, labeled as OPC-H 2 O-H 2 O, met the recommended strength at 28 days of 42.5 MPa as depicted in Figure 1 [26]. ere was an observed increase in compressive strength of the mortar prisms as curing period increased for all mortars irrespective of the curing regime with the highest observed at 90 days as shown in Figure 1. It was observed that the increase was statistically significant from 14 th to 28 th day of curing for all categories of mortar prisms. is was expected because cement hydration reactions progresses with time of curing period [29]. It was observed that the increase in compressive strength increased from 28 th to 90 th day as the control mortars was statistically insignificant. is was attributed due to the fact that OPC develops strength early and gains over 90% of its strength by the 28 th day [29]. Similar observations were made by iyagarajan et al. [30]. It was observed that microbial mortars labeled as OPC-H 2 O-BC, OPC-BC-H 2 O, and OPC-BC-BC recorded a higher compressive strength compared to the control mortars at all the curing ages as depicted in Figure 1. is was attributed to the metabolic activities of Bacillus cohnii. It was observed that across all the curing periods, OPC-BC-BC mortar category registered the highest compressive strength. e highest was recorded as 65.8 MPa at 90 days as shown in Figure 1. is was attributed to more Ca 2+ availed by both the mix media and curing media as compared to other mortar categories. is would then imply more MICP, hence more calcium carbonate and more CSH. e bacteria aided in the formation of more CSH and precipitating calcium carbonate through MICP process [31].

Compressive Strength.
is calcium carbonate acted as a sealant and was able to plug the pore within the microstructure, and this improved compressive strength [32]. Mortars cast and cured in microbial solution recorded the highest compressive strength across all the mortar categories. Mutitu et al. [33] made similar observation using e mortar cast with distilled water and cured in microbial solution recorded the lowest compressive strength. is observation was consistent with the findings made by Jonkers et al. [34], Chaurasia et al. [31], and Van Tittelboom et al. [35]. It was noted that compressive strength of the microbial mortars did not differ statistically significantly between the 28 th and 56 th day. is was attributed to the stationary phase of bacterial growth as explained by Skarstad et al. [36] and Zwietering et al. [37] and also reduction of microbial population as the microbes were being coated with a layer of CaCO 3 as a result of MICP process [11,31]. However, there was an observed statistically significant difference in the increase of compressive strength of all the microbial mortars between the 56 th and 90 th day.
is was attributed to log/exponential phase of the bacteria as explained by Skarstad et al. [36] and Zwietering et al. [37]. e bacteria at that point is said to experience optimal growth and multiply faster [30]. Table 4 presents the results for Bacillus cohnii percentage gain in compressive strength of mortars.
Percentage gain in compressive strength was exhibited across all mortar categories in the range of 1.1% to 28.3%.
is was found to increase as curing period increased [29]. e highest percentage gain was noted on the 90 th day as 28.3%. e difference in gain compressive strength was statistically significant between the 14 th day and 90 th day, and this was noted with all mortar categories.
is was attributed to microbial activity of MICP process. e lowest gain in strength was recorded at 14 days. is was attributed to probably inability of the microbe to precipitate substantive amount of calcium carbonate and also aid in formation of CSH as it was adapting to the new environmental conditions within the cement mortar matrix [33]. is is the lag phase of bacterial growth, and as reported by Skarstad et al. [36], the bacteria are adapting to the new environment. However, gain in strength progressively increased after 14 days, and this could imply that the microbe had adapted to the new environment. e presence of other products such as calcium carbonate in addition to CSH, which is majorly responsible for strength of cement, meant that more voids were sealed/plugged [29]. From SEM images, Figures 4-6, they show massive deposits of CaCO 3 and more CSH as compared to Figure 3. e additional products might have been deposited in the voids, thus sealing them [9,10]. e images show the presence of calcium silicate hydrate, C 3 S 2 H 3 , (CSH), calcium carbonate, (CaCO 3 ), ettringite (C 6 AS 3 H 32 , needle-like crystals), and calcium hydroxide (Portlandite, CH). Some ettringite was observed as needlelike-shaped crystals, and this was attributed to gypsum added to cement to control flash setting. eir formation is given as follows [34]: e ettringite is shown in Figures 3 and 4. e figures represent the control mortar denoted as OPC-H 2 O-H 2 O and mortar cast using distilled water and cured in microbial solution, denoted as OPC-H 2 O-BC, respectively. is was attributed to perhaps the gypsum added during the manufacture of cement to control flash setting and improve on cement workability. However, ettringite was not observed in Figures 5 and 6. is was attributed to the bacterial activity in forming more calcium carbonate that may have probably deposited on any ettringite formed. While calcium carbonate and CSH was observed across all the microbial mortars, OPC-BC-BC mortar image exhibited massive deposits of calcium carbonate and more CSH as shown in Figure 6.
is could account for the higher compressive strength observed with this category of mortars as compared to others. e SEM image shows a more refined microstructure and more dense cement matrix with this category of mortar compared with others. is was attributed to more microbial cells and more Ca 2+ from the feed available for metabolic activity of the bacteria. From the results, it can be elucidated that incorporating the bacteria in the mix media had more benefits than using distilled water.
During microbial cultivation, calcium acetate present in the feed brought in extra Ca 2+ ions into the cement matrix.
is enhanced the formation of more calcium silicate hydrate (CSH) and precipitation of more calcium carbonate. e cell wall of the bacteria acted as a nucleation site and in the presence of Ca 2+ ions availed by calcium acetate from the bacterial feed, crystallized out as CSH when they combined with precipitated CO 3 2− ions [33,38]. Bacillus cohnii in this investigation improved compressive strength of OPC, and this could improve durability properties and sustainability of cement-based materials in long run. MICP of calcium carbonate in this case was attributed to oxidation of acetate by Bacillus cohnii. is is given as follows [33]:

Journal of Chemistry
Generally, integrating bacterium in the cement matrix had a beneficial effect on the compressive strength development.
is, in long run, is expected to enhance other physical and mechanical aspects of microbial cement-made materials. Flexural strength, resistivity to chloride, and sulphate ingress would be improved due to reduced permeability [39]. us, incorporation of Bacillus cohnii in the cement matrix improved the physicomechanical aspects of the material.

Conclusion
Bacillus cohnii precipitated substantive quantity of calcium carbonate capable of enhancing compressive strength and microstructure of the mortar matrix. e highest compressive strength gain was noted with OPC-BC-BC mortar category, mortars cast and cured in microbial cultivated solution. Using microbial solution both as the mix and curing media yielded the highest benefits of microbial mortars. Bacillus cohnii enhanced the nucleation cites for MICP process resulting in enhanced overall microstructure of the mortar. e calcium acetate incorporated as a feed during bacterial cultivation introduced more Ca 2+ within the mortar matrix which enhanced the formation of calcium carbonate which plugged into pores as shown by the SEM micrographs/ images enhancing compressive strength. e bacteria did not influence the soundness of cement. e setting time of cement was significantly accelerated.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest. Mean values followed by the same small letter within the same column do not differ significantly from one another (one-way ANOVA, α � 0.05, SNK-test).     Journal of Chemistry