Ground improvement with single treatment using Mg 2+ modified all-in-one MICP solution: 1m sand column

Ground improvement with single treatment using Mg 2+ modified all-in-one MICP solution: 1m sand column | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ground improvement with single treatment using Mg 2+ modified all-in-one MICP solution: 1m sand column Seyed Mohammad Javad Hosseini, Dawei Guan, Liang Cheng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4449151/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The potential of microbially induced carbonate precipitation (MICP) for soil bio-improvement has been widely studied as an alternative to traditional cementation by Portland cement. While multiple-phase injection techniques are commonly used for MICP treatment, they impose complexities and require a high number of injections. One of the latest developments in the biocementation research area is using the one-phase-low-pH MICP method as a more effective and efficient alternative to the traditional two-phase method. The published studies in one-phase MICP used 1M concentration and injected all-in-one solution several times. So, this study primarily investigated the possibility of soil improvement by a single injection of high-concentration all-in-one solution in 1m columns. This high concentration can impose a toxic effect on bacterial activity and hinder urea conversion. Also, a high concentration of salts such as calcium or magnesium chloride can increase the ionic strength and decrease the uniformity of carbonate precipitation. The effect of 20% magnesium substitution and decreasing the initial temperature of substances were studied. The experiments in aquatic steps demonstrated that these magnesium cations and low temperatures can prolong the lag phase. The collected precipitation from magnesium-included solutions showed an enhancement in the crystal structure of calcium carbonate formations. The transportability of all-in-one solutions was examined by injection of 6 pore volume solution through a 20 cm sand column and comparing the optical density of effluent to the influent. Solutions with magnesium contents and low temperature demonstrated a higher transportability. Eventually, 1 m sand columns were treated with all-in-one solution and the most homogeneous urea conversion and calcium carbonate precipitation were observed in the column injected with a low temperature of magnesium substituted solution. Microbially induced carbonate precipitation (MICP) Biocementation All-in-one solution Magnesium effects Temperature effects Hydrophobicity Large-scale experiments Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 1 Introduction Microbially induced carbonate precipitation (MICP) has been developed as an alternative soil improving technique to curb the environmental challenges of traditional cementation projects. During the MICP process, a ureolytic bacteria produces a urease enzyme to catalyze urea hydrolysis, as represented in Eq. 1 \(CO{(N{H_2})_2}+2{H_2}O\xrightarrow{{Urease}}CO_{3}^{{2 - }}+2NH_{4}^{+}\) Eq. 1 The ammonium (NH 4 + ) released from urea hydrolysis results in a pH rise and commences the precipitation of calcium carbonate. Calcite is precipitated through the combination of carbonate ions (CO 3 2− ) from the hydrolysis of urea and the calcium cation (Ca 2+ ) from the supplied calcium source(Cheng & Cord-Ruwisch, 2012 ; Lu et al., 2023 ; Mujah et al., 2017 ; Pakbaz et al., 2018 ): \(C{a^{2+}}+C{O_3}^{{2 - }} \to CaC{O_3} \downarrow\) Eq. 2 The calcium carbonate (CaCO 3 ) generated from the biochemical reactions is responsible for the biocementation and improvement of the soil strength(Behzadipour & Sadrekarimi, 2023 ; Pakbaz et al., 2022 ; Shahin et al., 2020 ). Therefore, for MICP reactions, the presence of urea, calcium ions, and a urease-producing bacteria is vital. A bacterial suspension often provides ureolytic bacteria, and the cementation solution (CS) includes dissolved urea and calcium salt(Ashkan et al., 2019; van Paassen et al., 2009 ; Whiffin, 2004 ). Bacterial suspension and CS need to be delivered into soil matrix to trigger in situ biocementation reactions(Gurbuz et al., 2015 ; He et al., 2016 ; H. J. Lai et al., 2021 ; Mahawish et al., 2018 ). In literature, CS is often an equimolar concentration of urea and calcium chloride, and the employed bacteria is Sporsarcina pasteurii (Cheng et al., 2020 ; Do, 2019 ; Karimian & Hassanlourad, 2022 ; Pegah & M., 2020). In MICP, the designed method for delivering bacteria and CS can impact the efficiency of the process and the engineering properties of bioimproved soil(Cheng et al., 2019 ; Cheng & Shahin, 2015 ; Harkes et al., 2010 ; Jamshidi-Zanjani & Khodadadi Darban, 2017 ). Therefore, scientists have established different injection strategies to meet the demands of engineering projects. Until now, a multiphase injection is a conventional method in MICP that researchers often implement (Muynck et al., 2010 ; T. et al., 2017; Wang et al., 2017 ). However, the implementation of multi-phase injection methods often poses challenges due to their inherent complexity and the unpredictable nature of phase interactions during the injection process. Therefore, it is preferable to simplify the process with an injection of only one solution, including all necessary ingredients(Cheng et al., 2019 ; Chuangzhou et al., 2019 ; H.-J. Lai et al., 2022 ; X. Liu, 2023 ; Yang et al., 2022 ). Recently, all-in-one solution strategy has shown its advantages in providing a low-cost and highly efficient MICP (Cheng et al., 2019 ; Chuangzhou et al., 2019 ; M. J. Cui et al., 2021 ; M.-J. Cui et al., 2021 ; H.-J. Lai et al., 2022 ; Yang et al., 2022 ). In the all-in-one solution strategy, CS and bacteria are mixed before injection. As this mixture can stimulate immediate precipitation, a lag phase in the cementation should be created to provide a time window for completing the injection process. Some researchers have made this lag phase in the carbonate precipitation by adding acid to the all-in-one solution and decreasing the initial pH of the all-in-one solution to about 4(Cheng et al., 2019 ; Chuangzhou et al., 2019 ; M. J. Cui et al., 2021 ; M.-J. Cui et al., 2021 ; H.-J. Lai et al., 2022 ; Yang et al., 2022 ). In another approach, Xiao et. al 2021 have tried to reduce the initial temperature of all-in-one solution, to minimize the urease activity of bacteria and consequently make a lag phase in cementation (Xiao et al., 2021 ). With this background, this study employed the simultaneous effect of low pH and cold temperature in prolonging the lag phase for all-in-one solutions. In the first step, to further enhance the homogeneity and efficiency of treated sand with all-in-one solution, some effects of magnesium (Mg 2+ ) substitution for calcium (Ca 2+ ) were investigated. Some reasons support the significance of studying magnesium substitution's effect in all-in-one solutions. Firstly, the impact of magnesium cations on biocementation is rarely explored and covered with some contradictory results(Gowthaman et al., 2021a ). For example, as given in Table 1 , while most researchers have mentioned the positive effect of Mg 2+ , others concluded that magnesium cations could decrease bio-mortar strength (Abdel Gawwad et al., 2016a ; Putra et al., 2016a ; Xu et al., 2020a ; Zhang & Dawe, 2000a ). Secondly, although a few studies have noted the impact of magnesium ions in biocementation, they often have studied enzyme-induced carbonate precipitation (EICP) (Chandra & Ravi, 2020a ; Imran et al., 2021a ; Lu, 2016 ; Putra et al., 2016a ; Sun et al., 2019a ). Since there are no urease-positive bacteria in EICP, the precipitated carbonate by EICP along a soil column is generally more uniform than that of MICP (Ashkan et al., 2019; Dilrukshi et al., 2018 ; T. et al., 2017). As the uniformity of treated soil is the main challenge in all-in-one solution strategy, the significance of magnesium substitution can be highlighted in this study. Table 1 Summary of the published literature on the effects of magnesium substitution Mechanism of effect Effect Reference 1 Precipitation of aragonite + (Xu et al., 2020a ) 2 Delaying agent in precipitation + (Putra et al., 2016a ) 3 Inhibitory on biofilm formation + (Oknin et al., 2015 ) 4 Urease activity +- (Sun et al., 2019a ; Sun & Miao, 2019a ) 5 Carbonate precipitation productivity - (Abdel Gawwad et al., 2016a ; Sun et al., 2019a ) Also, the context of hydrophobicity and its significance in MICP is discussed in this study. Cell surface hydrophobicity (CSH) is a biophysical measurement of a cell's affinity for a hydrophobic versus hydrophilic environment(Gargiulo et al., 2008 ; Jain & Arnepalli, 2020 ; G. Liu et al., 2020 ; Rosenberg, 2006 ; Xia et al., 2021 ). Bacteria with higher CSH prefer to attach to the sand grains and show a lower transportability along a sand column. In this case, a higher population of bacterial cells will be stopped near the injection point and lead to an inhomogeneous distribution of bacteria through the column (Huysman & Verstraete, 1993 ; G. Liu et al., 2020 ; Redman et al., 2004 ; Xia et al., 2021 ; Zhong et al., 2016 ). Consequently, a high CSH forms bioclogging near the injection point and a low efficient MICP treatment on the farthest part of the column. Generally, changes in pH can alter the charge and conformation of cell surface molecules, such as lipids and proteins, which can affect the hydrophobicity of the cell surface(Eschlbeck et al., 2017 ; Mirani et al., 2018 ; Zhong et al., 2017 ). A decrease in pH (acidic conditions) can lead to an increase in cell surface hydrophobicity, as the protonation of functional groups on the cell surface can enhance hydrophobic interactions(Eschlbeck et al., 2017 ; Mirani et al., 2018 ; Zhong et al., 2017 ). Also, a decrease in temperature may lead to a decrease in cell surface hydrophobicity, as lower temperatures can cause the cell membrane to become more rigid and reduce the exposure of hydrophobic regions(Gallardo-Moreno et al., 2003 ; Gharabaghi et al., 2015 ; Kim & Walker, 2009 ; McCaulou et al., 1995 ). As in this study all-in-one solution is injected at low pH and low temperature, investigation on the role of CSH seems essential. 2 Material and methods In the first step, an experiment series was done on aqueous mediums for S1-S6 all-in-one solutions presented in Table 2 to assess pH variation and crystallography of derived precipitations. X-ray diffraction (XRD) tests provide a microscale point of view into the effect of magnesium on all-in-one solutions. As the second step, a group of short sand columns was treated with the injection of S1-S6 solutions, and the percentage of urea conversion and calcium carbonate was determined. In the third step, four columns of 1 meter in length were injected with S1-S4 solutions to evaluate the efficiency of all-in-one solutions in large-scale projects. Table 2 Composition of the evaluated all-in-one solutions Concentration of chemical substances (M) Bacteria pH Temperature CaCl 2 MgCl 2 Urea S1 2.00 0.00 2.00 10 U 4 RT S2 2.00 0.00 2.00 10 U 4 CT S3 1.60 0.40 2.00 10 U 4 RT S4 1.60 0.40 2.00 10 U 4 CT S5 1.60 0.00 2.00 10 U 4 RT S6 1.60 0.00 2.00 10 U 4 CT The initial pH of all the solutions was adjusted to about 4 by mixing adequate acetic acid with the reagents. 2.1 Bacterial culture Sporsarcina pasteurii was employed in the current study as a urease-producing bacteria. This bacterium was cultivated in a sterile aerobic batch growth medium (200 mL growth medium in a 1-L flask shaken at 180 rpm), including 20 g/L yeast extract, 15 g/L ammonium chloride, and 0.1 mM NiCl 2 , adjusted at pH = 9.2. The harvested bacterial culture had a urease activity of 20 ± 1U/ ml. 2.2 pH adjustment An adequate volume of 3 M acetic acid was added to the 15 ml CS in a 50 ml beaker to adjust the initial pH of the all-in-one solution. A pH meter monitored the pH variation while a magnetic stirrer mixed the acid-added CS. Then, 15 ml bacteria were added to 15 ml acid-added CS to achieve pH4. The adequate volume of acetic acid was determined with trial and error. 2.3 Soil type Natural silica sand with a hydraulic conductivity of 1.88×10 -4 m/s was used to fill 1m columns. The aggregation curve of the sand is given in Figure 1 . Following the Unified Soil Classification System (USCS), the sand used has been classified as poorly graded (SP). 2.4 Urease activity and Urea conversion Urease activity was determined via electrical conductivity (EC) in the absence of calcium cations. 1 mL of the bacterial cell suspension was added to 5 mL of 3 M urea and 4 mL of DI water (reaction concentration 1.5 M urea). Variations of the relative electrical conductivity were recorded over 5 minutes at 25 ± 1℃. In the presence of calcium ions, a modified Nessler method indicates urea conversion rate from the produced ammonia concentration in Equation 1 (Cheng et al., 2016). 2.5 Hydrophobicity There are several methods to analyze CSH, including Microbial Adhesion to Hydrocarbons (MATH), Contact Angle Measurement, Bacterial Adhesion to Solvents (BATS), Salt Aggregation Test (SAT), Hydrophobic Grid Membrane Filter (HGMF). Each method has its advantages and limitations, and the choice of a method depends on the specific requirements of the study and the characteristics of the tested bacteria. In literature, the most well-defined method for CSH measurement is microbial adherence to hydrocarbons (MATH) method (Kaczorek et al., 2018; Mozes & Rouxhet, 1987; Rosenberg, 2006; Rosenberg et al., 1980) . Therefore, in this study, the MATH method was employed to measure CSH of Sporsarcina pasteurii at room temperature (25º C) and cold temperature (4º C) and three pHs 9.2, 7, and 4. Here are the general steps for CSH measurements (Rosenberg et al., 1980, 1991): 1- Culture Preparation: Growing bacteria in the culture medium until they reach the desired growth phase (according to section 2.2) . 2- Washing: Centrifuging the culture and discarding the supernatant. Resuspend the bacterial pellet in a pH-adjusted phosphate buffer solution to get OD 400 below 1 by spectrophotometer. Keeping 2 ml of this bacterial suspension for measurement step (A 0 ). 3- Preparation of Hydrocarbon: Adding 1 ml of a hydrocarbon, such as xylene, to the 5 ml bacterial suspension. 4- Mixing: Vortexing the mixture vigorously for 2 minutes to allow the interaction between bacteria and the hydrocarbon. 5- Phase Separation: Allowing the mixture to be kept undisturbed for 15 minutes for the hydrocarbon and aqueous phases to separate. 6- Measurement: Measuring the optical density (OD 400 ) of the aqueous phase (A 1 ) and bacterial suspension (A 0 ). The decrease in OD represents the bacteria that have adhered to the hydrocarbon phase. 7- Calculation: Determining hydrophobicity by: 100 * (1 - OD of the aqueous phase (A 1 )/OD of the cell suspension (A 0 )). 2.6 Sampling from all-in-one solution In specific time intervals, disposable 1 ml syringes have been used for sampling from the all-in-one solutions in beakers and sand columns. To stop the bacterial activities of the samples, they were filtered by using 0.22µm disposable syringe filters and kept in a freezer (-22℃) until the analysis time. 2.7 Unconfined compressive strength (UCS) After the end of the treatment process for 1m columns, 10 pore volume (10 PV) tap water was injected through the 1m columns to wash out unconsumed substances. Then, columns were opened, and each column was divided into 10 sections with diameter-to-height ratios of 1:1.5 to 1:2 and air-dried for 48 h before the UCS test. UCS tests on bio-cemented soil columns were carried out according to the method stated in ASTM D2166 with a constant loading rate of 1.0 mm/min(ASTM, 2013). 2.8 Carbonate content measurement The weight of precipitated carbonate was obtained using a gravimetric acid washing process. This method weighed a fraction of an oven-dried sand column before and after washing with 1M HCl acid. The difference in weight before and after acid washing was considered the amount of precipitated carbonate (Cheng & Shahin, 2019) . Carbonate content percentage (CC%) is the amount of precipitated carbonate divided by the weight of sand after acid washing. 2.9 Microstructure analysis Fractions of biotreated sand columns were examined by SEM (Model: JSM-7800F). All samples were flushed with distilled water and dried at 60 °C for 48 hours before the microscopy investigation. XRD patterns were recorded on a BRUKER D8 ADVANCE machine using a scanning range from 2θ= 15°–70°(Cheng et al., 2014). The dried precipitated materials were observed to evaluate the effect of the magnesium substitution chloride on the mineralogical characteristics. Phase identification was carried out by searching the Inorganic Crystal Structure Database (ICSD) powder diffraction database by Xpert HighScore Plus (version 3.0e)(Cheng et al., 2014). 2.10 Experimental procedures 2.10.1 Aqueous test The pH variations and urea conversion on all S1-S4 solutions in Table 2 were monitored to investigate the effects of magnesium substitution and temperature. Solutions have been prepared individually by adding 15 ml bacteria to 15 ml pH-adjusted CS on a 50 ml beaker. The current study used calcium chloride anhydrous and magnesium chloride hexahydrate to source Ca 2+ and Mg 2+ , respectively. The pH variations on aqueous solutions were investigated while all-in-one solution was stirred at 400 rpm. This monitoring can indicate the lag phase of biochemical reactions at the aqueous solutions that can lead to the selection of the most desirable solutions for sand column treatment. 2.10.2 Short sand columns 50 ml syringe was used as short columns and filled with clean silica sand (mentioned in Figure 1 ) at a dry density of about 1.6 g/cm3 and 20 ml pore volume (PV). After the preparation of all-in-one solution in a beaker, it was immediately injected by a peristaltic pump from the top to the bottom at the flow rate of 20 ml/min. The columns were kept at room temperature in a fully saturated condition for 72 hours. Then, the columns were washed out with tap water, and 1 ml of the outflow was sampled for urea conversion measurement. The injected tap water also can wash out the excess reagents on the sand columns and prevent misleading results on UCS and CC%. 2.10.3 Bacterial transport in sand columns A semi-long column was designed to simulate the distribution of all-in-one solutions in long columns. Therefore, PVC columns were used for bacterial transport experiments with a 25 cm length and 4.5 cm inner diameter. The sand (defined in Figure 1 and section 2.3 ) was dry packed into the columns under vibration. Figure 2 shows a schematic of the setup for this experiment. Each bacterial transport experiment was conducted as the following procedure. First, the sand column was washed out and saturated by tap water. Then 6 PVs of the solutions were adjusted and introduced into the vertically oriented column at a 45 mL/min flow rate. The effluent from the column was collected every 1 min in 10-mL tubes. The optical density of all samples was measured with a spectrophotometer INESA N2S model at 600nm (OD 600 ). Prior researches show that OD 600 is on correlation to cell colony-forming-unit concentration (C, CFU/mL) for bacteria (Babakhani, 2019; G. Liu et al., 2020; Redman et al., 2004; Xia et al., 2021), (for Sporosarcina pasteurii , biomass concentration is C (g/L) = 0.438 × OD600, Ref.(Cheng et al., 2019)). Therefore, the ratio between OD 600 for effluent samples and the influent can represent the ratio of cell concentrations (C/C 0 ). All experiments were accomplished at a room temperature of∼25 ◦ C. 2.10.4 1m sand columns The large-scale experiments of this study have been carried out in 1m sand columns. The sand columns were a PVC tube with 100 cm length and 4.55 cm diameter and 10 sampling holes with 10 cm intervals ( Figure 3 ). The columns were filled with silica sand, as presented in Figure 1 and saturated by pumping tap water. The pore volume of each column is about 600 ml, and the total volume of pumped solution in each injection was 720 ml (1.2 PV). To evaluate the capability of solutions S1-S4 in Table 2, 1m columns were treated by an arrangement presented in Table 3 . This arrangement and set-up in Figure 3 help to simulate the injection of S1-S4 solutions for 1m columns. The pH-adjusted CS and bacteria suspension were simultaneously pumped at the same rate from the top end to the bottom by a T-shaped hose connector ( Figure 3a ). The pH of influent in the columns is 4 and sampling for measuring urea conversion has been done 72 hours from the injection time. Eventually, the columns were washed out with the injection of 10 PV tape water and opened. Table 3 Treatment arrangement of 1m columns Column Bacteria CS UC (U) Temp Volume CaCl 2 (M) MgCl 2 (M) Urea (M) Temp Volume pH S1 20 RT 0.6 PV 4 0 4 RT 0.6 PV ad S2 20 CT 0.6 PV 4 0 4 CT 0.6 PV ad S3 20 RT 0.6 PV 3.2 0.8 4 RT 0.6 PV ad S4 20 CT 0.6 PV 3.2 0.8 4 CT 0.6 PV ad ad: adjusted; RT: Room Temperature (25º Celsius); CT: Cold Temperature (4º Celsius); PV: Pore volume 3 Results and discussion 3.1 Aqueous solution: To prepare all-in-one solutions S1-S6 presented in Table 2 , 15 ml bacteria and 15 ml pH-adjusted CS were mixed in a 50 ml beaker while the beaker was in a magnetic stirrer. A pH meter measured the pH variations in the aquatic solution. As a result of urea conversion and ammonium production in Eq. 1 , pH increased gradually until a peak and then showed a sudden drop. Alongside this pH fall, some carbonate crystals appear in the solution, changing its color to white. The time interval from the start of the biochemical reactions until this pH drop is called a lag phase (LP in Fig. 4 ). Figure 4 shows the variation of pH and urea conversion in S1-S6 solutions. As it was explained in section 3.2 and Table 2 , S1 and S2 solutions in Fig. 4a , S3 and S4 solutions in Fig. 4b , and S5 and S6 solutions in Fig. 4c , have similar elements but with two different initial temperatures. Figure 4a demonstrates the lag phase of S1 and S2 solutions are 27 minutes and 85 minutes, respectively. As, S1 and S2 solutions have only a different initial temperature, the prolonged lag phase can be attributed to the effect of the initial temperature. S1 and S2 solutions also show a big difference in urea conversion rate. For example, after 30 minutes from the start of the reactions, while the urea conversion of S1 is about 2.4% it is about 0% for S2. Figure 4b illustrates the effect of magnesium substitution on lag phase and urea conversion. Solutions S3 and S4 are created by replacing 20% of calcium chloride with magnesium chloride in solutions S1 and S2 (Table 2 ). The lag phase for solutions S3 and S4 are 53 and 61 minutes. About 15% longer lag phase in S4 than in S3, shows decreasing the initial temperature of reagents in S3 can prolong the lag phase. In Fig. 3 c solutions S5 and S6 are created by removing 20% of calcium chloride in S1 and S2 solutions. The lag phase of S5 and S6 solutions are 27 and 30 minutes, respectively. After 30 minutes from the start of reactions, the urea conversion in S5 is about 10% higher than S6, and it is because of the higher initial temperature and higher urease activity of S5 than that of S6. Generally, Fig. 4 illustrates a low temperature decreases urease activity and prolongs the lag phase. Also, the lag phase of S2 and S4 solutions, respectively 85 and 61 minutes, are higher than the other ones. However, it is vital to investigate the final urea conversion of S1-S6 solutions. It is because there is a possibility that a solution provides a long lag phase and seems it can provide a uniform carbonate precipitation but because of the low final urea conversion the amount of carbonate precipitation is lower than the other ones. Thus, the final urea conversion of S1-S6 solutions was investigated after 72 hours from the start of reactions. As Fig. 5 demonstrates the final urea conversion in all the solutions except S2 is 100%. The reason for this low final urea conversion on S2 solution was investigated by measuring the in-situ urease activity of S1-S4 solutions after overcoming the associated lag phase. The results in Fig. 6 show that the toxic effect of high concentration and low pH make a harsh condition that decreases the in-situ urease activity. While in-situ urease activity of S1 solution is about 9.30 U/ml this is approximately 2.95 U/ml for S2. As this urease activity is low it cannot convert whole urea properly. The prolonged lag phase observed in S3 and S4 is a result of the presence of magnesium cations. Magnesium cations have a smaller ionic radius than calcium cations, which means that they can fit into the crystal lattice of calcium carbonate in place of calcium cations(Abdel Gawwad et al., 2016b ; Imran et al., 2021b ; Lv et al., 2022 ; Park et al., 2008 ; Putra et al., 2016b ; Sun et al., 2019b ; Sun & Miao, 2019b ; Xu et al., 2020b , 2021 ; Zhang & Dawe, 2000b ). Thus, the substitution of calcium cations with magnesium cations can cause interference with the crystal lattice and a consequent slowing down of calcium carbonate precipitation (Berner, 1975 ; Chandra & Ravi, 2020b ; Folk, 1974 ; Gowthaman et al., 2021b ; Xu et al., 2020b , 2021 ). 3.2 XRD analysis Figure 7 demonstrates the XRD spectrum of collected precipitations from aquatic reactions of S1-S6 solutions in Table 2 . It shows the morphology of calcium precipitation in S1, S2, S5, and S6 is entirely vaterite. The crystal structure S3 and S4 precipitations are vaterite and aragonite. Thus, replacing 20% of calcium chloride with magnesium chloride in S3 and S4 solutions can change calcium carbonate's morphology from vaterite to aragonite. In solutions S5 and S6, instead of magnesium chloride substitution, distilled water was substituted for 20% calcium chloride. As Fig. 7 e and Fig. 7 f show, there aren’t aragonite’s crystal structures in the precipitations of S5 and S6, and it can be explained by the lack of magnesium in substances. Figure 7 also demonstrates the effect of magnesium on changing the morphology regardless of the initial temperature of all-in-one solution. It is in line with the previous studies on carbonate precipitation that have explained the aragonite promotion as an effect of magnesium presence (Achal, n.d.; Chandra & Ravi, 2020b ; Gowthaman et al., 2021b ; Imran et al., 2021b ; Putra et al., 2016b ; Xu et al., 2020b , 2021 ). As the hardness of aragonite is higher than vaterite, this tendency to precipitate aragonite can improve the strength of columns treated with a magnesium-included all-in-one solution. 3.3 Short columns As explained in section 2.10.2 , 72 hours after the injection of solutions S1-S4 the percentage of urea conversion in the short sand columns is measured. Then the columns were opened and submerged in 10 PV water for 24 hours to ensure the byproduct and not-consumed reagents cannot affect the result of UCS test. Then, the sand columns were air-dried and CC% test was accomplished. Figure 8 shows that the final urea conversion in all the sand columns except S2 is 100%. In S2 short columns, the converted urea is about 45 percent, and it is much more 3.75% urea conversion for S2 solution (Fig. 5) in the beaker. The reason for the higher urea conversion in column S2 than S2 solution is the temperature. Although the initial temperature of reagents for S2 solution and S2 column are similar, the temperature of solution increases rapidly in sand column because sand in the column is at room temperature. This rapid increase in temperature increases the urease activity of bacteria and helps bacteria to pass the harsh conditions of high concentrations of calcium carbonate and low pH. As Fig. 9 shows the amount of carbonate in S2 is less than half of S1, and it is in line with 100% and 45% urea conversion in S1 and S2, respectively. The lower amount of CC% in S3-S6 is because magnesium does not participate in precipitation. 3.4 SEM analysis Figure 10 shows SEM photos from precipitated carbonate in short sand columns S1, S3-S5. As the precipitated carbonate in short column S2 is just 2%, this column was too loose and it wasn’t possible to make a sample for SEM analysis. Figure 10 shows the number of trapped bacteria in precipitations for samples from S1, S3, and S5 is higher than S4 and S5. It shows in room temperature more precipitation occurred around the bacteria. This precipitation can increase the size of bacteria cells several times and decrease their capability to pass through sand grains. The lower transportability of bacteria cells will decrease the homogeneity of carbonate precipitation in large-scale soil improvements. 3.5 Semi-long columns As mentioned in section 2.10.3 , experiments in semi-long columns have been carried out to investigate bacterial transportability in sand columns. The capability of S1-S4 solutions, in Table 2 , in transporting through a long column was evaluated. To do this, it was necessary to modify S1-S4 solutions for the bacterial transportability test. Hence, urea was omitted from the ingredients of S1-S4 solutions to make S'1-S'4, respectively. Table 4 shows the components of S'1-S'4 and the Control Solution. The absence of urea on S'1-S'4, and Control Solution prevents carbonate precipitation in effluent that can interrupt OD measurements. Figure 11 shows the normalized effluent concentration (C/C 0 ) against the number of pore volumes passed through the column. It is essential to mention that a solution with higher C/C 0 has a lower bacterial attachment close to the injection point of sand columns; consequently, it has a higher potential to distribute uniformly on long columns. Figure 11 shows a remarkable difference in C/C 0 curves for Control Solution and S'1. Since both S'1 and Control Solution don't have urea in the ingredient, there is no carbonate precipitation and lag time in these solutions. Therefore, the significant difference between the transportability of the Control Solution and S'1 can only be related to the effect of decreased pH by adding acetic acid. In other words, the pH adjustment by acid in this study not only has provided a lag phase but also dramatically improved bacteria transportability through sand columns. A comparison between the transportability of S'1 and S'3 in Fig. 11 exhibits that the amount of C/C 0 for S'3 is higher than for S'1. It shows that substituting magnesium chloride for 20% calcium chloride can improve the transportability of all-in-one solution and, consequently, enhance the homogeneity of precipitation in long columns. Also, the amount of C/C 0 for S'2 and S'4 is often higher than that of S'1 and S'4, respectively. Therefore, the low initial temperature of solutions can improve their transportability through sand columns. In conclusion and light of the results presented in Fig. 4 and Fig. 11 , magnesium substitution and low temperature of solutions prolong the lag phase and improve the homogeneity of precipitation. Table 4 Ingredients of solutions injected in the bacterial transport experiment Concentration of Ca 2+ (M) Concentration of Mg 2+ (M) Concentration of urea Bacterial activity (U/ml) Temperature pH Control Solution 2 2 0 10 RT Unadjusted S´1 2 0 0 10 RT 4 S´2 2 0 0 10 CT 4 S´3 1.6 0.4 0 10 RT 4 S´4 1.6 0.4 0 10 CT 4 3.6 Hydrophobicity To investigate the reason for the higher transportability of bacteria through sand columns at a low temperature, the effect of temperature on hydrophobicity has been investigated. The hydrophobicity of bacteria is checked as explained in section 2.5 . As Fig. 12 demonstrates, CSH will increase with the decrease in pH and increase in temperature. In pH 9.2, the initial pH of culture medium, CSH is -12.4 and − 6.6 at 25º C and 4ºC temperatures. However, in this study, as explained in Table 2 , the bacteria suspension, CS, and acetic acid are mixed and made all-in-one solution with pH 4. Therefore, the amount of CSH in pH 4 is the critical issue in this study. Figure 12 shows that at pH 4, CSH is 77.2% and 20.3% for 25º C and 4º C, respectively. Therefore, at a given pH, decreasing the initial temperature of bacterial suspension will decrease CSH. This lower bacterial surface hydrophobicity can lead to the higher transportability of solutions S2 and S4, which have a low initial temperature. 3.7 1m sand column: As demonstrated in Fig. 12 and section 3.4 , injection of S1-S4 solution to the 1m columns has been carried out. The percentage of urea conversion after 72 hours from the injection was checked, as demonstrated in Fig. 12b and explained in section 2.4 . Then 10 P.V tap water was injected through the sand columns to wash out the unconsumed reagents and remove the byproduct of carbonate precipitation. Figure 13 shows that urea conversion and calcium carbonate precipitation have a close relationship. The low initial temperature of injected solutions and magnesium substitution have developed the treatment results. Columns S2 and S4 have a more uniform treatment than S1 and S3, respectively, because the initial temperature of the injected solution in columns S2 and S4 are lower than S1 and S3. Columns S3 and S4 were injected with solutions including magnesium (Table 3 ), and this presence of magnesium in the solution improved the uniformity of precipitations. Also, decreasing the initial temperature of solutions and magnesium substitution showed a synergistic effect. Therefore, column S1 which was treated at room temperature of injected solutions and without magnesium substitution (Table 3 ) has the lowest efficiency of carbonate precipitation. Oppositely, column S4 was injected at a low initial temperature of substances with a solution that included magnesium cations and provided the most homogenous carbonate precipitation. 4 Conclusions The contribution of this research is upgrading and evaluating 2 M all-in-one solution in three steps. For the first step, two modifications have been made to 2 M all-in-one solution: 1- decreasing the initial temperature of all-in-one solution, 2- substituting 20% of calcium with magnesium. The findings of this study convinced that this magnesium substitution changes the morphology of carbonate from vaterite to aragonite. As the hardness of aragonite is higher than vaterite, this change in the crystal structure of precipitation will lead to a higher strength of bioimproved soil. Magnesium substitution and the low initial temperature of all-in-one solution prolonged the lag phase. These two modifications showed a synergistic effect, so the longest lag phase was for the magnesium-included all-in-one solution with a low initial temperature (S4). For the second step, the transportability of 2M all-in-one solutions through the sand columns was evaluated. The low initial temperature of all-in-one solution and magnesium substitution for calcium improved the transportability of bacteria. Thus, the magnesium-included solution with low temperature provided the best transportability. To explain the more uniform spread of bacteria at low temperatures, the variation of CSH against temperature and pH was determined. It showed that at pH 4, CSH decreased from 77.2 to 22.3 while temperature decreased from 25º C to 4ºC. This decrease in CSH can improve the transportability and distribution of bacteria in 1m sand columns. For the third step, the capability of 2M all-in-one solutions in the treatment of 1m long columns was examined. Columns treated with a lower temperature of substances and magnesium substitution have more uniformity of urea conversion and calcium carbonate. In general, this study verified that 2 M all-in-one solution can be converted entirely in 3 days and provide carbonate precipitation. Lowering the initial temperature of reagents will improve the lag phase, and transportability of bacteria and reduce the CSH. Magnesium substitution can prolong lag phases, develop bacterial transportability, and lead to precipitation of aragonite. Declarations Data Availability Statement All data, photos, and analyses presented in this paper are available from the corresponding author by request. Acknowledgments The work was supported by NSFC Major International Joint Research Project POW3M (51920105013), Jiangsu Distinguished Professor Program, and Jiangsu Province ‘‘Six Talent Peak’’ program (XCL-111). 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Colloids and Surfaces B: Biointerfaces, 139 , 244–248. https://doi.org/https://doi.org/10.1016/j.colsurfb.2015.11.024 Zhong, H., Liu, G., Jiang, Y., Yang, J., Liu, Y., & Yang, X. (2017). Transport of bacteria in porous media and its enhancement by surfactants for bioaugmentation: A review. 35 , 490–504. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4449151","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":308056832,"identity":"8cbe246a-c846-47bd-9f1b-150b6368d4a2","order_by":0,"name":"Seyed Mohammad Javad Hosseini","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Mohammad Javad","lastName":"Hosseini","suffix":""},{"id":308056836,"identity":"9c2328ba-0144-476b-9cb8-8b6d7faf16ed","order_by":1,"name":"Dawei Guan","email":"","orcid":"","institution":"Hohai University","correspondingAuthor":false,"prefix":"","firstName":"Dawei","middleName":"","lastName":"Guan","suffix":""},{"id":308056838,"identity":"f3770ea6-9c1a-4da4-b4e9-b25a83fac69a","order_by":2,"name":"Liang Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYDACdgY2ECXHwHAASLERo4UZosyYdC2JDWAeMVr4m5mfPfi4ozZ9w8HjDxg+lB1m4J/dgF+LxGE2c8OZZ47nbjhwxoBxxrnDDBJ3DuDXYsDMwybN23YMpIWBmbftMIOBRAIRWv62HUs3OHD8AfNforUwttUkGBw4YMDMSIwWoF/MJHvbDhjOBPrlYM+5dB6JGwS08Lc3P5P42VYnz3fj+MMHP8qs5fhnENACBYeB9h0ARyYPUeqBoA5oXwOxikfBKBgFo2CkAQA4XURAwZ7JJQAAAABJRU5ErkJggg==","orcid":"","institution":"Jiangsu University","correspondingAuthor":true,"prefix":"","firstName":"Liang","middleName":"","lastName":"Cheng","suffix":""}],"badges":[],"createdAt":"2024-05-20 12:36:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4449151/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4449151/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57502117,"identity":"536a704e-20b5-4d51-bae4-bd0977b0c64e","added_by":"auto","created_at":"2024-05-31 14:09:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":78653,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size distribution of sand used in this study\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/a90ac1bda495a104b86d2dba.png"},{"id":57502125,"identity":"92355678-1af3-4cb4-a008-9d635837d201","added_by":"auto","created_at":"2024-05-31 14:09:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":384662,"visible":true,"origin":"","legend":"\u003cp\u003ethe designed setup to evaluate bacterial transport through sand columns \u003cstrong\u003e(a)\u003c/strong\u003e schematic representation; and \u003cstrong\u003e(b)\u003c/strong\u003e the real setup erected in the laboratory. CS solution and bacteria suspension were injected parallelly by two peristaltic pumps that were set at a 22.5 ml/min injection rate.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/9d880f9e8cec59dbcddd2fe6.png"},{"id":57502609,"identity":"b7910476-bc0c-428d-8113-3e4aa2c055aa","added_by":"auto","created_at":"2024-05-31 14:17:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":259983,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e schematic representation of the designed setup for the treatment of 1m sand column; \u003cstrong\u003e(b)\u003c/strong\u003e embedded sampling points in 1m columns; \u003cstrong\u003e(c)\u003c/strong\u003e a photo from the 1 m sand column.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/ba1f9de38b49228265449d88.png"},{"id":57502608,"identity":"ae0c1ddb-f1da-46c9-ac75-47512060d6c8","added_by":"auto","created_at":"2024-05-31 14:17:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":84133,"visible":true,"origin":"","legend":"\u003cp\u003eThe initial pH variations of S1-S4 all-in-one solutions. The interval time from the start of reactions until the peak of pH shows the lag phase (LP) period.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/6b65e8d2605d094890c9be41.png"},{"id":57502607,"identity":"0e599206-c09a-4e68-a66f-8b4432310f3e","added_by":"auto","created_at":"2024-05-31 14:17:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11298,"visible":true,"origin":"","legend":"\u003cp\u003eThe final urea conversion of S1-S6 solutions in beakers\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/4a440d3cabc593a70761853d.png"},{"id":57502119,"identity":"6155f364-9d53-4f1f-85d2-296532750fa1","added_by":"auto","created_at":"2024-05-31 14:09:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":22834,"visible":true,"origin":"","legend":"\u003cp\u003eIn-situ urease activity of S1-S4 solutions after the lag phase\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/61c8ff86e0376151b0b3683d.png"},{"id":57502121,"identity":"bc1f87cc-e80f-40d4-ae97-082bbd8e90d3","added_by":"auto","created_at":"2024-05-31 14:09:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":205521,"visible":true,"origin":"","legend":"\u003cp\u003eXRD analysis of precipitations on: \u003cstrong\u003e(a)\u003c/strong\u003e S1 solution, \u003cstrong\u003e(b)\u003c/strong\u003e S2 solution, \u003cstrong\u003e(c)\u003c/strong\u003eS3 solution, \u003cstrong\u003e(d)\u003c/strong\u003e S4 solution, \u003cstrong\u003e(e)\u003c/strong\u003e S5 solution, \u003cstrong\u003e(f)\u003c/strong\u003e S6 solution. A represents aragonite, and V represents vaterite.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/79a57f3ba74d4ba3197d25d6.png"},{"id":57503151,"identity":"8bf51b08-722d-4cca-bbba-775e46dbea4a","added_by":"auto","created_at":"2024-05-31 14:25:47","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":35631,"visible":true,"origin":"","legend":"\u003cp\u003eThe final urea conversion of S1-S6 solutions in sand columns and solutions\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/3a8e05ab7805217579fda0c9.png"},{"id":57503517,"identity":"b5b84ad7-b629-4b51-bd33-a140f5c977e8","added_by":"auto","created_at":"2024-05-31 14:33:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":21071,"visible":true,"origin":"","legend":"\u003cp\u003eThe final urea conversion of S1-S6 solutions in beakers\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/8a21c8b9a8a706493d133adb.png"},{"id":57502611,"identity":"1c06a0a4-bad1-4f62-9836-1ddcb677f490","added_by":"auto","created_at":"2024-05-31 14:17:47","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":498496,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/63a464e5e80fa0a9944af35c.png"},{"id":57502128,"identity":"90907de0-d426-4c05-b6a6-81c3a1766105","added_by":"auto","created_at":"2024-05-31 14:09:47","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":46501,"visible":true,"origin":"","legend":"\u003cp\u003eBreakthrough curves for bacterial transport of solutions S1-S4. C/C\u003csub\u003e0\u003c/sub\u003e shows the ratio of bacterial cell concentration of effluent to influent.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/bd77aa1616b82f17bf1f76ac.png"},{"id":57502126,"identity":"f729f529-d89f-4285-8791-82c00270fe46","added_by":"auto","created_at":"2024-05-31 14:09:47","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":25018,"visible":true,"origin":"","legend":"\u003cp\u003echanges of microbial surface hydrophobicity against pH and temperature. RT represents Room temperature (25°C) and CT represents Cold temperature (4° C)\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/235c61b7f2be434e635c18bd.png"},{"id":57502612,"identity":"bd2f4e3d-f352-4651-ba94-3e915ffbc393","added_by":"auto","created_at":"2024-05-31 14:17:47","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":99291,"visible":true,"origin":"","legend":"\u003cp\u003eThe log of urea conversion percentage and calcium carbonate precipitation in 1m columns S1-S4\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/fa39812bc8f24f0e6bdf8642.png"},{"id":59067698,"identity":"a6ef9eca-31e1-48ae-a4fa-517001874bad","added_by":"auto","created_at":"2024-06-26 03:35:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2651985,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4449151/v1/3d066857-6ac2-47e3-b881-f986b2cdda65.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ground improvement with single treatment using Mg 2+ modified all-in-one MICP solution: 1m sand column","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eMicrobially induced carbonate precipitation (MICP) has been developed as an alternative soil improving technique to curb the environmental challenges of traditional cementation projects. During the MICP process, a ureolytic bacteria produces a urease enzyme to catalyze urea hydrolysis, as represented in \u003cb\u003eEq.\u0026nbsp;1\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(CO{(N{H_2})_2}+2{H_2}O\\xrightarrow{{Urease}}CO_{3}^{{2 - }}+2NH_{4}^{+}\\)\u003c/span\u003e \u003c/span\u003e \u003cb\u003eEq.\u0026nbsp;1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) released from urea hydrolysis results in a pH rise and commences the precipitation of calcium carbonate. Calcite is precipitated through the combination of carbonate ions (CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e) from the hydrolysis of urea and the calcium cation (Ca\u003csup\u003e2+\u003c/sup\u003e) from the supplied calcium source(Cheng \u0026amp; Cord-Ruwisch, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mujah et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pakbaz et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e):\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(C{a^{2+}}+C{O_3}^{{2 - }} \\to CaC{O_3} \\downarrow\\)\u003c/span\u003e \u003c/span\u003e \u003cb\u003eEq.\u0026nbsp;2\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e) generated from the biochemical reactions is responsible for the biocementation and improvement of the soil strength(Behzadipour \u0026amp; Sadrekarimi, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pakbaz et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shahin et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, for MICP reactions, the presence of urea, calcium ions, and a urease-producing bacteria is vital. A bacterial suspension often provides ureolytic bacteria, and the cementation solution (CS) includes dissolved urea and calcium salt(Ashkan et al., 2019; van Paassen et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Whiffin, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Bacterial suspension and CS need to be delivered into soil matrix to trigger in situ biocementation reactions(Gurbuz et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; He et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; H. J. Lai et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mahawish et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In literature, CS is often an equimolar concentration of urea and calcium chloride, and the employed bacteria is \u003cem\u003eSporsarcina pasteurii\u003c/em\u003e(Cheng et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Do, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Karimian \u0026amp; Hassanlourad, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pegah \u0026amp; M., 2020).\u003c/p\u003e \u003cp\u003eIn MICP, the designed method for delivering bacteria and CS can impact the efficiency of the process and the engineering properties of bioimproved soil(Cheng et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Cheng \u0026amp; Shahin, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Harkes et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Jamshidi-Zanjani \u0026amp; Khodadadi Darban, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, scientists have established different injection strategies to meet the demands of engineering projects. Until now, a multiphase injection is a conventional method in MICP that researchers often implement (Muynck et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; T. et al., 2017; Wang et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the implementation of multi-phase injection methods often poses challenges due to their inherent complexity and the unpredictable nature of phase interactions during the injection process. Therefore, it is preferable to simplify the process with an injection of only one solution, including all necessary ingredients(Cheng et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chuangzhou et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; H.-J. Lai et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; X. Liu, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecently, all-in-one solution strategy has shown its advantages in providing a low-cost and highly efficient MICP (Cheng et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chuangzhou et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; M. J. Cui et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; M.-J. Cui et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; H.-J. Lai et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the all-in-one solution strategy, CS and bacteria are mixed before injection. As this mixture can stimulate immediate precipitation, a lag phase in the cementation should be created to provide a time window for completing the injection process. Some researchers have made this lag phase in the carbonate precipitation by adding acid to the all-in-one solution and decreasing the initial pH of the all-in-one solution to about 4(Cheng et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chuangzhou et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; M. J. Cui et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; M.-J. Cui et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; H.-J. Lai et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In another approach, Xiao et. al \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2021\u003c/span\u003e have tried to reduce the initial temperature of all-in-one solution, to minimize the urease activity of bacteria and consequently make a lag phase in cementation (Xiao et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). With this background, this study employed the simultaneous effect of low pH and cold temperature in prolonging the lag phase for all-in-one solutions.\u003c/p\u003e \u003cp\u003eIn the first step, to further enhance the homogeneity and efficiency of treated sand with all-in-one solution, some effects of magnesium (Mg\u003csup\u003e2+\u003c/sup\u003e) substitution for calcium (Ca\u003csup\u003e2+\u003c/sup\u003e) were investigated. Some reasons support the significance of studying magnesium substitution's effect in all-in-one solutions. Firstly, the impact of magnesium cations on biocementation is rarely explored and covered with some contradictory results(Gowthaman et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). For example, as given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, while most researchers have mentioned the positive effect of Mg\u003csup\u003e2+\u003c/sup\u003e, others concluded that magnesium cations could decrease bio-mortar strength (Abdel Gawwad et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e; Putra et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Zhang \u0026amp; Dawe, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2000a\u003c/span\u003e). Secondly, although a few studies have noted the impact of magnesium ions in biocementation, they often have studied enzyme-induced carbonate precipitation (EICP) (Chandra \u0026amp; Ravi, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Imran et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e; Lu, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Putra et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). Since there are no urease-positive bacteria in EICP, the precipitated carbonate by EICP along a soil column is generally more uniform than that of MICP (Ashkan et al., 2019; Dilrukshi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; T. et al., 2017). As the uniformity of treated soil is the main challenge in all-in-one solution strategy, the significance of magnesium substitution can be highlighted in this study.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of the published literature on the effects of magnesium substitution\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMechanism of effect\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEffect\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrecipitation of aragonite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(Xu et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDelaying agent in precipitation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(Putra et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInhibitory on biofilm formation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(Oknin et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrease activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(Sun et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Sun \u0026amp; Miao, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarbonate precipitation productivity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(Abdel Gawwad et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAlso, the context of hydrophobicity and its significance in MICP is discussed in this study. Cell surface hydrophobicity (CSH) is a biophysical measurement of a cell's affinity for a hydrophobic versus hydrophilic environment(Gargiulo et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Jain \u0026amp; Arnepalli, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; G. Liu et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rosenberg, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Xia et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bacteria with higher CSH prefer to attach to the sand grains and show a lower transportability along a sand column. In this case, a higher population of bacterial cells will be stopped near the injection point and lead to an inhomogeneous distribution of bacteria through the column (Huysman \u0026amp; Verstraete, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; G. Liu et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Redman et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Xia et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhong et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Consequently, a high CSH forms bioclogging near the injection point and a low efficient MICP treatment on the farthest part of the column.\u003c/p\u003e \u003cp\u003eGenerally, changes in pH can alter the charge and conformation of cell surface molecules, such as lipids and proteins, which can affect the hydrophobicity of the cell surface(Eschlbeck et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mirani et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhong et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A decrease in pH (acidic conditions) can lead to an increase in cell surface hydrophobicity, as the protonation of functional groups on the cell surface can enhance hydrophobic interactions(Eschlbeck et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mirani et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhong et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Also, a decrease in temperature may lead to a decrease in cell surface hydrophobicity, as lower temperatures can cause the cell membrane to become more rigid and reduce the exposure of hydrophobic regions(Gallardo-Moreno et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Gharabaghi et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kim \u0026amp; Walker, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; McCaulou et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). As in this study all-in-one solution is injected at low pH and low temperature, investigation on the role of CSH seems essential.\u003c/p\u003e"},{"header":"2 Material and methods","content":"\u003cp\u003eIn the first step, an experiment series was done on aqueous mediums for S1-S6 all-in-one solutions presented in \u003cstrong\u003eTable 2\u003c/strong\u003e to assess pH variation and crystallography of derived precipitations. X-ray diffraction (XRD) tests provide a microscale point of view into the effect of magnesium on all-in-one solutions. As the second step, a group of short sand columns was treated with the injection of S1-S6 solutions, and the percentage of urea conversion and calcium carbonate was determined. In the third step, four columns of 1 meter in length were injected with S1-S4 solutions to evaluate the efficiency of all-in-one solutions in large-scale projects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e Composition of the evaluated all-in-one solutions\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 1.2371%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 59.3815%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration of chemical substances (M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTemperature\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 1.2371%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.4124%;\"\u003e\n \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMgCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eUrea\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.2371%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.4124%;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10 U\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.2371%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.4124%;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10 U\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.2371%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.4124%;\"\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10 U\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.2371%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.4124%;\"\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10 U\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.2371%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.4124%;\"\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10 U\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eS6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.2371%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.4124%;\"\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10 U\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cul\u003e\n \u003cli\u003eThe initial pH of all the solutions was adjusted to about 4 by mixing adequate acetic acid with the reagents.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003e2.1 Bacterial culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSporsarcina pasteurii\u003c/em\u003e was employed in the current study as a urease-producing bacteria. This bacterium was cultivated in a sterile aerobic batch growth medium (200 mL growth medium in a 1-L flask shaken at 180 rpm), including 20 g/L yeast extract, 15 g/L ammonium chloride, and 0.1 mM NiCl\u003csub\u003e2\u003c/sub\u003e, adjusted at pH = 9.2. The harvested bacterial culture had a urease activity of 20 \u0026plusmn; 1U/ ml.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 pH adjustment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn adequate volume of 3 M acetic acid was added to the 15 ml CS in a 50 ml beaker to adjust the initial pH of the all-in-one solution. A pH meter monitored the pH variation while a magnetic stirrer mixed the acid-added CS. Then, 15 ml bacteria were added to 15 ml acid-added CS to achieve pH4. The adequate volume of acetic acid was determined with trial and error.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 \u0026nbsp;Soil type\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNatural silica sand with a hydraulic conductivity of 1.88\u0026times;10\u003csup\u003e-4\u003c/sup\u003e m/s was used to fill 1m columns. The aggregation curve of the sand is given in \u003cstrong\u003eFigure 1\u003c/strong\u003e. Following the Unified Soil Classification System (USCS), the sand used has been classified as poorly graded (SP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Urease activity and Urea conversion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUrease activity was determined via electrical conductivity (EC) in the absence of calcium cations. 1 mL of the bacterial cell suspension was added to 5 mL of 3 M urea and 4 mL of DI water (reaction concentration 1.5 M urea). Variations of the relative electrical conductivity were recorded over 5 minutes at 25 \u0026plusmn; 1℃. In the presence of calcium ions, a modified Nessler method indicates urea conversion rate from the produced ammonia concentration in \u003cstrong\u003eEquation 1\u003c/strong\u003e(Cheng et al., 2016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 \u0026nbsp;Hydrophobicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are several methods to analyze CSH, including Microbial Adhesion to Hydrocarbons (MATH), Contact Angle Measurement, Bacterial Adhesion to Solvents (BATS), Salt Aggregation Test (SAT), Hydrophobic Grid Membrane Filter (HGMF). Each method has its advantages and limitations, and the choice of a method depends on the specific requirements of the study and the characteristics of the tested bacteria. In literature, the most well-defined method for CSH measurement is microbial adherence to hydrocarbons (MATH) method \u003csup\u003e(Kaczorek et al., 2018; Mozes \u0026amp; Rouxhet, 1987; Rosenberg, 2006; Rosenberg et al., 1980)\u003c/sup\u003e. Therefore, in this study, the MATH method was employed to measure CSH of \u003cem\u003eSporsarcina pasteurii\u0026nbsp;\u003c/em\u003eat room temperature (25\u0026ordm; C) and cold temperature (4\u0026ordm; C) and three pHs 9.2, 7, and 4. Here are the general steps for CSH measurements (Rosenberg et al., 1980, 1991):\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1- Culture Preparation:\u003c/strong\u003e Growing bacteria in the culture medium until they reach the desired growth phase (according to \u003cstrong\u003esection 2.2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2- Washing:\u003c/strong\u003e Centrifuging the culture and discarding the supernatant. Resuspend the bacterial pellet in a pH-adjusted phosphate buffer solution to get OD\u003csub\u003e400\u003c/sub\u003e below 1 by spectrophotometer. Keeping 2 ml of this bacterial suspension for measurement step (A\u003csub\u003e0\u003c/sub\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3- Preparation of Hydrocarbon:\u003c/strong\u003e Adding 1 ml of a hydrocarbon, such as xylene, to the 5 ml bacterial suspension.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4- Mixing:\u003c/strong\u003e Vortexing the mixture vigorously for 2 minutes to allow the interaction between bacteria and the hydrocarbon.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5- Phase Separation:\u003c/strong\u003e Allowing the mixture to be kept undisturbed for 15 minutes for the hydrocarbon and aqueous phases to separate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6- Measurement:\u003c/strong\u003e Measuring the optical density (OD\u003csub\u003e400\u003c/sub\u003e) of the aqueous phase (A\u003csub\u003e1\u003c/sub\u003e) and bacterial suspension (A\u003csub\u003e0\u003c/sub\u003e). The decrease in OD represents the bacteria that have adhered to the hydrocarbon phase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7- Calculation:\u003c/strong\u003e Determining hydrophobicity by: 100 * (1 - OD of the aqueous phase (A\u003csub\u003e1\u003c/sub\u003e)/OD of the cell suspension (A\u003csub\u003e0\u003c/sub\u003e)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Sampling from all-in-one solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn specific time intervals, disposable 1 ml syringes have been used for sampling from the all-in-one solutions in beakers and sand columns. To stop the bacterial activities of the samples, they were filtered by using 0.22\u0026micro;m disposable syringe filters and kept in a freezer (-22℃) until the analysis time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Unconfined compressive strength (UCS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;After the end of the treatment process for 1m columns, 10 pore volume (10 PV) tap water was injected through the 1m columns to wash out unconsumed substances. Then, columns were opened, and each column was divided into 10 sections with diameter-to-height ratios of 1:1.5 to 1:2 and air-dried for 48 h before the UCS test. UCS tests on bio-cemented soil columns were carried out according to the method stated in ASTM D2166 with a constant loading rate of 1.0 mm/min(ASTM, 2013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8\u0026nbsp; Carbonate content measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe weight of precipitated carbonate was obtained using a gravimetric acid washing process. This method weighed a fraction of an oven-dried sand column before and after washing with 1M HCl acid. The difference in weight before and after acid washing was considered the amount of precipitated carbonate\u003csup\u003e(Cheng \u0026amp; Shahin, 2019)\u003c/sup\u003e. Carbonate content percentage (CC%) is the amount of precipitated carbonate divided by the weight of sand after acid washing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Microstructure analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFractions of biotreated sand columns were examined by SEM (Model: JSM-7800F). All samples were flushed with distilled water and dried at 60 \u0026deg;C for 48 hours before the microscopy investigation. XRD patterns were recorded on a BRUKER D8 ADVANCE machine using a scanning range from 2\u0026theta;= 15\u0026deg;\u0026ndash;70\u0026deg;(Cheng et al., 2014). The dried precipitated materials were observed to evaluate the effect of the magnesium substitution chloride on the mineralogical characteristics. Phase identification was carried out by searching the Inorganic Crystal Structure Database (ICSD) powder diffraction database by Xpert HighScore Plus (version 3.0e)(Cheng et al., 2014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Experimental procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10.1 Aqueous test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pH variations and urea conversion on all S1-S4 solutions in \u003cstrong\u003eTable 2\u003c/strong\u003e were monitored to investigate the effects of magnesium substitution\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eand temperature. Solutions have been prepared individually by adding 15 ml bacteria to 15 ml pH-adjusted CS on a 50 ml beaker. The current study used calcium chloride anhydrous and magnesium chloride hexahydrate to source Ca\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e, respectively. The pH variations on aqueous solutions were investigated while all-in-one solution was stirred at 400 rpm. This monitoring can indicate the lag phase of biochemical reactions at the aqueous solutions that can lead to the selection of the most desirable solutions for sand column treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10.2 Short sand columns\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e50 ml syringe was used as short columns and filled with clean silica sand (mentioned in \u003cstrong\u003eFigure 1\u003c/strong\u003e) at a dry density of about 1.6 g/cm3 and 20 ml pore volume (PV). After the preparation of all-in-one solution in a beaker, it was immediately injected by a peristaltic pump from the top to the bottom at the flow rate of 20 ml/min. The columns were kept at room temperature in a fully saturated condition for 72 hours. Then, the columns were washed out with tap water, and 1 ml of the outflow was sampled for urea conversion measurement. The injected tap water also can wash out the excess reagents on the sand columns and prevent misleading results on UCS and CC%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10.3 Bacterial transport in sand columns\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA semi-long column was designed to simulate the distribution of all-in-one solutions in long columns. Therefore, PVC columns were used for bacterial transport experiments with a 25 cm length and 4.5 cm inner diameter. The sand (defined in \u003cstrong\u003eFigure 1\u003c/strong\u003e and \u003cstrong\u003esection 2.3\u003c/strong\u003e) was dry packed into the columns under vibration. \u003cstrong\u003eFigure 2\u003c/strong\u003e shows a schematic of the setup for this experiment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEach bacterial transport experiment was conducted as the following procedure. First, the sand column was washed out and saturated by tap water. Then 6 PVs of the solutions were adjusted and introduced into the vertically oriented column at a 45 mL/min flow rate. The effluent from the column was collected every 1 min in 10-mL tubes. The optical density of all samples was measured with a spectrophotometer INESA N2S model at 600nm (OD\u003csub\u003e600\u003c/sub\u003e). Prior researches show that OD\u003csub\u003e600\u003c/sub\u003e is on correlation to cell colony-forming-unit concentration (C, CFU/mL) for bacteria (Babakhani, 2019; G. Liu et al., 2020; Redman et al., 2004; Xia et al., 2021), (for \u003cem\u003eSporosarcina pasteurii\u003c/em\u003e, biomass concentration is C (g/L) = 0.438 \u0026times; OD600, Ref.(Cheng et al., 2019)). Therefore, the ratio between OD\u003csub\u003e600\u003c/sub\u003e for effluent samples and the influent can represent the ratio of cell concentrations (C/C\u003csub\u003e0\u003c/sub\u003e). All experiments were accomplished at a room temperature of\u0026sim;25\u003csup\u003e◦\u003c/sup\u003eC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10.4 1m sand columns\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe large-scale experiments of this study have been carried out in 1m sand columns. The sand columns were a PVC tube with 100 cm length and 4.55 cm diameter and 10 sampling holes with 10 cm intervals (\u003cstrong\u003eFigure 3\u003c/strong\u003e). The columns were filled with silica sand, as presented in \u003cstrong\u003eFigure 1\u003c/strong\u003e and saturated by pumping tap water. The pore volume of each column is about 600 ml, and the total volume of pumped solution in each injection was 720 ml (1.2 PV).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the capability of solutions S1-S4 in \u003cstrong\u003eTable 2,\u003c/strong\u003e 1m columns were treated by an arrangement presented in \u003cstrong\u003eTable 3\u003c/strong\u003e. This arrangement and set-up in \u003cstrong\u003eFigure 3\u003c/strong\u003e help to simulate the injection of S1-S4 solutions for 1m columns. The pH-adjusted CS and bacteria suspension were simultaneously pumped at the same rate from the top end to the bottom by a T-shaped hose connector (\u003cstrong\u003eFigure 3a\u003c/strong\u003e). The pH of influent in the columns is 4 and sampling for measuring urea conversion has been done 72 hours from the injection time. Eventually, the columns were washed out with the injection of 10 PV tape water and opened. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e Treatment arrangement of 1m columns\u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.286677908937605%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eColumn\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.24451939291737%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eBacteria\u003c/strong\u003e\u003c/p\u003e\u0026nbsp;\u0026nbsp;\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.468802698145026%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eCS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.849624060150376%\"\u003e\n \u003cp\u003eUC\u003c/p\u003e\n \u003cp\u003e(U)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.969924812030076%\"\u003e\n \u003cp\u003eTemp\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.466165413533835%\"\u003e\n \u003cp\u003eVolume\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.278195488721805%\"\u003e\n \u003cp\u003eCaCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.466165413533835%\"\u003e\n \u003cp\u003eMgCl\u003csub\u003e2\u003c/sub\u003e (M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.654135338345865%\"\u003e\n \u003cp\u003eUrea\u003c/p\u003e\n \u003cp\u003e(M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.022556390977444%\"\u003e\n \u003cp\u003eTemp\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.466165413533835%\"\u003e\n \u003cp\u003eVolume\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.827067669172933%\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e\u003cstrong\u003eS1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.322091062394604%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.63575042158516%\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0.6 PV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.118043844856661%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.45531197301855%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.094435075885329%\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0.6 PV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.227655986509275%\"\u003e\n \u003cp\u003ead\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e\u003cstrong\u003eS2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.322091062394604%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.63575042158516%\"\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0.6 PV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.118043844856661%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.45531197301855%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.094435075885329%\"\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0.6 PV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.227655986509275%\"\u003e\n \u003cp\u003ead\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e\u003cstrong\u003eS3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.322091062394604%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.63575042158516%\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0.6 PV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.118043844856661%\"\u003e\n \u003cp\u003e3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.45531197301855%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.094435075885329%\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0.6 PV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.227655986509275%\"\u003e\n \u003cp\u003ead\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e\u003cstrong\u003eS4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.322091062394604%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.63575042158516%\"\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0.6 PV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.118043844856661%\"\u003e\n \u003cp\u003e3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.45531197301855%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.094435075885329%\"\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.286677908937605%\"\u003e\n \u003cp\u003e0.6 PV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.227655986509275%\"\u003e\n \u003cp\u003ead\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cul\u003e\n \u003cli\u003ead: adjusted; RT: Room Temperature (25\u0026ordm; Celsius); CT: Cold Temperature (4\u0026ordm; Celsius); PV: Pore volume\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Aqueous solution:\u003c/h2\u003e\n \u003cp\u003eTo prepare all-in-one solutions S1-S6 presented in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, 15 ml bacteria and 15 ml pH-adjusted CS were mixed in a 50 ml beaker while the beaker was in a magnetic stirrer. A pH meter measured the pH variations in the aquatic solution. As a result of urea conversion and ammonium production in \u003cstrong\u003eEq.\u0026nbsp;1\u003c/strong\u003e, pH increased gradually until a peak and then showed a sudden drop. Alongside this pH fall, some carbonate crystals appear in the solution, changing its color to white. The time interval from the start of the biochemical reactions until this pH drop is called a lag phase (LP in \u003cstrong\u003eFig.\u0026nbsp;4\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 4\u003c/strong\u003e shows the variation of pH and urea conversion in S1-S6 solutions. As it was explained in section \u003cspan class=\"InternalRef\"\u003e3.2\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, S1 and S2 solutions in \u003cstrong\u003eFig.\u0026nbsp;4a\u003c/strong\u003e, S3 and S4 solutions in \u003cstrong\u003eFig.\u0026nbsp;4b\u003c/strong\u003e, and S5 and S6 solutions in \u003cstrong\u003eFig.\u0026nbsp;4c\u003c/strong\u003e, have similar elements but with two different initial temperatures. Figure 4a demonstrates the lag phase of S1 and S2 solutions are 27 minutes and 85 minutes, respectively. As, S1 and S2 solutions have only a different initial temperature, the prolonged lag phase can be attributed to the effect of the initial temperature. S1 and S2 solutions also show a big difference in urea conversion rate. For example, after 30 minutes from the start of the reactions, while the urea conversion of S1 is about 2.4% it is about 0% for S2. Figure 4b illustrates the effect of magnesium substitution on lag phase and urea conversion. Solutions S3 and S4 are created by replacing 20% of calcium chloride with magnesium chloride in solutions S1 and S2 (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The lag phase for solutions S3 and S4 are 53 and 61 minutes. About 15% longer lag phase in S4 than in S3, shows decreasing the initial temperature of reagents in S3 can prolong the lag phase. In Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec solutions S5 and S6 are created by removing 20% of calcium chloride in S1 and S2 solutions. The lag phase of S5 and S6 solutions are 27 and 30 minutes, respectively. After 30 minutes from the start of reactions, the urea conversion in S5 is about 10% higher than S6, and it is because of the higher initial temperature and higher urease activity of S5 than that of S6.\u003c/p\u003e\n \u003cp\u003eGenerally, \u003cstrong\u003eFig.\u0026nbsp;4\u003c/strong\u003e illustrates a low temperature decreases urease activity and prolongs the lag phase. Also, the lag phase of S2 and S4 solutions, respectively 85 and 61 minutes, are higher than the other ones.\u003c/p\u003e\n \u003cp\u003eHowever, it is vital to investigate the final urea conversion of S1-S6 solutions. It is because there is a possibility that a solution provides a long lag phase and seems it can provide a uniform carbonate precipitation but because of the low final urea conversion the amount of carbonate precipitation is lower than the other ones. Thus, the final urea conversion of S1-S6 solutions was investigated after 72 hours from the start of reactions. As \u003cstrong\u003eFig.\u0026nbsp;5\u003c/strong\u003e demonstrates the final urea conversion in all the solutions except S2 is 100%. The reason for this low final urea conversion on S2 solution was investigated by measuring the in-situ urease activity of S1-S4 solutions after overcoming the associated lag phase. The results in \u003cstrong\u003eFig.\u0026nbsp;6\u003c/strong\u003e show that the toxic effect of high concentration and low pH make a harsh condition that decreases the in-situ urease activity. While in-situ urease activity of S1 solution is about 9.30 U/ml this is approximately 2.95 U/ml for S2. As this urease activity is low it cannot convert whole urea properly.\u003c/p\u003e\n \u003cp\u003eThe prolonged lag phase observed in S3 and S4 is a result of the presence of magnesium cations. Magnesium cations have a smaller ionic radius than calcium cations, which means that they can fit into the crystal lattice of calcium carbonate in place of calcium cations(Abdel Gawwad et al., \u003cspan class=\"CitationRef\"\u003e2016b\u003c/span\u003e; Imran et al., \u003cspan class=\"CitationRef\"\u003e2021b\u003c/span\u003e; Lv et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Park et al., \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e; Putra et al., \u003cspan class=\"CitationRef\"\u003e2016b\u003c/span\u003e; Sun et al., \u003cspan class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Sun \u0026amp; Miao, \u003cspan class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Xu et al., \u003cspan class=\"CitationRef\"\u003e2020b\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang \u0026amp; Dawe, \u003cspan class=\"CitationRef\"\u003e2000b\u003c/span\u003e). Thus, the substitution of calcium cations with magnesium cations can cause interference with the crystal lattice and a consequent slowing down of calcium carbonate precipitation (Berner, \u003cspan class=\"CitationRef\"\u003e1975\u003c/span\u003e; Chandra \u0026amp; Ravi, \u003cspan class=\"CitationRef\"\u003e2020b\u003c/span\u003e; Folk, \u003cspan class=\"CitationRef\"\u003e1974\u003c/span\u003e; Gowthaman et al., \u003cspan class=\"CitationRef\"\u003e2021b\u003c/span\u003e; Xu et al., \u003cspan class=\"CitationRef\"\u003e2020b\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 XRD analysis\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e demonstrates the XRD spectrum of collected precipitations from aquatic reactions of S1-S6 solutions in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. It shows the morphology of calcium precipitation in S1, S2, S5, and S6 is entirely vaterite. The crystal structure S3 and S4 precipitations are vaterite and aragonite. Thus, replacing 20% of calcium chloride with magnesium chloride in S3 and S4 solutions can change calcium carbonate\u0026apos;s morphology from vaterite to aragonite. In solutions S5 and S6, instead of magnesium chloride substitution, distilled water was substituted for 20% calcium chloride. As Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee and Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ef show, there aren\u0026rsquo;t aragonite\u0026rsquo;s crystal structures in the precipitations of S5 and S6, and it can be explained by the lack of magnesium in substances. Figure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e also demonstrates the effect of magnesium on changing the morphology regardless of the initial temperature of all-in-one solution. It is in line with the previous studies on carbonate precipitation that have explained the aragonite promotion as an effect of magnesium presence (Achal, n.d.; Chandra \u0026amp; Ravi, \u003cspan class=\"CitationRef\"\u003e2020b\u003c/span\u003e; Gowthaman et al., \u003cspan class=\"CitationRef\"\u003e2021b\u003c/span\u003e; Imran et al., \u003cspan class=\"CitationRef\"\u003e2021b\u003c/span\u003e; Putra et al., \u003cspan class=\"CitationRef\"\u003e2016b\u003c/span\u003e; Xu et al., \u003cspan class=\"CitationRef\"\u003e2020b\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). As the hardness of aragonite is higher than vaterite, this tendency to precipitate aragonite can improve the strength of columns treated with a magnesium-included all-in-one solution.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Short columns\u003c/h2\u003e\n \u003cp\u003eAs explained in section \u003cspan class=\"InternalRef\"\u003e2.10.2\u003c/span\u003e, 72 hours after the injection of solutions S1-S4 the percentage of urea conversion in the short sand columns is measured. Then the columns were opened and submerged in 10 PV water for 24 hours to ensure the byproduct and not-consumed reagents cannot affect the result of UCS test. Then, the sand columns were air-dried and CC% test was accomplished. Figure 8 shows that the final urea conversion in all the sand columns except S2 is 100%. In S2 short columns, the converted urea is about 45 percent, and it is much more 3.75% urea conversion for S2 solution (Fig. 5) in the beaker. The reason for the higher urea conversion in column S2 than S2 solution is the temperature. Although the initial temperature of reagents for S2 solution and S2 column are similar, the temperature of solution increases rapidly in sand column because sand in the column is at room temperature. This rapid increase in temperature increases the urease activity of bacteria and helps bacteria to pass the harsh conditions of high concentrations of calcium carbonate and low pH. As Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e shows the amount of carbonate in S2 is less than half of S1, and it is in line with 100% and 45% urea conversion in S1 and S2, respectively. The lower amount of CC% in S3-S6 is because magnesium does not participate in precipitation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 SEM analysis\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 10\u003c/strong\u003e shows SEM photos from precipitated carbonate in short sand columns S1, S3-S5. As the precipitated carbonate in short column S2 is just 2%, this column was too loose and it wasn\u0026rsquo;t possible to make a sample for SEM analysis. \u003cstrong\u003eFigure 10\u003c/strong\u003e shows the number of trapped bacteria in precipitations for samples from S1, S3, and S5 is higher than S4 and S5. It shows in room temperature more precipitation occurred around the bacteria. This precipitation can increase the size of bacteria cells several times and decrease their capability to pass through sand grains. The lower transportability of bacteria cells will decrease the homogeneity of carbonate precipitation in large-scale soil improvements.\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Semi-long columns\u003c/h2\u003e\n \u003cp\u003eAs mentioned in section \u003cspan class=\"InternalRef\"\u003e2.10.3\u003c/span\u003e, experiments in semi-long columns have been carried out to investigate bacterial transportability in sand columns. The capability of S1-S4 solutions, in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, in transporting through a long column was evaluated. To do this, it was necessary to modify S1-S4 solutions for the bacterial transportability test. Hence, urea was omitted from the ingredients of S1-S4 solutions to make S\u0026apos;1-S\u0026apos;4, respectively. \u003cstrong\u003eTable\u0026nbsp;4\u003c/strong\u003e shows the components of S\u0026apos;1-S\u0026apos;4 and the Control Solution. The absence of urea on S\u0026apos;1-S\u0026apos;4, and Control Solution prevents carbonate precipitation in effluent that can interrupt OD measurements. Figure 11 shows the normalized effluent concentration (C/C\u003csub\u003e0\u003c/sub\u003e) against the number of pore volumes passed through the column. It is essential to mention that a solution with higher C/C\u003csub\u003e0\u003c/sub\u003e has a lower bacterial attachment close to the injection point of sand columns; consequently, it has a higher potential to distribute uniformly on long columns. Figure 11 shows a remarkable difference in C/C\u003csub\u003e0\u003c/sub\u003e curves for Control Solution and S\u0026apos;1. Since both S\u0026apos;1 and Control Solution don\u0026apos;t have urea in the ingredient, there is no carbonate precipitation and lag time in these solutions. Therefore, the significant difference between the transportability of the Control Solution and S\u0026apos;1 can only be related to the effect of decreased pH by adding acetic acid. In other words, the pH adjustment by acid in this study not only has provided a lag phase but also dramatically improved bacteria transportability through sand columns. A comparison between the transportability of S\u0026apos;1 and S\u0026apos;3 in \u003cstrong\u003eFig.\u0026nbsp;11\u003c/strong\u003e exhibits that the amount of C/C\u003csub\u003e0\u003c/sub\u003e for S\u0026apos;3 is higher than for S\u0026apos;1. It shows that substituting magnesium chloride for 20% calcium chloride can improve the transportability of all-in-one solution and, consequently, enhance the homogeneity of precipitation in long columns. Also, the amount of C/C\u003csub\u003e0\u003c/sub\u003e for S\u0026apos;2 and S\u0026apos;4 is often higher than that of S\u0026apos;1 and S\u0026apos;4, respectively. Therefore, the low initial temperature of solutions can improve their transportability through sand columns. In conclusion and light of the results presented in \u003cstrong\u003eFig.\u0026nbsp;4\u003c/strong\u003e and \u003cstrong\u003eFig.\u0026nbsp;11\u003c/strong\u003e, magnesium substitution and low temperature of solutions prolong the lag phase and improve the homogeneity of precipitation.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e Ingredients of solutions injected in the bacterial transport experiment\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tabe\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration of Ca\u003csup\u003e2+\u003c/sup\u003e (M)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration of Mg\u003csup\u003e2+\u003c/sup\u003e (M)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConcentration of urea\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBacterial activity (U/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl Solution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnadjusted\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u0026acute;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u0026acute;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u0026acute;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u0026acute;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Hydrophobicity\u003c/h2\u003e\n \u003cp\u003eTo investigate the reason for the higher transportability of bacteria through sand columns at a low temperature, the effect of temperature on hydrophobicity has been investigated.\u003c/p\u003e\n \u003cp\u003eThe hydrophobicity of bacteria is checked as explained in section \u003cspan class=\"InternalRef\"\u003e2.5\u003c/span\u003e. As \u003cstrong\u003eFig.\u0026nbsp;12\u003c/strong\u003e demonstrates, CSH will increase with the decrease in pH and increase in temperature. In pH 9.2, the initial pH of culture medium, CSH is -12.4 and \u0026minus;\u0026thinsp;6.6 at 25\u0026ordm; C and 4\u0026ordm;C temperatures. However, in this study, as explained in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, the bacteria suspension, CS, and acetic acid are mixed and made all-in-one solution with pH 4. Therefore, the amount of CSH in pH 4 is the critical issue in this study. Figure\u0026nbsp;12 shows that at pH 4, CSH is 77.2% and 20.3% for 25\u0026ordm; C and 4\u0026ordm; C, respectively.\u003c/p\u003e\n \u003cp\u003eTherefore, at a given pH, decreasing the initial temperature of bacterial suspension will decrease CSH. This lower bacterial surface hydrophobicity can lead to the higher transportability of solutions S2 and S4, which have a low initial temperature.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 1m sand column:\u003c/h2\u003e\n \u003cp\u003eAs demonstrated in \u003cstrong\u003eFig.\u0026nbsp;12\u003c/strong\u003e and section \u003cspan class=\"InternalRef\"\u003e3.4\u003c/span\u003e, injection of S1-S4 solution to the 1m columns has been carried out. The percentage of urea conversion after 72 hours from the injection was checked, as demonstrated in \u003cstrong\u003eFig.\u0026nbsp;12b\u003c/strong\u003e and explained in section \u003cspan class=\"InternalRef\"\u003e2.4\u003c/span\u003e. Then 10 P.V tap water was injected through the sand columns to wash out the unconsumed reagents and remove the byproduct of carbonate precipitation.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e shows that urea conversion and calcium carbonate precipitation have a close relationship. The low initial temperature of injected solutions and magnesium substitution have developed the treatment results. Columns S2 and S4 have a more uniform treatment than S1 and S3, respectively, because the initial temperature of the injected solution in columns S2 and S4 are lower than S1 and S3. Columns S3 and S4 were injected with solutions including magnesium (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), and this presence of magnesium in the solution improved the uniformity of precipitations. Also, decreasing the initial temperature of solutions and magnesium substitution showed a synergistic effect. Therefore, column S1 which was treated at room temperature of injected solutions and without magnesium substitution (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) has the lowest efficiency of carbonate precipitation. Oppositely, column S4 was injected at a low initial temperature of substances with a solution that included magnesium cations and provided the most homogenous carbonate precipitation.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eThe contribution of this research is upgrading and evaluating 2 M all-in-one solution in three steps. For the first step, two modifications have been made to 2 M all-in-one solution: 1- decreasing the initial temperature of all-in-one solution, 2- substituting 20% of calcium with magnesium. The findings of this study convinced that this magnesium substitution changes the morphology of carbonate from vaterite to aragonite. As the hardness of aragonite is higher than vaterite, this change in the crystal structure of precipitation will lead to a higher strength of bioimproved soil. Magnesium substitution and the low initial temperature of all-in-one solution prolonged the lag phase. These two modifications showed a synergistic effect, so the longest lag phase was for the magnesium-included all-in-one solution with a low initial temperature (S4).\u003c/p\u003e \u003cp\u003eFor the second step, the transportability of 2M all-in-one solutions through the sand columns was evaluated. The low initial temperature of all-in-one solution and magnesium substitution for calcium improved the transportability of bacteria. Thus, the magnesium-included solution with low temperature provided the best transportability. To explain the more uniform spread of bacteria at low temperatures, the variation of CSH against temperature and pH was determined. It showed that at pH 4, CSH decreased from 77.2 to 22.3 while temperature decreased from 25\u0026ordm; C to 4\u0026ordm;C. This decrease in CSH can improve the transportability and distribution of bacteria in 1m sand columns.\u003c/p\u003e \u003cp\u003eFor the third step, the capability of 2M all-in-one solutions in the treatment of 1m long columns was examined. Columns treated with a lower temperature of substances and magnesium substitution have more uniformity of urea conversion and calcium carbonate.\u003c/p\u003e \u003cp\u003eIn general, this study verified that 2 M all-in-one solution can be converted entirely in 3 days and provide carbonate precipitation. Lowering the initial temperature of reagents will improve the lag phase, and transportability of bacteria and reduce the CSH. Magnesium substitution can prolong lag phases, develop bacterial transportability, and lead to precipitation of aragonite.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eAll data, photos, and analyses presented in this paper are available from the corresponding author by request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by NSFC Major International Joint Research Project POW3M (51920105013), Jiangsu Distinguished Professor Program, and Jiangsu Province \u0026lsquo;\u0026lsquo;Six Talent Peak\u0026rsquo;\u0026rsquo; program (XCL-111).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. The first draft of the manuscript was written by Seyed Mohammad Javad Hosseini, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdel Gawwad, H. A., Mohamed, S. A. E. A., \u0026amp; Mohammed, S. A. (2016a). Impact of magnesium chloride on the mechanical properties of innovative bio-mortar. 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Transport of bacteria in porous media and its enhancement by surfactants for bioaugmentation: A review. \u003cem\u003e35\u003c/em\u003e, 490\u0026ndash;504.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Microbially induced carbonate precipitation (MICP), Biocementation, All-in-one solution, Magnesium effects, Temperature effects, Hydrophobicity, Large-scale experiments","lastPublishedDoi":"10.21203/rs.3.rs-4449151/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4449151/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe potential of microbially induced carbonate precipitation (MICP) for soil bio-improvement has been widely studied as an alternative to traditional cementation by Portland cement. While multiple-phase injection techniques are commonly used for MICP treatment, they impose complexities and require a high number of injections. One of the latest developments in the biocementation research area is using the one-phase-low-pH MICP method as a more effective and efficient alternative to the traditional two-phase method. The published studies in one-phase MICP used 1M concentration and injected all-in-one solution several times. So, this study primarily investigated the possibility of soil improvement by a single injection of high-concentration all-in-one solution in 1m columns. This high concentration can impose a toxic effect on bacterial activity and hinder urea conversion. Also, a high concentration of salts such as calcium or magnesium chloride can increase the ionic strength and decrease the uniformity of carbonate precipitation. The effect of 20% magnesium substitution and decreasing the initial temperature of substances were studied. The experiments in aquatic steps demonstrated that these magnesium cations and low temperatures can prolong the lag phase. The collected precipitation from magnesium-included solutions showed an enhancement in the crystal structure of calcium carbonate formations. The transportability of all-in-one solutions was examined by injection of 6 pore volume solution through a 20 cm sand column and comparing the optical density of effluent to the influent. Solutions with magnesium contents and low temperature demonstrated a higher transportability. Eventually, 1 m sand columns were treated with all-in-one solution and the most homogeneous urea conversion and calcium carbonate precipitation were observed in the column injected with a low temperature of magnesium substituted solution.\u003c/p\u003e","manuscriptTitle":"Ground improvement with single treatment using Mg 2+ modified all-in-one MICP solution: 1m sand column","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-31 14:09:42","doi":"10.21203/rs.3.rs-4449151/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9bf5046f-d5c6-4d7c-a04b-12ced0275381","owner":[],"postedDate":"May 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-26T03:27:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-31 14:09:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4449151","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4449151","identity":"rs-4449151","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}