Improving the Strength of Sandy Soils via Ureolytic CaCO3 Solidification by Sporosarcina ureae

55 'Microbial induced carbonate precipitation' (MICP) is a biogeochemical process that can be applied to strengthen materials. The hydrolysis of urea by microbial catalysis to form carbonate is a commonly studied example of MICP. In this study, Sporosarcina ureae, a ureolytic organism, was compared to other ureolytic and non-ureolytic organisms of Bacillus and Sporosarcina in the assessment of its ability to produce carbonates by ureolytic MICP for ground reinforcement. It was found that S. ureae grew optimally in alkaline (pH ~9.0) conditions which favoured 60 MICP and could degrade urea (30.28 U/mL) at levels similar to S. pasteurii (32.76 U/mL), the model ureolytic MICP organism. When cells of S. ureae were concentrated (OD600 ~15-20) and mixed with cementation medium containing 0.5 M calcium chloride (CaCl2) and urea into a model sand, repeated treatments (3 x 24 h) were able to improve the confined direct shear strength of samples from 15.77 kPa to as much as 135.8 kPa. This was more than any other organism observed in the study. Imaging of the reinforced samples with scanning electron microscopy and 65 energy dispersive spectroscopy confirmed the successful precipitation of calcium carbonate (CaCO3), organized as calcite, across sand particles by S. ureae. Treated samples were also tested experimentally according to model North American climatic conditions to understand the environmental durability of MICP. No significant (p < 0.05) change in strength was observed for samples that underwent freeze-thaw cycling or flood-like simulations. However, samples fell to 29.2 % of untreated controls following acid-rain simulations. Overall, the species S. ureae 70 was found to be an excellent organism for MICP by ureolysis to achieve ground strengthening. However, the feasibility of MICP as a durable reinforcement technique is limited by specific climate conditions (i.e. acid rain).

S. ureae experiment yielded very high NH3-NH4+ values. What was the NH3-NH4+ concentration at time zero? There appears to be significant ureolytic activity at 1 hour (Figures 2a & b), but there was very little change in the solution pH (Figures 2c & d). Based on ureolytic reactions on page 2 (line 63), I would have expected NH3-NH4+ production to be concurrent to changes in alkalinity." 10 (2) Author"s Response Thank you for your insightful comments. We have taken the time to consider, arrange and incorporate the relevant data to clarify changes in (1) pH and (2)  In addition we have also made edits to line 130 and 155 of the original PDF file to better explain the starting cell density and method to generate NH3-NH4 + production values, over time, used to generate Figure 2. 20 We welcome any further comments regarding the technical aspects of the project. Biomediated calcium carbonate (CaCO 3 ) production is the process by which organisms induce the precipitation of calcium carbonate. With reference to bacterial CaCO 3 precipitation, also known as, 'microbial induced carbonate precipitation', 'microbial induced calcite precipitation' (MICP) and 'microbial induced calcium carbonate 85 precipitation' (MICCP), the phenomenon is well documented (Stocks-Fischer et al., 1999;Dejong et al., 2006;Whiffin et al., 2007;van Paassen et al., 2010). For example, cyanobacteria precipitate CaCO 3 in microbial processes related to the shedding of the S-layer, forming the stalagmites and stalactites in limestone caves and adding to the rocky sediments of coral reefs (Southam 2000). Crystal aggregation of CaCO 3 in the kidney, urinary tract or gallbladder have been shown to be induced by microorganisms such as Proteus mirabilis, a urease positive organism 90 due to secondary infection (Worcester and Coe 2008). Ureolytic soil organisms of the species Sporosarcina or Bacillus, can also induce CaCO 3 . For example, in their cycling of nitrogen with a urease enzyme (Hammes et al., 2003;Gower 2008;Worcester and Coe 2008). This last group of MICP producers has peeked recent engineering interests to apply them in a bioengineering and repair context. MICP biotechnology utilizing ureolytic soil organisms, most notably Sporosarcina pasteurii, has been shown to 95 directly reinforce or restore engineered or natural structures, such as the repair of historical monuments (Le Métayer-Levrela et al., 1999; see also Webster and May 2006), marble slabs (Li and Qu 2011) and stone heritage sites (Rodriquez-Navaro et al., 2012) and reduce weathering of soil embankments (Chu et al., 2012). The enzyme urease (urea amidohydrolase, E.C. 3.5.1.5) initiates the process, catalyzing the breakdown of urea to raise local pH and produce CaCO 3 in a solution of calcium ions often supplied as calcium chloride (CaCl 2 ), as summarized in equations 1 and 2 (eq. [1, 2]). The produced CaCO 3 fills 100 structural gaps or bridges materials (i.e., soils grains, etc.) to form a cemented product with unconfined strengths of up to 20 MPa (Whiffin et al., 2007).
These gram positive organisms offer other attractive features such as spore forming capability allowing for long term capsule storage in cements (Jonkers 2011) and exopolysaccharide (EPS) secretion for improved material bonding (Bergdale 2012). 110 The application of MICP in industry as a biotechnology is proposed to help reduce the need for current structure repair practices such as chemical grouting, which have been found to be environmentally detrimental in its permanence (DeJong et al., 2010) and, in some cases, posing serious human health risks (Karol 2003). That said, ureolytic MICP does produce excess ammonia which can be harmful (van Paassen et al., 2010). The use of nitrifying and denitrifying bacteria could help solve this issue by oxidizing ammonia to nitrate and later nitrogen gas without 115 affecting MICP. In fact, the work of Gat et al. (2014) has shown co-cultures of ureolytic and non-ureolytic bacteria can actually be beneficial to MICP. Alternatively, denitrifying bacteria can be used to directly induce MICP to avoid ammonia toxicity, though the level of CaCO 3 is comparatively less to ureolytic MICP and harmful nitrites can build up in solution (van Paassen et al. 2010). Other pathways to achieve MICP have also been explored with B. megaterium and B. spahericus (Li et al., 2015; see also Kang et al., 2015). 120 Problems on large scale application of the MICP technology have occurred too and remain unsolved. Research by van Paassen et al. (2009) found poor sample homogeneity of MICP as well as decreasing biomass and urease-inducing CaCO 3 activity over time and increasing soil depth in a pilot 100 m 3 sand study using Sporosarcina pasteurii, attributing these heterogeneities mostly to the application process. Alternative metabolisms and bacteria for large scale applications in biomineralization of CaCO 3 have also been investigated by the group (van Paassen et al., 2010). Indeed, it has been 125 commented that the type of bacteria utilized is one of the major considerations and potential limitations in large scale geotechnical operations (Mitchell and Santamarina, 2005). Therefore, the search for new bacteria by which to achieve viable levels of MICP is important for optimizing the protocol best suited (in terms of performance, economics and environmental impact) for marketing in green industry (Cheng and Cord-Ruwisch 2012;Patel 2015;van Paassen et al., 2010). Following a literature review of the nine documented species of 130 Sporosarcina (Claus and Fahmy, 1986), seven species were found to be urease positive and distinct from Sporosarcina pasteurii as alterative ureolytic MICP sources. While no candidate improves on some of the short comings of ureolytic MICP (i.e., ammonia toxicity), each candidate was found to be poorly investigated in the current MICP technology, despite fitting the ureolytic model for MICP. One candidate, Sporosarcina ureae was selected at random for investigation as it was deemed appropriate to explore the feasibility of a single candidate species in thorough comparison to other, already published 135 species applied in ureolytic MICP.
Thus, the primary goal of this study was to investigate the suitability of S. ureae as a MICP organism in material improvement by testing it experimentally against the previously investigated species of Sporosarcina pasteurii, Bacillus megaterium and Bacillus sphaericus. In its assessment, a parallel investigation was also performed to assess how the MICP technology, utilizing S. ureae as the candidate MICP organism, can perform under various environmental conditions 140 including acid rain, flooding and freeze-thaw cycling concurrent with colder North American climates. supplemented with 15 g/L agar (BD Difco TM ). Long term stocks of all cultures were prepared as described (Moore and Rene, 1975) but using dry ice as the freezing agent. Corning Falcon © tube. The overnight stock was combined with 200 mL of appropriate culture medium in a 500 mL Erlenmeyer flask and cultured at 175 RPM. When OD 600 reached ~ 0.5, the culture was spun down at 5000 RPM for 5 minutes followed by a pellet re-suspension in 50 mL Tris buffered saline (TBS; 50 mM Tris-base [Trizma © , Sigma-Aldrich], 150 mM NaCl [Molecular biology grade, Sigma-Aldrich], pH 7.5). The process was repeated with final resuspension (OD 600 ~ 0.2) in 200 mL of a urea broth (UB) medium in a 500 mL Corning PYREX © round glass media storage 170 bottle containing a modified Stuart's Broth (Stuart et al., 1945) as follows: 20 g/L Urea (BioReagent, Sigma-Aldrich), 5 g/L Tris-Base (Trizma © , Sigma-Aldrich), 1 g/L glucose (Reagent grade, Sigma-Aldrich), pH 8.0, with (UB-1) or without (UB-2) 10 g/L yeast extract (YE) (BD Difco TM ). A negative control included a medium only condition. All steps were performed aseptically with preparations run at 200 RPM at 30 °C in triplicate for each medium condition: UB-1 and UB-2. Each culture for a medium condition was staggered 10 min apart and observed for 12 h, with duplicate 2.5 mL aliquots aseptically 175 withdrawn every 1hr, beginning at time zero (t = 0 h). The entire protocol was performed twice for a total of 6 data sets (n = 6), measured in duplicate, per culture in a single medium condition.

180
To evaluate different cell parameters efficiently, duplicate aliquots (2.5mL) were taken for pH tracking, OD 600 absorbance and sample storage for NH 3 -NH 4 + analysis. In brief, first, whole aliquot volume pH was taken with a SB20 symphony pH probe (VWR). Next, a 1mL volume was removed for OD 600 reading using a BioMate 3 UV-Vis Spectrophotometer (Thermoscientific). Finally, a 500 uL sample for NH 3 -NH 4 + analysis was retrieved and diluted in 500 uL of ddH 2 O and stored as described by HACH Inc. (Hach Co. 2015) with the following modifications: -20 o C 185 storage, 1 drop 5 N H 2 SO 4 . To avoid errors in volume delivery by micropipette, measurements were taken as mass over an analytical balance, and volumes calculated assuming a density of 1 g/mL.

6
Samples were thawed and neutralized with 5 N NaOH as described by HACH Inc. (Hach Co. 2015). NH 3 -NH 4 + measurements were then performed as outlined (HACH Co. 2015) using a portable DR2700 HACH spectrophotometer after samples were brought to a measureable range (0.01 to 0.50 mg/L NH 3 -N). All measurements for appropriate dilutions were made by mass and corrected to volume as described above. Final values were reported as units (U = mol of NH 3 -NH 4 + produced per minute) per mL of culture starting from t = 1 h. 195

200
Industrial quality, pure coarse silica sand (Unimin Canada Limited) was examined with the following grain distribution where D 10 , D 5o , D 60 are 10 %, 50 % and 60 % of the cumulative mass: D 10 = 0.62 mm, D 50 = 0.88 mm, D 60 = 0.96 mm. The uniformity coefficient, C u was 1.55 indicating a uniform, poorly graded sand as designated by the Unified Soil Classification System (USCS) (ASTM 2011). A poorly graded soil was used as a model due to its undesirable geotechnical characteristics in construction (i.e., settling) and tendency for instability in nature (i.e., 205 liquefaction) (Nakata et al., 2001;Scott 1991).

Cementation medium (CM) and culture
Cells of each strain were grown in 1L of their respective medium split into two 1 L Erlenmeyer flasks containing 210 500 mL medium each at 175 RPM to an OD 600 of ~ 1.5 -2.0. Cells were then harvested and successively concentrated over three runs to 50 mL. Runs involved a spin down at 5000 RPM for 5 min followed by a pellet re-  (Cruz-Ramos et al., 1997). A positive control with S. pasteurii (ATCC 11859), a ureolytic organism capable of ureolytic MICP, (van Paassen et al., 2009) was also run. The procedure was repeated every 24 h to provide fresh sample inoculate for injection during cementation trials. 220

Sample preparation and cementation trial
Triplicate test units were constructed from aluminum ( Fig. 1), each housing a triplicate set of sample moulds measuring 60 x 60 x15mm. Moulds were sized according to the sample intake for the direct shear apparatus 225 (Model: ELE-26-2112/02) utilized in confined shear tests. Each mould had equipped to it a drainage valve for media replacement. Filter paper was placed over the drainage valve holes during sand packing to prevent material loss. Silica (autoclaved; dry cycle, 120 o C, 15 min) was packed to a dry density of 2.50 -2.55 g/cm 3 and injected Comment [L2]: Data was collected to calculate NH3-NH4 production from t = 0 h and reported as a production amount, over time (i.e., U), per mL of culture, beginning from t = 1 h as can be seen on Figure 2 0 panels (c) and (d) with 25 mL CM suspension containing bacteria. Volumes were drained and replaced 3 times, each at 24 h periods.
Thereafter, at the beginning and end of each 24 h incubation period, 1 mL of solution was reserved and serially 230 diluted using TBS (50 mM Tris-base [Trizma © , Sigma-Aldrich], 150 mM NaCl [Molecular biology grade, Sigma-Aldrich], pH 7.5) onto appropriate agar plates (as described above) laced with 0.1 mg/L Ampicillin (Sigma-Aldrich) to measure biomass as colony forming units (CFU). Many species of Bacillus were found to be resistant at these Ampicillin concentrations (Environment Canada 2015), but otherwise lethal to most contaminant bacteria. In-lab tests observed more than 95 % survival rates for all considered Bacillus and Sporsosarcina strains compared to a 235 less than 0.1 % survival rate among a model E. coli (DH5α TM , Thermofisher). Ambient temperatures of treated sands were maintained at 22 o C, reflective of average sub-surface soil temperatures of central North American climate in the summer (Mesinger et al., 2006).

Confined direct shear tests
Treated samples were drained, flushed twice with 50 mL of ddH 2 O and dried in an oven at 65 o C for 48 h prior to 260 removal from the moulds. The shear strength tests were performed in a direct shear machine as detailed above.
Unless otherwise specified, shear tests were performed on samples with an applied normal stress of 25 kPa. Shear stress was then applied to failure at a rate of 2.5 mm/min under dry and drained conditions. Stress-strain curves were acquired via LabView data acquisition software.

Scanning electron microscopy (SEM) observation
Visualization of silica grains from the surface layer of treated sands was carried out to confirm the crystalline nature of the resulting precipitates using a JEOL6610LV scanning electron microscope (5 kV). Elemental composition of surface structures was analyzed, in parallel, by energy dispersive x-ray spectroscopy (EDS). Prior to microscopy 270 analysis, samples were dried at 65 o C for 48 h.

Water flushing 275
The ability for cured samples to perform following long-term saturation was tested over a one month trial. Treated sands were incubated with ddH 2 O over 6 periods of incubation. Each period involved injection of 25 mL of ddH 2 O followed by a 5 day treatment under ambient temperature of 22 o C. Volumes were replaced at the end of each period. No aliquots for colony counts were taken. 280

Ice-water cycling
To understand the degree to which cemented trials could withstand ice cycling, a selected number of samples were treated over 6 periods of ddH 2 O incubation as described immediately above. However, each period began with a 285 freezing at -20 o C for 24 h, holding for 3 days at -20 o C, followed by a thawing for 24 h at 22 o C. The selected maximum and minimum temperatures reflect those capable of being reached in Ontario winters and summer (Canada), respectively, according to Environment Canada (Climatic station: Ottawa CDA) (Government of Canada 2017).

Statistical processing
All statistical manipulations were performed in Excel (2007). Sample means were reported alongside the standard error of the mean (SE) or standard deviation (SD). Normality of all data sets were confirmed with the Anderson-Darling test (α = 0.05). The Student's t-test (unpaired, two-tailed; α = 0.05) was utilized to compare sample means 305 of experimental conditions for statistical significance. Prior to each t-test, homogeneity of variances for data sets were determined using a F-test (α = 0.05). Where variances were statistically observed as unequal, a Welch's t-test was adapted to test statistical significance between two sample means. in both media types. When urea in medium moved from the sole (i.e., UB-2) to a co-contributor (i.e., UB-1) for nitrogen provision, NH 3 -NH 4 + production dropped to near zero values (Fig. 2) for B. subtilis (0.44 U/mL), B. megaterium (0.56 U/mL) and L. sphaericus (1.20 U/mL) (p < 0.05). However, isolates of S. ureae and S. pasteurii 320 observed no significant (p > 0.05) decrease; a rise in production (t = 0-5 h) followed by a levelling off in value (t = 6-12 h) as the general trend observed in UB-1 and in UB-2 (Fig. 2).
Additionally, the final OD600 (t = 12 h) achieved for all strains in UB-2 medium was significantly decreased (p < 0.05) compared to UB-1 medium values. In UB-2 medium, bacterial communities of L. sphaericus, B. megaterium 385 and B. subtilis had sessile growth patterns observed as early as 10 h (p > 0.05, L. sphaericus); however, continual and significant (p < 0.05) increases in optical density were observed when comparing identical times for these cultures in UB-1 medium. Growth cessation occurred for S. ureae and S. pasteurii in both conditions but later in UB-1 (t = 11 h) compared to UB-2 (t = 9-10 h) medium (Fig. 2).

Changes in pH
The alkalinity increased with the increase in time for the strains of S. ureae and S. pasteurii studied, in both UB-1 (8.99, 9.2) and UB-2 (8.74, 8.8) medium. The lowest final pH values were observed in L. sphaericus (7.88; 8.16), B. megaterium (7.85 ; 7.93) and B. subtilis (7.70 ; 7.81) in UB-1 and UB-2 medium, at the end of 12 h (Fig. 2). 395 While pH continued to rise (p < 0.05) for S. pasteurii and S. ureae in either UB-1 or UB-2 medium, it was constant (p > 0.05) for L. sphaericus, B. megaterium and B. subtilis after time in UB-1 medium as early as 6 h (L. sphaericus) and 7 h (B. subtilis) in UB-2 medium. While final pH values for L. sphaericus, B. megaterium and B. subtilis reached significantly (p < 0.05) higher final (t = 12 h) values in UB-2 medium compared to UB-1 the opposite was true for S. pasteurii and S. ureae; values in UB-2 were significantly (p < 0.05) lower compared to UB-1. In general, 400 acidity increased with the increase in time for L. sphaericus, B. megaterium and B. subtilis in UB-1 medium to a critical value. This was also true in UB-2 medium except for L. sphaericus which showed an increase in pH over time.

Microstructure investigation
The precipitation of calcium as CaCO 3 via MICP was visualized. Sand granules from approximately the first 1cm of sands treated with MICP solution (i.e., CM-1) combined with S. ureae are shown (Fig. 5) where rhombohedra shaped crystals arranged in rosette peaks (20-40 μm) can be seen across the surface of a sand grain (Fig. 5).  shaped structures (40-80 μm) can also be visualized, though less commonly, across grain surfaces (Fig. 5). Calcium, carbon and oxygen peaks captured by EDS analysis for the rhombohedra crystals suggest CaCO 3 precipitation whereas calcium peaks present in the rod-shaped formations support amorphous calcium precipitation.

Environmental durability of MICP
Destruction of MICP sands with S. ureae inoculations was evident following exposure to acid rain as direct shear strengths reduced to 39.7 kPa (Fig. 6) or 29.2 % compared to those with no such treatment (Fig. 3). There was a significant increase (p > 0.05) in durability (i.e., strength retention) of treated sands under flooding (111.7 kPa) or 530 freeze thaw (93.5 kPa) rounds compared to acidified states. In fact, no severe mechanical damage was significantly (p > 0.05) incurred by samples treated under simulated flooding or freeze-thaw cycles (Fig. 6) compared to sands tested under ideal (i.e., non-environmental) conditions (Fig. 3).

Discussion
In characterizing S. ureae as a ureolytic organism in MICP, it was chief to understand: (1) its ability to degrade urea over time relative to other commonly applied MICP bacterial isolates and (2) its preference for urea as a 550 nitrogen source. The strain (BGSC 70A1) was consistent in its total nitrogen (NH 3 -NH 4 + ) production (p > 0.05) regardless of source nitrogen availability as yeast extract or urea. This can be attributed to mostly urea catabolism in UB-1 medium and entirely so in UB-2 medium as urea was the sole source of nitrogen. It is important to note that minor mineralization of the yeast extract components in UB-1 medium would likely have contributed ammonium (Gat et al., 2014) in this medium condition. This is supported by data recorded for the negative control (medium-555 only) in UB-1 medium with production as high as 0.12 U/mL (Fig. 2). Also, degradation of amino acids from bacterial metabolism, such as ornithine, particularly supplied in UB-1 medium via yeast extract, could also contribute to total nitrogen in solution for this condition (Cruz-Ramos et al., 1997). For both mediums (UB-1 and UB-2) dissolution of ammonium as ammonia into the atmosphere would have reduced available nitrogen for measurement, over time. Thus, a quantitative urea hydroylsis rate cannot be determined from the data collected, as 560 nitrogen production over extended periods of time is a complex collection of some or all of these processes.
However, overall, the total nitrogen production over time draws support for S. ureae as a promising MICP candidate in biocement as over the time period measured it was able to produce a consistent amount of nitrogen as ammoniaammonium in UB-1 or UB-2 medium and ammonia production has been found to be directly proportional to CaCO 3 production (Reddy et al., 2010) and soil stabilization (Park et al., 2012). As mentioned, the production of nitrogen 565 by S. ureae in medium is due mostly, or completely, to urea catabolism and this process is likely driven chiefly by its urease enzyme (Mobley and Hausinger, 1989; see also Gruninger and Goldman, 1988). Alternatively, an unknown urea-degrading enzyme other than urease could produce or contribute to the result. Notably, all Bacillus strains observed a significant decrease in total ammonia production (p < 0.05), when yeast extract was available (i.e., UB-1). This was not observed for S. ureae (p > 0.05) much like S. pasteurii. Urea is a nitrogen source for 570 bacterial growth, often catabolised by urease, (Lin et al., 2012) which has been found to be controlled by nitrogen levels and pH as well as other factors which can differ between bacterial species (Mobley et al., 1995; see also Mobley et al., 2001). Our observations indicate that S. ureae selects for urea in a metabolic pattern potentially similar to S. pasteurii and quite differently from the Bacillus strains investigated here, which appear to have medium-dependent metabolism of urea. This is particularly interesting for B. subtilis as it has been applied as a non-575 ureolytic control organism in previous literature (Stocks-Fischer et al., 1999;Gat et al., 2014). However, it had significant (p < 0.05), non-zero total ammonia activity especially in UB-2 medium. These observations are consistent with previously published literature linking total ammonia production to urea breakdown from urease, when urea is the sole source of nitrogen and urease is the assumed main catabolic enzyme; the enzyme expressed constitutively in species of Sporosarcina (Mobley et al., 1995) but in a repressible manner (i.e., activated in the 580 absence of NH 4 + and other forms of nitrogen [i.e., NO 3 -] and urea being the sole nitrogen source) in strains such as B. megaterium (Mobley and Hausinger, 1989) and B. subtilis (Cruz-Ramos et al.,1997; see also Atkinson and Fisher, 1991). This is indeed suggested by our data as it was observed for B. subtilis, B. megaterium and L. sphaericus that increased total ammonia production reached significantly (p<0.05) higher values in UB-2 media compared to near zero values in UB-1 with yeast extract as a co-nitrogen source. In fact, in UB-2 medium peaks were reached within 585 3-6 h from near zero values (t = 0-1 h) for all Bacillus species, further suggesting an increase in processes related to urea hydrolysis, such as urease expression, overtime following a reduction in genetic repression. This also corroborates well with growth patterns. Biphasic growth of a comparatively slow (t = 6-12 h) rate following a brief plateau (t = 5-6 h) from a comparatively fast (t = 0-5 h) rate of growth was observed for these strains, in general ( Fig. 2). An increase in urease, or other urea hydrolysis processes, may account for an ability to grow still further 590 following plateau as nitrogen was made available by increased urea degradation, though slower as glucose carbon was more depleted. Alternatively, the decreased growth could be due to decreased oxygen content for respiration and/or an increase in harmful metabolites in solution over time; each Bacillus species switching to a slower, anaerobic growth pattern. Taken together, this has significance as while B. megaterium and L. sphaericus have been investigated as candidates in ureolytic MICP, this has not been extensively the case for B. subtilis which in this 595 study shows ureolytic capability under specific conditions. This may guide future research on ureolytic MICP with B. subtilis, particularly where cementation media do not contain nutrient rich additives such as yeast extract. This has been the case in some literature solutions for inducing ureolytic MICP (Cheng et al., 2013;van Paassen et al., 2010). In this study B. subtilis was included in sand solidification as a non-ureolytic strain control as cementation media contained yeast extract, intended for maximum biomass support and CaCO 3 production rates (van Paassen et 600 al., 2010). Returning to S. ureae, it is very clear that it prefers an alkaline environment, like S. pasteurii and quite different from other isolates in trials, as in all growth conditions samples grew not only exponentially but towards an increased pH. Urea hydrolysis, driven potentially by urease, in this species, may maintain high levels for production of the highly alkaline environment to which it is suited for growth as an alkalophile and for its role as a nitrogen cycler (Gruninger and Goldman 1988). This is also found important for CaCO 3 production (Whiffen et al., 2007) 605 (Fig. 2). The species S. ureae may use the proton gradient for energy production (Jahns 1996) to support growth energetically (i.e., ATP) as well as materially (i.e., nitrogen source). This may partly account for S. ureae and S. pasteurii having the smallest change in growth between UB-1 and UB-2 medium by having the material but also energetic means to multiply. This is extremely promising as van Paassen et al. (2010) determined the CaCO 3 precipitation rate is positively correlated to the number of viable micro-organisms in solution. Thus, taken together, 610 the ureolytic, pH and growth data of this study support S. ureae as superior in ureolytic action to every Bacillus strain considered except S. pasteurii. Instead, S. ureae and S. pasteurii may be considered co-capable MICP candidates. This should prompt interest for further differential investigations between the two strains on such parameters as protease activity, exopolysaccharide production and biofilm levels, also connected to MICP capability, so as to identify the superior candidate. Some work in this direction has already been done (Achal et al., 615 2009).
To understand the macroscopic engineering aspects of S. ureae in MICP application, efforts of this study were instead focused on measuring and assessing its ability to strengthen model sands via urea hydrolysis to form CaCO 3 .
In experiments with a model silica sand featuring poor geotechnical characteristics (i.e., uniform sand profile) for high susceptibility to settling and static strength decreases (Conforth 2005), it was clearly shown that the S. ureae 620 treatment led to consolidation of the medium in only 48 h with a significant improvement in strength (p < 0.05) approximately eight times that for the treated (135.77 kPa) versus the control (15.76 kPa) samples (Fig. 3). In addition, while average consolidation strengths showed no significant change (p < 0.05) between species of S. ureae and S. pasteurii, the peak sample strength recorded for an S. ureae mould (175.8 kPa) exceeded the maximum sample strength recorded for S. pasteurii (165.7 kPa), the typical model ureolytic organism in MICP soil strengthening. It was 625 also well above peak average strength recorded for B. subtilis (28.1 kPa) (Fig. 3). This is as expected; B. subtilis is a non-ureolytic organism in the 'good nitrogen' (Atkinson and Fisher, 1991)  The presence of crystals as rhombohedra was observed (Fig. 5) along sand granules treated with S. ureae providing credence to the idea that it is capable of inducing prevalent organized formation of secondary minerals 635 (Fig. 5). The crystals were analyzed by EDS and the results provided strong evidence for CaCO 3 formation.
Rhombehedra organization also dictates calcite crystallization (Anthony et al., 2003). Media and B. subtilis treated sands gave no discernible crystal CaCO 3 formation. This provides evidence of superficial strengthening in shear tests for these treatments based on natural biofilm excretion (B. subtilis) or sporadic mineral crystallization. Of note, amorphous calcium deposits were widespread among S. ureae treated sand granules (Fig. 4). These large 640 amorphous precipitates likely indicate inefficiencies in conversion of calcium to crystalline CaCO 3 perhaps due to high calcium concentrations which have been found to hinder crystal formation (Al Qabany et al., 2012).
Investigators may be prompted to test alternative calcium concentrations to increase calcium to CaCO 3 conversion efficiency among S. ureae inoculates applied to MICP.
Interestingly, in analyzing cell viability of inoculates before and after incubation in treated sands, it was found 645 that S. ureae maintained significantly higher (p < 0.05) post-incubation (2.56 x 10 7 CFU) colony numbers compared > 0.05) different strengths in sands versus S. pasteurii, which both had approximately the same total NH 3 -NH 4 + production, the microbial ureolytic MICP may have been driven by nucleation in a non-linear fashion based on a number of factors. For example, the ability for individual cells to precipitate CaCO 3 , which would include their ability to act as effective nucleation sites for crystal formation, can be hindered when an abundance of cells injected 665 into porous material (i.e., sands) lead to pore plugging from the organic matter (i.e., cells) based on a sup-optimal spreading process. This has been seen to lead to a varied amount of CaCO 3 precipitation throughout the volume of a mould (van Paassen et al., 2009). Where cells are distributed more evenly to prevent clogging of this nature, nucleation may be beneficial for MICP (Hommel et al., 2015) and reveal a positive, linear relation between cell count and CaCO 3 precipitation. Collectively, this may work to explain why S. ureae with a near identical NH 3 -NH 4 + 670 activity to S. pasteurii did not outperform it on average in undrained, direct shear strength tests despite a higher colony total on average (p < 0.05). It may also explain the broader range of strengths achieved in S. ureae (Fig. 3).
For example, a sub-optimal spreading mechanism could have hindered strength achievement in some moulds of S. ureae treatment where pore plugging by organic matter (i.e., cells) occurred. Optimization of treatment protocols may prove S. ureae to be the superior candidate compared to S. pasteurii more consistently as it has increased total 675 cell numbers (Fig. 3) to support more regular nucleation of CaCO 3 overtime, in tandem with a NH 3 -NH 4 + production, proximal to that of the highly ureolytic strain S. pasteurii. However, it is important to note that S. ureae cells are significantly smaller than cells of S. pasteurii (Claus and Fahmy, 1986) and that, therefore, the total cellular surface area available for nucleation of CaCO 3 would be quite proximal. This provides a ready explanation for why no significant differences in strength are expected if total cellular surface area for nucleation, regardless of 680 whether it is spread over a relatively high number of smaller cells (i.e., S. ureae) or fewer number of larger cells (i.e., S. pasteurii) was most important for indicating CaCO 3 nucleation and thus strength enhancement potential, where NH 3 -NH 4 + production is similar.
It was the current authors" focus to also apply tests in conditions reflective of a Canadian environment and with a novel bacterial isolate (S. ureae). Sands treated with S. ureae and which underwent short-term flooding (111.67 685 kPa) or freeze-thaw cycling (93.47 kPa) showed non-significant (p > 0.05) strength decreases compared to in-lab (135.77 kPa) conditions (Fig. 6). It has been shown that MICP treated sands remain some porosity in materials (Cheng and Cord-Ruwisch 2012;Chu et al., 2012) and that good strength maintenance in seasonal water saturation and freeze-thaw is possible with porous materials (Cornforth 2005). Further studies may wish to investigate the permeability of hardened sands via S. ureae at various levels of CaCO 3 precipitation to strike a balance between 690 porosity, peak strength and endurance overtime in weather simulations.
Predictably, it was seen that the acid rain model, reflective or a Northern Ontario rain pH (4.4), eroded the shear strength of sands (Fig. 6) to 35.5 % of originally observed values (Fig. 3). This is a result of the reaction of acid with CaCO 3 producing units of H 2 O, CO 2 and salt, known as weathering. A study by Cheng and Cord-Ruwisch (2013) reported similar results with a Bacillus sphaericus model. This prompts the idea that a MICP strength model, 695 regardless of the bacteria treatment selected (S. ureae, S. pasteurii, etc.) for strength enhancement, would require a time-based repair of treated volumes. This realistically limits its geotechnical and economical practicality in the industry. However, it does prompt interest to test the ability of natural buffers, such as limes and sodas, to increase the life-span of MICP induced strength enhancement by reducing acid rain degradation.

Conclusions
This study has worked to verify that S. ureae is a suitable organism to be applied in the soil hardening technology currently being developed via ureolytic MICP. The authors designate it a close ureolytic MICP candidate, in performance, to the well studied S. pasteurii and a superior one to several other Bacillus strains. As larger scale 705 simulations are employed, it is strongly encouraged by the authors that further optimization in the treatment procedure, regardless of the MICP organism selected, be undergone including ideal soil buffering to reduce certain climatic effects (i.e., acid rain) and optimum volume porosity in the space to be treated to assure an economical application in industry.

Competing Interests
The authors state they have no conflict of interest.