Biomediated calcium carbonate (CaCO3) production is the process by which organisms induce the precipitation of calcium carbonate. With reference to bacterial CaCO3 precipitation – also known as “microbially induced carbonate precipitation”, “microbially induced calcite precipitation” (MICP) and “microbially induced calcium carbonate 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 CaCO3 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 CaCO3 in the kidney, urinary tract or gallbladder have been shown to be induced by microorganisms such as Proteus mirabilis, a urease-positive organism, due to secondary infection (Worcester and Coe, 2008). Ureolytic soil organisms of the genera Sporosarcina or Bacillus can also induce CaCO3, 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 piqued 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 directly reinforce or restore engineered or natural structures – such as the repair of historical monuments (Le Métayer-Levrela et al., 1999; Webster and May, 2006), marble slabs (Li and Qu, 2011) and stone heritage sites (Rodriquez-Navaro et al., 2012) – and to reduce weathering of soil embankments (Chu et al., 2012). The enzyme urease (urea amidohydrolase, EC 3.5.1.5) initiates the process, catalyzing the breakdown of urea to raise local pH and produce CaCO3 in a solution of calcium ions often supplied as calcium chloride (CaCl2), as summarized in Eqs. (1) and (2). The produced CaCO3 fills structural gaps or bridges materials (e.g. soil grains) to form a cemented product with unconfined strengths of up to 20 MPa (Whiffin et al., 2007). CO(NH2)2+2H2O↔2NH4++CO32-(ureahydrolysis)2NH4++CO32-+CaCl2↔CaCO3+2NH4Cl(CaCO3formation)

Bacterial species such as Bacillus sphaericus (van Tittelboom et al., 2010) and Bacillus megaterium (Krishnapriya et al., 2015) have also been applied in material or volume strengthening. The aforementioned ureolytic soil organisms are attractive for MICP as they are “generally regarded as safe” (GRAS) bacteria with accessible substrates (i.e. urea) and an aerobic metabolism applicable to most engineering and terrestrial environments (DeJong et al., 2006).

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).

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 their permanence (DeJong et al., 2010), 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 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 CaCO3 is comparatively less than that of 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. subtilis, B. megaterium and B. sphaericus (Kang et al., 2015; Li et al., 2015).

Problems in 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 CaCO3 activity over time and increasing soil depth in a pilot 100 m3 sand study using Sporosarcina pasteurii, attributing these heterogeneities mostly to the application process. Alternative metabolisms and bacteria for large-scale applications in biomineralization of CaCO3 have also been investigated by the group (van Paassen et al., 2010). Indeed, it has been 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 industries (van Paassen et al., 2010; Cheng and Cord-Ruwisch, 2012; Patel, 2015). Following a literature review of the nine documented species of Sporosarcina (Claus and Fahmy, 1986), seven species were found to be urease positive and distinct from Sporosarcina pasteurii as alternative ureolytic MICP sources. While no candidate improves on some of the shortcomings 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 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 including acid rain, flooding and freeze–thaw cycling concurrent with colder North American climates.

In characterizing S. ureae as a ureolytic organism in MICP, the goals of the study were 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 nitrogen source. The strain (BGSC 70A1) was consistent in its total nitrogen (NH3–NH4+) production regardless of whether the nutrient medium included (i.e. UB-1) or did not include (i.e. UB-2) yeast extract. 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-only) in UB-1 medium with production as high as 0.12 U mL-1 (Fig. 2a, b). 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 media (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 hydrolysis rate cannot be determined from the data collected, as nitrogen production over extended periods of time is a complex collection of some or all of these processes. This limits the conclusions able to be drawn as only the broad bacterial activity in medium, as regards preferences for urea as a nitrogen source, over time can be considered. For a quantitative method determining urease rates a robust protocol is presented by Lauchnor et al. (2015). Also, urea-hydrolysis-induced CaCO3 precipitation rates can be determined by measuring the decrease in dissolved Ca2+ ions over time (Harbottle et al., 2016). 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 ammonia–ammonium in UB-1 or UB-2 medium, and ammonia production has been found to be directly proportional to CaCO3 production (Achal et al., 2009) and soil stabilization (Park et al., 2012).

As mentioned, the production of nitrogen by S. ureae in medium is due mostly, or completely, to urea catabolism, and this process is likely driven chiefly by its urease enzyme (Gruninger and Goldman, 1988; Mobley and Hausinger, 1989). Alternatively, an unknown urea-degrading enzyme other than urease could produce or contribute to the result. Notably, all Bacillus strains observed a decrease in total ammonia production when yeast extract was available (i.e. UB-1). This was not observed for S. ureae, much like S. pasteurii. Urea is a nitrogen source for bacterial growth, often catabolized 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, 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.

The observation that the investigated Bacillus strains have medium-dependent metabolism of urea is particularly interesting for B. subtilis as it has been applied as a non-ureolytic control organism in previous literature (Stocks-Fischer et al., 1999; Gat et al., 2014). In UB-2 medium, a non-zero total ammonia activity was measured for this strain (Fig 2a, b). This is 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 absence of NH4+ and other forms of nitrogen (i.e. NO3-), with urea being the sole nitrogen source) in strains such as B. megaterium (Mobley and Hausinger, 1989) and B. subtilis (Atkinson and Fisher, 1991; Cruz-Ramos et al.,1997). This is indeed suggested by our data as it was observed for B. subtilis (and also for B. megaterium and L. sphaericus) that increased total ammonia production reached higher values in UB-2 medium compared to near-zero values in UB-1 medium with yeast extract as an alternative nitrogen source. In fact, in UB-2 medium peaks were reached within 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, over time following a reduction in genetic repression (Fig 2a, b). This also corrolates well with growth patterns. A comparatively slow growth rate occurred (t= 8–12 h) after a comparatively fast (t= 3–7 h) rate of growth following a lag period (t= 0–2 h) for these strains, in general (Fig. 2a, b). An increase in urease, or other urea hydrolysis processes, may account for an ability to grow quickly (t= 3–7 h) despite nitrogen limitation in UB-2, as ureolysis would provide nitrogen for growth-related processes. However, growth could have been restricted, over time, due to other nutrient limitations such as glucose depletion. This would explain a continued but reduced growth rate (t= 8–12 h) (Fig. 2a, b). Alternatively, or in addition, the decreased growth could be due to decreased dissolved oxygen content in medium over time, which is required for aerobic respiration, such that each Bacillus species switched to a slower anaerobic growth pattern. An increase in harmful metabolites such as organic acids in solution over time could also have hindered growth. There is evidence that this occurred for these species in UB-1 medium as a decrease in pH over time was observed which correlates with organic acid production (Fig. 2c). 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 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 (van Paassen et al., 2010; Cheng et al., 2013). In this study B. subtilis was included in sand solidification as a non-ureolytic strain control as the cementation medium contained yeast extract, intended for maximum biomass support and CaCO3 production rates (van Paassen et al., 2010).

It is clear that S. ureae prefers an alkaline environment, like S. pasteurii and quite different from the other isolates in trials, as in both growth conditions samples grew not only exponentially but towards an increased pH (Fig. 2c, d). Urea hydrolysis, driven potentially by urease, in this species may maintain ureolytic activity for production of the highly alkaline environment to which it is suited for growth as an alkaliphile and for its role as a nitrogen cycler (Gruninger and Goldman, 1988). These conditions are also important for CaCO3 production (Whiffin et al., 2007). It can also use the charge gradient generated from ammonium production for energy (Jahns, 1996) to support growth. A diagram of this adenosine triphosphate (ATP)-generating system coupled to ureolysis is available in the work of Jahns (1996) and Whiffin (2004). Additionally, the ammonium is an accessible nutrient (i.e. nitrogen) source (Gruninger and Goldman, 1988). 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 CaCO3 precipitation rate is positively correlated with the number of viable microorganisms in solution. Thus, taken together, 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. Indeed, the work of Harbottle et al. (2016) likewise found S. ureae and S. pasteurii to be about as efficient in terms of ureolytic activity. Given the current data, S. ureae and S. pasteurii are comparable as candidates for ureolytic MICP. This should prompt interest for further investigations differentiating between the two strains on such parameters as protease activity, exopolysaccharide production and biofilm levels, which are also connected to MICP capability (Achal et al., 2010), so as to identify the superior candidate. Some differential work has already been done (Sarmast et al., 2014).

To understand the macroscopic engineering aspects of S. ureae in MICP application, efforts of this study were focused on measuring and assessing its ability to strengthen model sands via urea hydrolysis to form CaCO3. 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 treatment led to consolidation of the medium in 48 h with an improvement in strength to 135.77 kPa. This was 8 times that of the control treatment (15.76 kPa) (Fig. 3). In addition, while average consolidation strengths had no statistically significant difference (p < 0.05, n= 3) between S. ureae and S. pasteurii, the peak sample strength recorded for a 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 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) nutrient conditions supplied by the yeast extract of CM-1 medium. Other Bacillus species were not tested under the assumption that they too would experience repressive urea hydrolysis expression in CM-1 medium and would produce similar observations as a result. This is supported by data provided by the groups of Al Qabany et al. (2012) and van Paassen et al. (2010), which found that CaCO3 precipitation, and by inference soil strength, improved with more suitable microorganisms in MICP. Taken together, this study provides evidence that S. ureae is capable of soil improvement by ureolytic MICP similarly to S. pasteurii.

The presence of crystals as organized “rosettes” and amorphous “rods” was observed (Fig. 5a, b) along sand granules treated with S. ureae, which is evidence that it is capable of inducing prevalent formation of secondary minerals. The structures were analyzed by EDS, and the results provide support for CaCO3 formation (Fig. 5c, d). Assuming that the solution was saturated with respect to CaCO3 and that the nucleation and crystallization of the calcite polymorph was thermodynamically favoured over time, the organized deposits should represent calcite (De Yoreo and Vekilov, 2003). However, fast nucleation and crystallization can result in amorphous CaCO3 structures and could explain the rod deposits that appear amorphous in morphology under SEM (Fig. 5a, b) (Addadi et al., 2003). This observation is limited, though, as SEM cannot discriminate among CaCO3 polymorphs which can have varying morphology based on the crystallization conditions (Ni and Ratner, 2008). The exact polymorph of CaCO3 for each structure could be distinguished with techniques such as X-ray diffraction (XRD) and/or Fourier transform infrared spectroscopy (FTIR) (Anthony et al., 2003; Ni and Ratner, 2008). Assuming the rod structures are amorphous precipitates, this indicates that the treatment conditions were potentially suboptimal for the maximum precipitation of crystalline CaCO3 such as calcite over time. This could be due to high local chemical concentrations (e.g. calcium) which have been found to hinder CaCO3 crystal formation as calcite (Al Qabany et al., 2012). Investigators may be prompted to test alternative calcium concentrations from those used in this study for injections so as to increase the efficiency of crystalline CaCO3 precipitation in MICP. Finally, medium and B. subtilis treated sands gave no discernible crystal CaCO3 formation (data not shown). This provides evidence of superficial strengthening in shear tests for these treatments based on natural biofilm excretion (B. subtilis) or sporadic mineral crystallization. Thus, overall, the microscopy evidence does support that S. ureae can precipitate CaCO3 for strength improvements in soil, which was part of the goal in studying S. ureae in MICP.

When analyzing the cell viability of injections before and after incubation in treated sands, it was found that S. ureae maintained higher post-incubation (2.56 × 107 CFU) cell abundance compared to S. pasteurii (1.21 × 107 CFU) and that these differences were statistically significant (p < 0.05, n= 9) (Fig. 5). Also, both species' cell abundance was lower and found to be statistically significantly different (p < 0.05, n= 9) compared to the cell abundance for B. subtilis (3.2 × 107 CFU). This difference could be due to the solution (i.e. TBS) utilized for serial dilution of the growth medium. The TBS did not include ammonium and was not buffered at a high pH, which are two necessary conditions for the survival of alkaliphilic genera such as Sporosarcina (Morsdörf and Kaltwasser, 1989). Thus, a deflated value for S. pasteurii and S. ureae would result. Also, moulds become mostly anaerobic over time below the subsurface and within the microenvironments of sand grains as oxygen is depleted by bacterial respiration (van Paassen et al., 2010). B. subtilis cells may have survived anaerobically (Clements et al., 2002) as opposed to the obligate aerobes S. ureae and textitS. pasteurii (Claus and Fahmy, 1986), leading to higher post-incubation cell abundance for B. subtilis. However, considering the percentage loss of cell abundance calculated as described (Sect. 3.4) is comparable between all three species, this indicates that neither species outperforms the other in cell survival while in the high-salt, high-urea CM-1 medium with incubation in treated sands. That being written, the total cell abundance in S. ureae is higher compared to S. pasteurii. This is important as cells provide nucleation points for CaCO3 formation. Indeed, the literature reports designate that strength enhancement by ureolytic MICP is driven not only by urea hydrolysis activity but also by the presence of bacteria acting as nucleation sites (Stocks-Fischer et al., 1999; Gat et al., 2014). While sand surfaces can also act as nucleation points, the negatively charged bacteria cell wall attracts positively charged cations (e.g. calcium) preferentially for the controlled nucleation of CaCO3 over time. In fact, it has been shown that cell abundance in MICP treatments positively correlate with the precipitation of CaCO3 in both the rate of production and crystal size (Mitchel and Ferris, 2006). The group of Hommel et al. (2015) have even developed a model showing that calcite precipitation is proportional to cell abundance (i.e. biomass) and potentially improved soil strengths. This model assumes that the features of the cells such as biofilm production around their cell walls favour and facilitate the precipitation of CaCO3. It follows that any intact cell wall part of the biofilm can facilitate the precipitation process whether the cell itself is alive or dead. Thus, in general, more cells equates to more CaCO3 precipitation. However, in this study, S. ureae gave rise to strengths in sands that were not statistically significantly different (p > 0.05, n= 3) versus S. pasteurii treatments. This is unexpected since S. ureae had comparable ureolytic activity to S. pasteurii but higher cell abundance over time in precipitation medium. Therefore, more CaCO3 precipitation should have occurred and led to a greater strength increase in sands in S. ureae treatments. This non-linear increase in strength compared to cell abundance can be a result of a number of factors. For example, the ability of cells to precipitate CaCO3 can be hindered when an abundance of cells injected into porous material (i.e. sands) leads to pore plugging from the organic matter (i.e. cells). This has been seen to lead to a varied amount of CaCO3 precipitation throughout the volume of a mould (van Paassen et al., 2009). Where cells are distributed more evenly, they can facilitate the precipitation of CaCO3 as nucleation points (Hommel et al., 2015). This may explain why S. ureae, having a comparable NH3–NH4+ activity to S. pasteurii, did not outperform it on average in undrained, direct shear strength tests despite having a higher cell abundance on average. It may also explain the broader range of strengths achieved in S. ureae (Fig. 3). For example, a suboptimal spreading mechanism could have hindered strength achievement in some moulds of S. ureae treatment where pore plugging by organic matter (i.e. cells) occurred. With this in mind, optimization of treatment protocols would help to determine whether or not S. ureae is the superior candidate compared to S. pasteurii given that it has consistently increased total cell abundance (Fig. 3) to support more nucleation of CaCO3 over time, in tandem with a NH3–NH4+ production comparable to that of S. pasteurii. However, it is important to note that S. ureae cells are significantly smaller than cells of S. pasteurii (Claus and Fahmy, 1986). Therefore, the total cellular surface area available for nucleation of CaCO3 would be similar for the two species. This provides a possible explanation for why no statistically significant differences in strength were observed because if total cellular surface area was most important for precipitating CaCO3 this means there would be no difference in strengths expected for the same total cellular surface area, whether it was spread over a relatively high number of smaller cells (i.e. S. ureae) or lower number of larger cells (i.e. S. pasteurii).

It was the current authors' focus to also apply tests in conditions reflective of a Canadian environment with a relatively novel bacterial isolate (S. ureae). Sands treated with S. ureae and which underwent short-term flooding (111.67 kPa) or freeze–thaw cycling (93.47 kPa) showed no statistically significant (p > 0.05, n= 3) strength difference compared to in-lab (135.77 kPa) conditions (Fig. 6). It has been shown that MICP-treated sands maintain 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 cycling is possible with porous materials (Litvan, 1980; Cornforth, 2005). Further studies may wish to investigate the permeability of hardened sands via S. ureae at various levels of CaCO3 precipitation to strike a balance between porosity, peak strength and endurance over time in weather simulations.

Predictably, it was seen that the acid rain model, reflective of a northern Ontario rain pH (Sect. 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 CaCO3 producing units of H2O, CO2 and salt, known as weathering. A study by Cheng et al. (2013) reported similar results with a Bacillus sphaericus model. This prompts the idea that a MICP strength model, 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.

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 simulations are employed, it is strongly encouraged by the authors that further optimization in the treatment procedure, regardless of the MICP organism selected, be undertaken 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.