Influence of native ureolytic microbial community on biocementation potential of Sporosarcina pasteurii

Microbially induced calcium carbonate precipitation (MICP)/Biocementation has emerged as a promising technique for soil engineering applications. There are chiefly two methods by which MICP is applied for field applications including biostimulation and bioaugmentation. Although bioaugmentation strategy using efficient ureolytic biocementing culture of Sporosarcina pasteurii is widely practiced, the impact of native ureolytic microbial communities (NUMC) on CaCO3 mineralisation via S. pasteurii has not been explored. In this paper, we investigated the effect of different concentrations of NUMC on MICP kinetics and biomineral properties in the presence and absence of S. pasteurii. Kinetic analysis showed that the biocementation potential of S. pasteurii is sixfold higher than NUMC and is not significantly impacted even when the concentration of the NUMC is eight times higher. Micrographic results revealed a quick rate of CaCO3 precipitation by S. pasteurii leading to generation of smaller CaCO3 crystals (5–40 µm), while slow rate of CaCO3 precipitation by NUMC led to creation of larger CaCO3 crystals (35–100 µm). Mineralogical results showed the predominance of calcite phase in both sets. The outcome of current study is crucial for tailor-made applications of MICP.

Microbially induced calcium carbonate precipitation (MICP) is a ubiquitously recorded process in nature and is responsible for creation of numerous geological formations in terrestrial and marine environments 1 . Recently this process has been replicated in lab conditions for numerous engineering applications, as it leads to the formation of carbonate cement at ambient temperature conditions by harnessing the cementation potential of living microorganisms. The major applications include improvement of mechanical properties of soil 2,3 , bioremediation of heavy metals and radio nucleotides [4][5][6] , enhancement of oil recovery 7 , repair of concrete cracks 8,9 , and sequestration of atmospheric CO 2 10 . The chief benefit of this bio-mimicked cementation process includes self-healing ability, eco-friendliness, recyclability, and low viscosity paving the way for deeper penetration 11 .
MICP/Biocementation occurs via various metabolic pathways of bacteria such as ureolysis, denitrification, sulfate reduction, and iron reduction 12 . Amongst the different pathways, MICP via ureolytic pathway is the most widely explored route because of its straightforwardness, efficacy, short time, and no excess production of protons 13,14 . In the microbial ureolytic pathway, urea is hydrolysed into ammonia and carbon dioxide by the action of urease 2 . Subsequently, these products equilibrate in water to form bicarbonate, ammonium, and hydroxide ions, which elevate the pH of the microenvironment around the bacteria (Eq. 1). An increase in pH favors the equilibrium shifts from bicarbonate ions to carbonate ions. The formed carbonate ions then precipitate as calcium carbonate on the bacterial surface in the presence of calcium 2 (Eq. 2).
For applications of MICP in soils, especially in the field, there are two modes by which calcifying bacteria are supplemented: biostimulation (enrichment of native population) or bioaugmentation (supplementation of efficient foreign bacteria). The biostimulation approach deals with the modification of existing field conditions

Results
Influence of the native ureolytic microbial community on the kinetics of calcium carbonate precipitation. To investigate the influence of native ureolytic microbial community (NUMC) on calcium carbonate precipitation at varying concentrations (0, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 OD), soluble calcium concentration in the cementation medium was monitored for up to 288 h (at an interval of 12 h). From Fig. 1a it can be observed that the soluble calcium concentration decreased over time in all the groups with varying rates except group A to which no NUMC was added. The calcium concentration decreased to 50% from the initial value for group B at 96th hour, for group C at 60th hour, for group D at 48th hour, for group E at 36th hour, and group F and G at the 24th hour. At the end of the process, the soluble calcium ions in all the sets were exhausted, except in set A. Kinetic constants (K cal ) of CaCO 3 precipitation were used to further investigate the effect of various parameters on carbonate precipitation 35 . The monitored profiles were computationally fitted using Eq. (4) to calculate K cal values (Fig. 1a). Table 1  www.nature.com/scientificreports/  www.nature.com/scientificreports/ where K x is a constant, X is the NUMC concentration, and K cal, max is the maximum kinetic constant for CaCO 3 precipitation. When K x is equal to X, the value of K cal is equal to half of the K cal, max . The observed values are K x = 1 OD and K cal , max = 0.1 h −1 in this study. Figure 1b shows MM type plot that relates K cal with NUMC concentration. Figure 1c shows the pH change over time in all the sets. In the cementation medium, pH was observed to be between 6.5 and 8.3 in all the groups throughout the process. It can be seen that the rate of pH change within the groups followed a similar trend except for the control group A.
Influence of the native ureolytic microbial community on augmented S. pasteurii. To investigate the influence of NUMC on S. pasteurii (bioaugmentation), soluble calcium concentration in the cementation medium was monitored over time and fitted with Eq. (4). Figure 2a shows both observed and fitted curves from groups 1 to 7. From this figure, an exponential decrease of soluble calcium concentration was observed in all the groups with immediate effect upon the addition of NUMC and S. pasteurii. The concentration was recorded to be around zero at the 6th hour. From the fitted curves, the values of the kinetic constants for calcium carbonate precipitation were calculated ( Table 2) and compared (Fig. 2b). From Table 2 it can be seen that the kinetic constant values are 0.64, 0.65, 0.64, 0.73, 0.67, 0.66, 0.70, and 0.78 h −1 for the groups 1 to 7, respectively, i.e., the values were distributed between 0.64 and 0.78 h −1 . The change in the pH values of the cementation medium was also monitored (Fig. 2c) and the observed values were found to be between 6.5 and 8 for all the groups.  26 . Hence, the shape and size of precipitated crystals were analysed via scanning electron micrography (Fig. 3). For groups 1 to 4, rhombohedral-shaped crystals of size 5-10 µm were observed for the samples collected at the 12th hour. For group 5, the size of the individual and clustered rhombohedral-shaped crystals was found to be 15-25 µm for the samples collected at the 12th hour. For groups 6 and 7, for the samples collected at the 12th hour the size of both the clustered rhombohedral-shaped crystals was 30-40 µm. SEM images showed a cluster of rhombohedral-shaped crystals for the samples collected at 288th hour for the groups B to G. The size of these crystals varied between 35 and 100 µm. The polymorph is a determining factor of strength and hardness of CaCO 3 in MICP. Therefore, the qualitative and quantitative information of the CaCO 3 crystals were obtained using the powdered XRD technique (for the groups B to G at 288th hour and the groups 1 to 7 at the 12th hour). Figure 4 shows the XRD spectrum of group B and the representative spectrums of all the other groups. Tables 3 and 4 show the morphology and phase analysis of native ureolytic microbial community and bioaugmentation studies. It was observed that only group B showed 2.3% of the vaterite phase of CaCO 3 crystals and all observed crystal phases of all the groups were of the calcite phase.

Discussion
In this study, we investigated the effect of NUMC at varying concentrations on the biocementation potential of the most widely used biocementing culture S. pasteurii. We also compared the CaCO 3 precipitation potential of NUMC at different concentrations in the presence and absence of S. pasteurii. The soluble calcium concentration was measured, and its kinetics was analysed using a logistic Eq. (4) to compare the biocementation potentials of NUMC and augmented S. pasteurii. pH was also monitored to identify the range that favours CaCO 3 precipitation. SEM and XRD analyses were performed, which revealed the morphology (size and shape) and mineralogy of the crystals formed. NUMC is capable of inducing CaCO 3 precipitation in their microenvironment 38 . In Fig. 1a, the soluble calcium concentration decreased in all the groups. It could be due to carbonate ions generated in the MICP process during urea hydrolysis, which facilitates precipitation of soluble calcium around the bacterial cell wall in a cementation medium 2 . The complete exhaustion in the soluble calcium ions in the groups (group B-G) indicates that all the calcium in the medium is converted into CaCO 3 . Moreover, the supplied equimolar concentration of urea is enough for the complete conversion of CaCO 3 . The observed decrease in CaCO 3 precipitation rate (Fig. 1a) is due to encapsulation of CaCO 3 on the bacterial surface that limits the transport of nutrients transport including urea across the bacterial membrane 39 . The rate of soluble calcium depletion was observed to increase on increasing the NUMC concentration in the cementation medium. Increasing the NUMC concentration increases the total urease activity of the system, which in turn increases the soluble calcium depletion rate 22 . Moreover, the results show a positive correlation between CaCO 3 precipitation rate and the cell concentration [22][23][24] . Furthermore, the relationship between K cal and NUMC concentration could be used to design and develop a similar process for field applications. The kinetic constant K cal, Max in the mathematical Eq. (3) denotes the maximum ability of the NUMC to achieve MICP at a faster rate, in this case, 0.1 h −1 . The kinetic constant K x is equal to 1 OD, which indicates the concentration of NUMC required to achieve half the value of K cal, Max .
S. pasteurii is a widely employed bacterial strain for bioaugmentation of soil consolidation and stabilization process because of its high urease-producing potential 40 . Hence, this bacterium was chosen as the model organism for this study. Supersaturation Index (SI) is one of the key parameters for the initiation of CaCO 3 precipitation 5 . Quick CaCO 3 precipitation was observed for groups 1-7 in the cementation medium. This indicates that the cementation medium has reached the required SI in a short time. pH also affects the SI, which is evident from the reported result 41 (Fig. 2c). Moreover, the ready availability of the positively charged calcium ions in the vicinity of the negatively charged bacterial surface could also favour quick CaCO 3 precipitation 3 .
The observed K cal value of group 1 (0.64 h −1 ) with S. pasteurii of 0.4 OD was sixfold higher than the K cal, Max (0.1 h −1 ) value of NUMC. This indicates that S. pasteurii has relatively high CaCO 3 precipitation potential compared to NUMC. However, the observed results are in contrast to the reported studies that suggest biostimulation is the best possible approach for biocementation 38,42 . This could be due to the presence of different NUMC and varying study conditions between different research groups. The influence of varying concentrations of NUMC on the bioaugmentation potential of S. pasteurii was also investigated. However, no significant changes in the K cal www.nature.com/scientificreports/ values were observed within the groups when K cal values were compared between groups 1 to 7 (Fig. 2b). This indicates that the presence of NUMC did not influence the CaCO 3 precipitation potential of S. pasteurii even at a concentration as high as eightfold (group 7) over a period of two weeks in this study. The pH of the cementation medium greatly influences the CaCO 3 precipitation and also affects bacterial urease production 41 . In this study, the pH of the cementation medium of all the groups irrespective of the group type varied between 6.5 to 8.3. This indicates that the CaCO 3 precipitation occurred between the observed pH range. Urease activity of the bacteria results in the generation of ammonium ions that in turn affects the pH of the cementation medium. The rate of pH change was observed to be comparatively high for groups 1 to 7, which could be attributed to the high urease activity of S. pasteurii 43 . However, the same was not observed in groups A to G which could be attributed to the low urease activity of NUMC. www.nature.com/scientificreports/ The molecular mechanism of CaCO 3 crystal nucleation, growth, and morphology (size and shape) in the biocementation process is a complex phenomenon. Nature of the bacterial community, solution chemistry of the cementation medium (supersaturation index), the concentration of nutrients, calcium, and Mg 2+ ions significantly influence the crystal growth kinetics and characteristics 44,45,47 . In this study, groups B to G with only NUMC at different concentrations showed a cluster of rhombohedral-shaped crystals, sized 35-100 µm at 288th hour. Whereas groups 1 to 4 with S. pasteurii in particular, yielded individual crystals of size 5-10 µm at 12th hour. A decrease in crystal size during bioaugmentation is due to the high driving force, which results in the fast attaining of the saturation state during CaCO 3 precipitation. According to the classical nucleation theory:   www.nature.com/scientificreports/ the nucleus size of the crystal decreases when the driving force to reach the saturation state for the precipitation increases 46 . This result is consistent with a previous study by Cuthbert and co-workers who reported that a higher initial saturation state influences the lower-sized crystals 39 . The generation of ammonium ions and inorganic carbon due to the effective urea hydrolysis increases the pH and alkalinity of the cementation medium. It develops the oversaturated cementation solution that leads to the spontaneous CaCO 3 precipitation 5 . It is possible to obtain different phases of CaCO 3 including aragonite, calcite, vaterite, and two hydrated crystalline phases as monohydric calcite and ikaite in the MICP process 1 . This is because the polymorphism of CaCO 3 is highly dependent on various parameters of the precipitation environment 47,[49][50][51] . In general, many studies reported that the phase transition from metastable vaterite phase to more stable calcite phase during the CaCO 3 precipitation process 22,26 . But, the specific phase preference by different bacterial cultures could depend on several parameters including the type of bacteria, specific amino acid sequences of urease, organic acid production, extracellular polymeric substances of the bacteria, the kinetics of the precipitation process, cementation medium composition, and other physicochemical parameters that affect supersaturation index of the solution [47][48][49][50][51] .
In this study, no visible CaCO 3 crystals were observed in group A due to a lack of bacterial metabolic activity that leads to the undersaturation of the system. In the case of group B, besides 97.7% of calcite, 2.3% of vaterite form of CaCO 3 crystals were formed at the end 288th hour. On the other hand, in all other groups including group C to G and group 1 to 7 only calcite form of CaCO 3 crystals was observed at the end of precipitation (Fig. 4).
From the results, it is evident that calcite is the predominant polymorph of CaCO 3 crystals in both cases. It is also evident that the presence of NUMC does not affect calcite formation. Moreover, the observed results follow the Ostwald rule of crystallization, which states that thermodynamically crystal formation favors the less soluble calcite than more soluble vaterite 27 . There could be a possible delay in the transformation of vaterite to calcite form when the rate of CaCO 3 precipitation is slow. Hence, this could be attributed to the slow transformation of vaterite to calcite in groups B to G 27 . Nevertheless, only rhombohedral-shaped calcite form of crystals was observed in all the groups despite different bacteria employed in this study at the end of the process. These calcite form crystals have superior engineering properties (strength and stiffness) compared to vaterite and aragonite forms of CaCO 3 crystals.

Conclusions
In this study, we investigated the influence of NUMC on the biocementation potential of the most widely used bacterial culture S. pasteurii. We evaluated the biogenic CaCO 3 precipitation kinetics of NUMC at varying concentrations in the presence and absence of S. pasteurii along with its impact on the morpho-mineralogical characteristics of the precipitated carbonates. The concentration of cells has a major impact on the CaCO 3 precipitation kinetics as well as morpho-mineralogical properties of precipitated carbonate crystals as observed in the case of NUMC. The rate of CaCO 3 precipitation in the case of NUMC is very slow compared to S. pasteurii; and this can have a major impact on its application. S. pasteurii is highly efficient in biocementation even in the presence of native ureolytic cultures at different concentrations. CaCO 3 precipitation kinetics of S. pasteurii was not found to impact significantly in the presence of NUMC; even when their concentration is eight folds higher. Although the rate of CaCO 3 is low in the case of NUMC, but it has a positive impact on the quality of crystals. The size of calcite crystals in the case of NUMC with low metabolic activity is much higher (6-10 times) compared to smaller crystals formed by S. pasteurii. This demonstrates that it is crucial to have fundamental knowledge on the biocementation potential of native communities and the need for alternatives such as supplementation of S. pasteurii. The observed results of the current study demonstrate, for the first time, that the quantitative and qualitative properties of biocement can be tailored utilising the information of CaCO 3 precipitation kinetics with native as well as augmented cultures. The findings of this study can pave way for several new possibilities for ureolysis driven biocementation in the area of advanced functional living materials.

Materials and methods
Bacteria, growth medium, and OD measurement. The bacteria used in this study are the Native Ureolytic Microbial Community (NUMC) 52 and S. pasteurii (ATCC 11859). The NUMC is a mixture of four different ureolytic bacteria (BS1, BS2, BS3, and BS4) in equal proportions isolated from Brahmaputra riverbank soil (India) and enriched using a medium containing 13 g/L nutrient broth and 5% urea. From the previous study 52 , the BLAST analysis of the 16S rRNA sequence results showed that BS1 and BS2 are close relatives of Sporosarcina siberiensis. Whereas BS3 and BS4 are close relatives of Sporosarcina pasteurii, and Sporosarcina soli, respectively. The selected bacteria turned the urea agar base from yellow to pink colour within 12 h in the qualitative urease test. The steps are included in Fig. 5. Both S. pasteurii and NUMC were grown in Ammonium -Yeast extract medium (ATCC 1376) contains yeast extract (20 g/L), ammonium sulphate (10 g/L), and 0.13 M tris base (pH 9) were maintained at 30 ºC and 180 rpm. The individual components of the growth medium were autoclaved and mixed after cooling under sterile conditions. To measure the concentration of the overnight grown NUMC and S. pasteurii, the media containing bacteria were centrifuged at 4500 rpm for 10 min and the optical density was measured using a spectrophotometer (Thermo scientific, Genesis 10S) at 600 nm with 0.85% sodium chloride solution as blank.
Cementation medium and conditions. The cementation medium provides required nutrients and cementation components for NUMC and S. pasteurii. 100 mL of cementation medium was prepared by mixing 65 mL of autoclaved distilled water containing 0.2 g of yeast extract followed by the addition of required concentrations of NUMC and S. pasteurii cell pellet obtained after centrifugation (4500 rpm for 10 min). Then 10 and Enumeration of bacterial concentration. The bacterial concentration was measured by the serial dilution method. Petri plates containing 1.5% agar in ATCC 1376 media were used to spread the bacteria; 1 OD of bacteria in saline was found to contain cells equivalent to 4.5 × 10 8 cells/mL.

Study design.
This study was designed to investigate the influence of NUMC on the biocementation potential of augmented S. pasteurii. The study was divided into two major groups. Each group is further subdivided into seven subgroups namely A to G and 1 to 7. Measurement of soluble calcium ions and pH. The soluble calcium ions were measured by using the complexometric titration procedure 53 . 40 µL of the sample was diluted to 10 mL followed by the addition of 400 µL 1 N sodium hydroxide solution and a few drops of hydroxy naphthol blue disodium salt (1% W/V) solution indicators. Then the mixture was titrated against 1 mM EDTA disodium salt solution until the colour change from pink to blue was observed. The slope of the standard (0-2.5 mM CaCl 2 ) was used to calculate the actual concentration of calcium ions in the sample. The change in pH during biocementation was recorded using a pH meter (Thermo scientific, Orion star, A211).
Morphology and phase analysis of CaCO 3 . The CaCO 3 precipitate from the cementation medium was analysed at the end of the process. 30 mL of sample was taken was centrifuged at 4500 rpm for 10 min. The pellets obtained were washed twice with distilled water and dried at 37 ºC overnight. Then the dried crystals were subjected to scanning electron microscopy and XRD.
Morphology (size and shape). The variable pressure electron microscope (VP-SEM, Zeiss, EVO 40-XVP, 2008) was used to observe the size and shape of the CaCO 3 precipitate. The samples were placed on carbonaluminum tape and coated using a carbon evaporative coater (Creissington, 2080C, 2011). The beam intensity and voltage were 8.0 and 10 kV, respectively with a working distance of around 15 mm. The secondary electron imaging was used to obtain scanning electron micrographs. The sizes of the crystals from the micrographs were obtained using IMAJEJ (1.8.0 172) software.
Phase. Bruker D8 advance diffractometer with Ni-filtered Cu Kα radiation (40 kV, 40 mA) over the range 7°-120° 2θ, with a step size of 0.015° was used to collect the XRD data. The powdered CaCO 3 was resuspended in ethanol and deposited onto low-background holders. Further, the phase identification was done in Bruker EVA 5.2 using the Crystallography Open Database (COD) (http:// www. cryst allog rphy. net/). The phase quantification www.nature.com/scientificreports/ was done in Topas Academic 7 using the Rietveld method. Also, the crystal structures were identified from the COD.

Calculation of kinetic constants for calcium carbonate precipitation. Non-linear regression
analysis was done using the curve fitting method. The exponential decrease of soluble calcium concentration over time was fitted with logistic Eq. (4) using the solver function in Excel (2016 MSO) to calculate the kinetic parameter called the kinetic constant of CaCO 3 precipitation (K cal ). In curve fitting, the least-square method was used. The sum of squared values of the difference between the experimental and predicted value was fixed as the objective function, and K cal is the variable. K cal values were further used to compare the kinetics of CaCO 3 precipitation at various operating conditions of this study. The logistic Eq. (4) is a slight modification of Eq. (3) from our previous study 57 .
where, C o is the initial concentration of calcium (mM), C cal (t) is the soluble calcium concentration (mM) at given time, t is the time (h) and, K cal is the kinetic constant of calcium carbonate precipitation (h −1 ). www.nature.com/scientificreports/