Fifty-two bacterial cultures were isolated from Poovar mangrove ecosystem sediment samples. Of these, 21 isolates gave urease positive results on urease agar. These bacterial strains were inoculated into urease broth and urease production was quantified, based on a spectrophotometric assay. The urease activity of these isolates ranged from 1.8 to 28 U/ml. The bacterial isolate with the highest activity was selected for the further experiments. The strains with high urease activity were identified as B. halodurans through 16S rRNA ribotyping. The urease-producing bacteria is ubiquitous in natural environments. However, other common urease-producing strains reported here, Helicobacter pylori, Proteus vulgaris, Staphylococcus aureus, and Pseudomonas aeruginosa, etc., are pathogenic or opportunistic pathogens to humans (Stabnikov et al. 2013). Additionally, many other bacterial strains that are used in microbial-induced calcium precipitation (MICP) with urease production Bacillus sp. VS1 and Bacillus sp. were reported (El-Bessoumy et al. 2009; Stabnikov et al. 2013). VUK5 has been extensively used in MICP studies (Stabnikov et al. 2013). In biocementation studies, spore-forming strains of urease-producing bacteria, were found to be more compatible to environments with high salt concentrations (Bachmeier et al. 2002; Stabnikov et al. 2013). For this reason, the optimization experiment in the present study was conducted for the B. halodurans, isolate PO15 alone. The major concern for environmental applications is the selection of an avirulent bacterial that has no adverse effect on humans or animals. Though many high urease-producing bacteria have been reported, they are mostly associated with human pathogenesis and cannot be used for any in situ environmental applications. In the present study, the mangrove bacterium, B. halodurans strain PO15, has no reported virulence and so could be employed in the optimization analysis.
In the present study, the most promising urease-producing isolate (B. halodurans, isolate PO15) was selected, based on urease activity. Figure 1 presents a schematic representation of urease production in the B. halodurans isolate P015, based on Box-Behnken experimental design. Initial urease activity of B. halodurans isolate PO15 was about 28 U/ml. This value was quite higher than the activity reported for Bacillus thuringiensis N2, a marine bacterium (3.53 U/ml) (El-Bessoumy et al. 2009). To determine the effect of different factors on urease production, culture conditions were optimized using a Box-Behnken experimental design. A maximum urease activity of 295.80 U/ml, a tenfold increase from initial activity, was achieved using the design (Table 1). Significant factors identified, based on the model, were the incubation period, pH, incubation temperature, and inoculum percentage. Though aeration was reported as a major factor in enzyme production, for urease production, oxygen concentration has no role except in MICP (Bakhtiari et al. 2006). The design predicted from the experiment was found to be significant (R2 value of 0.9961). Equation 1 represents the quadratic model regression equation describing the predicted model. Khodadadi and Bilsel (2015) reported that the conditions favouring the urease production of S. pasteurii, the amount of urea hydrolyzed, and the rate of hydrolysis all inhibited bacterial cell growth and the specific hydrolysis rate of urea and vice versa. However, in this study, a better urease production using B. halodurans PO15 was achieved. However, it is difficult to draw a comparison with other studies as most of them reported on the urea hydrolysis rate rather than urease production.
Figure 1. Representation of urease production using Bacillus halodurans isolate PO15 based on Box-Behnken experimental design
Run Actual Val Predicted Val 1 234.05 242.13 2 258.91 239.63 3 202.08 208.55 4 225.25 205.97 5 234.05 242.13 6 209.50 177.22 7 231.50 250.97 8 234.05 242.13 9 234.05 242.13 10 234.05 242.13 11 234.05 242.13 12 234.05 242.13 13 234.05 242.13 14 234.05 242.13 15 241.41 211.13 16 234.05 242.13 17 234.05 242.13 18 248.50 267.97 19 300.66 268.38 20 234.05 242.13 21 267.25 284.72 22 234.05 242.13 23 234.05 242.13 24 298.58 279.30 25 220.75 238.22 26 248.58 266.05 27 176.83 196.30 28 233.83 253.30 29 204.75 211.22 30 262.91 243.63 31 253.41 223.13 32 316.66 284.38 33 234.05 242.13 34 289.25 295.72 35 268.75 238.47 36 234.05 242.13 37 326.08 295.80 38 234.05 242.13 39 234.05 242.13 40 234.05 242.13 41 162.08 179.55 42 270.83 238.55 43 246.58 253.05
Table 1. Optimization of urease production using Bacillus halodurans isolate PO15 based on Box-Behnken experimental design
The fitted model is represented as Eq. 1
The interaction between the various medium components and factors for achieving maximum urease production are shown in contour plots (Fig. 2). From the interaction between the variables, it was found that the pH of the production medium is a critical factor for urease production. When the medium pH vs. incubation period was tested, maximum urease activity of ~ 140 U/ml was achieved on the 6th day of incubation. For temperature vs. incubation period, urease activity reached a maximum after the 5th day only. Inoculum percentage vs incubation period achieved a maximum activity of 167 U/ml after 5 days. This suggests that a minimum of 5 days of incubation with a pH 5-7 is ideal for urease production, irrespective of all tested inoculum percentages and incubation temperatures. In another study on urease optimization of A. niger PTCC 5011, using conventional one-factor method, a maximum activity of 2.44 U/ml was obtained (Bakhtiari et al. 2006). Significant factors identified, based on the model, were incubation period, pH, incubation temperature, and inoculum percentage. The production of the enzyme depended on process variables such as nutrients, pH, temperature, incubation period, inoculum level and inducer concentration (Sharma et al. 2009). Optimization of medium by the classical methods involved changing one independent variable (i.e., nutrient, pH, temperature) while keeping all other variables constant. However, one-factor optimization is extremely time-consuming, expensive for a large number of variables (Okyay and Rodrigues 2014) and often results in wrong conclusions. Use of statistical-based approaches, such as response surface methodology (RSM), could overcome the limitations of the single-factor optimization process. Also, the RSM-based approach requires fewer trials to calculate the different variables and their interactions, compared to other optimization methods (Managamuri et al. 2017; Peng et al. 2018).
Figure 2. Contour plot showing response of variable influencing urease production using Bacillus halodurans isolate PO15 based on Box-Behnken experimental design. The interaction between variables are shown: a incubation period vs. pH; b incubation period vs. temperature; c incubation period vs. inoculum (%); d pH vs. temperature; e pH vs. inoculum (%); f temperature vs. inoculum (%)
Except for the orthogonal array design-based approach for urease production using A. niger (Bakhtiari et al. 2006) and response surface methodology (RSM) (Khan et al. 2019), there are no statistical optimization-based reports available for other bacteria. This is the first report on the statistical optimization of urease production of B. halodurans. From the ANOVA results, the model F value of 6.65 and the P values < 0.005 indicate that the model was significant (Supplementary Table S1). The statistical model was validated through detection of 295 U/ml urease activity with optimized factors. Recently, El-Bessoumy et al. (2009) reported extracellular urease production from B. thuringiensis N2, however, the enzyme activity was very low.
A specific activity of 62.34 U/mg was observed for purified urease with 5.6-purification fold and a yield of 87%. The specific activity of the isolate PO15 was higher than those derived from Aspergillus creatinolyticus (32.74 U/mg), Lactobacillus reuteri (13.0 U/mg) (Kakimoto et al. 1989), A. niger (0.325 U/mg) (Smith et al. 1993), and R. oryzae (0.18 U/mg) (Geweely 2006). Based on the LB plot, a Vmax of 333.33 mmol L-1 mg-1 min-1 with Km values of 1.7 mmol/L was observed for UA. The LB plot showing the enzyme kinetics is shown in Fig. 3a. The UA enzyme was tested for its thermostability and was found to be stable up to 60 ℃ (Fig. 3b). In another study, the fungal urease of Aspergillus exhibited maximum production and urease activity at 35 ℃ and the least activity was at 50 ℃ (Khan et al. 2019). Others reported 35 ℃ and 40 ℃ as the optimum temperature for urease activity (Danial et al. 2015; Fathima and Jayalakshmi 2012). In this study, however, a maximum urease activity at 35 ℃ was observed, the enzyme had no significant decrease in urease activity even at 60 ℃. This clearly validates the thermostable property of the urease enzyme.
Figure 3. Characterization of UA enzyme from B. halodurans isolate PO15 and evaluation of its biomineralization ability. (a) LB plot showing enzyme kinetics; (b) enzyme activity at different temperature; (c) enzyme activity at different pH; (d) Relative reduction of free Ca2+ in the media using urease enzyme produced by the Bacillus halodurans isolate PO15 [values expressed as mean ± S.D of triplicate experiment]
Similarly, the optimal pH for maximum urease activity was found to be pH 7 (Fig. 3c). There was an increase in urease activity with an increase of pH from 3.0-9.0 (Khan et al. 2019). Some bacterial ureases exhibited high activity in alkaline conditions (pH of 9.0) (Phang et al. 2018), while some had maximum urease activity at pH 8 (Danial et al. 2015; Mirbod et al. 2002). Two fungal isolates of the genus Aspergillus had an optimum pH of 8.0 and 8.5 (Kappaun et al. 2018). In general, the fungal urease had their maximum activity in the basic medium, while bacterial urease tended to be more variable (Khan et al. 2019). The higher thermostability favours the application of UA in a variety of industrial and environmental engineering applications.
To understand the rate at which CO2 is trapped as carbonates, a calcium carbonate precipitation study was carried out. The relative reduction of free calcium in the media is shown in Fig. 3d. B. halodurans PO15 was able to achieve a reduction of (82.8 ± 0.17)% free Ca2+. Maximum reduction was observed after 48 h incubation. For the bioremediation of CO2, the microbial biomineralization ability is of great importance (Silva-Castro et al. 2015). Application of urease derived from Sporosarcina pasteurii for processes of biomineralization and co-precipitation of CaCO3 was reported by Whiffin et al. (2007) and Al-Thawadi (2011). This process of urease-aided CaCO3 mineralization has a great potential in environmental engineering applications as well as for remediation and cementation in in situ conditions (Krajewska 2018). Bibi et al. (2018) reported indigenous Bacillus bacteria with biomineralization capability that could enhance soil stabilization isolated from Qatari soil. A similar report observed that B. licheniformis was able to precipitate calcium carbonate by ureolysis (Helmi et al. 2016). This precipitation process uses carbonate ions released during urea hydrolysis and a pH shift to highly alkaline condition. It was found that the ureolytic property of Bacillus sp. is high with respect to any other genus and that this might be due to their physiological ability to adapt to stressed conditions (Helmi et al. 2016). Moreover, this also facilitates bioremediation of toxic metals and radionuclides through solid-phase capture (Fujita et al. 2000).