Application of response surface methodology for carbonate precipitation production induced by a mutant strain of Sporosarcina pasteurii

 Meng Li1, Hongxian Guo2, Xiaohui Cheng3

1Department of Civil Engineering, Tsinghua University, Beijing, China; PH (086) 010-62795080; email: limeng@tsinghua.edu.cn 2 Department of Civil Engineering, Tsinghua University, Beijing, China; PH (086) 010-62788615; email: guohx@tsinghua.edu.cn 3 Department of Civil Engineering, Tsinghua University, Beijing, China; PH (086) 010-62782019; email: chengxh@tsinghua.edu.cn

ABSTRACT

The application of microbial technology has brought new opportunities for the development of civil engineering. Microbial induced carbonate precipitation (MICP), which use microbial catalyzed hydrolysis of urea to produce calcium carbonate crystals chemically and physically similar to sandstone, could increase strength and stiffness of sands or stones. In this work we investigated the process of calcium carbonate precipitation induced by Sporosarcina pasteurii. To improve the yield of calcium carbonate precipitation, the strain of S. pasteurii was isolated and screened after NTG (N-methyl-N’’-nitroso-N-nitrosoguanidine) mutated. Mutant strain B-20 gave the highest MICP production. The urease activity was tested on different medium with byproducts; soyabean meal was the selected major components of medium. Then, the optimization of culture medium for enhanced urease activity was conducted employing response surface methodology. Plackett-Burman design was firstly used to screen the most important variables, and subsequently central composite design was adopted to investigate the optimum value of the selected factors for achieving maximum urease activity and MICP yield. INTRODUCTION

Bacterial processes that bind metals and form minerals are widespread and

represent a fundamental part of key biogeochemical cycles (DeJong et al., 2006). Microbially-mediated carbon cycling, especially as related to the precipitation and dissolution of carbonate minerals, is one of the fundamental research foci in the rapidly expanding field of biogeology (Mitchell et al., 2005). Carbonate precipitation is an important aspect of biomineralization, and has been investigated extensively due to its wide range of technological implications. In comparison with inorganically produced minerals, biominerals often have their own specific properties including unique morphological, size, crystallinity, isotopic and trace element compositions (Ivanov et al., 2008). Applications of carbonate mineralization by bacteria include the production of biomimetic materials and bioremediation through leaching, solid-phase capture of inorganic contaminants or plugging-cementation in rock and other porous media fissures (Hoffman et al., 1999). Specifically, CaCO3 production by bacteria has led to the exploration of the process in the field of construction materials and ground reinforcement for applications including conservation and restoration of ancient masonry buildings, remediation of concrete cracks and improving the compressive strength of ground bases and foundations. There are many microbial processes which

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can lead to the precipitation of CaCO3. Currently many studies focus on microbially catalyzed hydrolysis of urea (Le Metayer et al., 1999; Murphy et al., 2000).

S. pasteurii could induce calcium carbonate precipitation by catalyzed hydrolysis of urea. In this process, CaCO3 was induced by hydrolysis of urea in a solution with calcium chloride. Urease was used to catalyze the hydrolysis of urea and produce ammonium and carbonate ions. In the presence of dissolved calcium ions, the produced carbonated ions will precipitate and form calcium carbon crystals.

The yield of calcium carbonate production is an important process variable. In preliminary experiments, we analyzed the parameter of the process in carbonate precipitation by S. pasteurii, and found that various carbon and nitrogen sources, inorganic salts in the culture medium, the inoculum age, and the concentration of urease and calcium ion in the reaction solution markedly influenced the productivity and economics of the carbonate precipitation bioprocesses (Shen et al., 2008).

Response surface methodology has eliminated and drawbacks of classical methods and has proved to be powerful and useful for the optimization of the target metabolites production. It can also be used to evaluate the relative significance of several variables simultaneously (Deepak et al., 2008).

The aim of this work was to apply the Plackett-Burman design, followed by the paths of steepest ascent and response surface methodology to optimize and culture medium composition for the yield of urease activity and calcium carbonate precipitation.

MATERIALS AND METHODS

1. Strain and culture medium

The majority of wild-type Sporosarcina pasteurii strains (ATCC11859) investigated came from American Type Culture Collection. The mutants generated by NTG treatment were grown on NH4-YE solid medium or urea agar medium, and subsequent fermentation was carried out using NH4-YE liquid medium.

The NH4-YE medium consisted of the following: yeast extract 20 g/l, (NH4)2SO4 10 g/l, NiCl2 2.68mg/l (Whiffin et al., 2005). All the media were autoclaved at 121℃for 20 min. S. pasteurii was cultured at 30� and 200 rpm. 2．Experimental protocol of microbial mutation

The experimental protocol for the treatment of S. pasteurii was as follow: The cells were washed twice with 0.5 M phosphate buffer (pH 8.0) and resuspended in 1 ml of the same buffer. The cells were diluted in phosphate buffer to obtain about 107 cfu/ml and mixed with 0.1% NTG at 15 min, 30 min, 45 min and 60 min, respectively. The colonies were randomly selected and transferred on urea agar base medium to check the production of urease based on the pink color. 3. Analytical method 3.1 Optical density (OD600)

During the course of the experiments the optical density measured at 600 nm was used as an indication of biomass concentration. 3.2 Urease activity

Urease activity was measured immediately after sampling. In the absence of calcium ions, urease activity was determined by a conductivity method. The urease reaction involves the hydrolysis of non-ionic substrate urea to ionic products thus generating a proportionate increase in conductivity under standard conditions. 1ml of bacterial suspension was added to 9 ml of 1.11M urea and the relative conductivity

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change in (mS.cm-1.min-1) was recorded over 5 min at 20℃. In the measured range of activities mS.cm-1.min-1 correlated with a hydrolysis activity of 11.1mM urea.min-1

(Whiffin et al., 2007). 4. Experimental design and data analysis

In preliminary experiments, various carbon and nitrogen sources, and inorganic salts were evaluated for their suitability to sustain good urease activity by strain W-17. The results revealed that the major variables affecting the performance of the culture in terms of urease activity were yeast extract, soybean meal, starch, NH4NO3，(NH4)2SO4，MgSO4，NiCl2，KH2PO4，Na2HPO4. These components were chosen for further optimization. 4.1. Plackett-Burman design (PBD)

PBD was employed for screening the most significant culture parameters affecting urease activity by S. pasteurii. Each independent variable was tested at two levels, high and low, which are denoted by (+) and (-), respectively. The experimental design with the name, symbol code, and actual level of the variables is shown in Table 2. Two dummy variables were studied in 12 experiments to calculate the standard error. Urease activity was carried out in duplication and the average value as taken as the response. The variables with confidence levels above 90% were considered to have significant effect on urease activity and thus used for further optimization. 4.2. Path of the steepest ascent experiment

To move rapidly towards the neighborhood of the optimum response, we used the method of steepest ascent. The experiments were adopted to determine a suitable direction by increasing or decreasing the concentrations of variables according to the results of PBD (Gheshlaghi et al., 2005). 4.3. Central composite designs (CCD) and response surface methodology

To describe the response surface in the optimum region, a central composite design and response surface methodology was performed. The low, middle and high levels of each variable were designated as -1.41421,-1, 0, and 1, 1.41421. 4.4. Statistical analysis

Minitab 15.0 (Minitab Inc., Pennsylvania, USA) was used for the experimental designs and subsequent regression analysis of the experimental data. Statistical analysis of the model was performed to evaluate the analysis of variance (ANOVA). The quality of the polynomial model equation was judged statistically by the coefficient of determination R2, and its statistical significance was determined by an F-test. The significance of the regression coefficients was tested by a t-test. RESULTS

1. Selection of appropriate wild-type strain and mutation

More than 100 wild-type S. pasteurii were screened for urease production in shake flask fermentation on NH4-YE media. The best strain was named as W-17 which was found to be an excellent urease activity and calcite carbonate producer; hence it was selected as the parent strain for the mutation experiments.

Strain improvement of W-17 was performed with NTG treatment. For effective mutation and screening of the mutants, it is desirable to have a high rate of cell lethality. Figure 1 shows the lethality rate of S. pasteurii treated by NTG with respect to treatment time. It can be seen that the lethality rates of the treated colonies increased to 91.7%, 93.8%, 99.0% and 99.7%, respectively, after treated with NTG for 15, 30, 45 and 60 min. When the sample was treated for 75 min under these

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conditions, no colony could survive. Based on the results shown in Fig. 1, the treatment time employed in this study was 30 min to obtain a desirable lethality rate.

Firstly, Urea agar medium plates were used to select positive mutants because of the pink color caused by the hydrolysis of urea products. High urease activity colonies exhibiting large red zones on were transferred to NH4-YE agar media. Pure colonies were evaluated in shake flask fermentation for urease production and induced calcite carbonate precipitation. NH4-YE medium was used in for selection of positive colonies. Biomass, urease activity and specific activity were measured to select the positive mutants. Altogether more than 40,000 mutant colonies were produced and 300 NTG mutants were selected for shake flask fermentation. 2. Urease activity and specific urease activity of mutants

Urease activity and specific urease activity of the untreated wild strains and the selected 10 mutant strains, was determined by fermentation experiments. For the untreated wild strain W-17, the corresponding values of urease activity and specific urease activity were 13.67 mM·min-1 and 3.30 mM·min-1·OD600

-1. The selected 10 mutant strains exhibited diverse fermentation productivities.

In fermentation the urease activity of 10 mutant strains, were in the range of 17.61-28.37 mM·min-1, which was an improvement of about 29-228% compared to the wild-type strain W-17. B-20, the urease activity was 28.37 mM·min-1, increased by 2.07 fold compared to the wild strain. Thus, the B-20 strain was used to examine the genetic stability after NTG treatment in terms of urease production (Fig. 2).

Figure 1. Variation of the lethality rate with different NTG treatment time (n=3).

Figure 2. Biomass (●), urease (■) and specific urease activity (▲) of S. pasteurii

W-17 (A) and the mutant S. pasteurii B-20 (B).

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3. The genetic stability of mutants The mutant B-20 was isolated to study its genetic stability (Table 1). After a

16-generation culture, the strain B-20 still maintained high specific urease activity and urease activity. Table 1. Values of Urease (mM•min-1) and Specific urease activity (mM•min-1•OD600-1) for B-20 for different generation cultures (n = 3)

Generation 2 3 4 10 12 16 Urease activity 28.44±1.03 29.56±0.25 27.68±0.98 29.07±1.20 27.91±1.25 26.34±0.96 Specific urease

activity 9.45±0.22 9.38±0.24 9.33±0.94 9.46±0.77 9.55±0.61 9.48±0.84

4. Optimization of an economical industrial medium

For large scale production of urease, it was necessary to find an inexpensive substrate for bacteria to grow on, that still produced a high level of urease activity. Several alternative protein sources were investigated to replace yeast extract. S. pasteurii was cultivated under aerobic batch conditions on 10g/l yeast extract, 10g/l (NH4)2SO4 and 10g/l of defined substrates (cottonseed protein, peanut meal, wheat protein, soy flour, molasses, soybean meal, beef extract). In all cultures, pH was adjusted to 8.5 with NaOH, and cultures were grown at 30�. The biomass, urease activity and specific urease activity were shown in Fig. 3. The high level of urease activity was produced in the soybean meal medium.

Figure 3. Biomass (black), urease (white) and specific urease activity (grey) of S. pasteurii B-20 in different medium.

5. Optimization the medium by PBD

The importance of the nine components, namely, Yeast extract, soybean meal, starch, NH4NO3，(NH4)2SO4，MgSO4，NiCl2，KH2PO4，Na2HPO4 for urease activity was investigated by PBD. Table 2 shows the effects of these components on the response and significant levels.

Table 2 The Plackett-Burman design for screening variables in Urease activity. Factors(g/l) Code Low

level (-1)

High level (+1)

Effect Coefficient t-value p-value

Yeast extract x1 10 30 -1.22 -0.611 -0.5 0.664 soybean meal x2 10 30 17.65 8.824 7.28 0.018

Starch x3 5 15 0.11 0.055 0.05 0.968

0

5

10

15

20

25

30

35

0

2

4

6

8

10

12

cottonseed protein

peanut meal

wheat protein

soya flour molasses soyabean meal

beef extract yeast extract

Ureaseactivity (m

M.m

in‐1)

Biom

ass (OD600)

specific u

reaseactivity

(mM.m

in‐1.OD600‐1)

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NH4NO3 x4 0 10 5.22 2.611 1.35 0.31 (NH4)2SO4 x5 8 24 -7.67 -3.836 -5.07 0.037

MgSO4 x6 0.025 0.25 2.86 1.432 1.18 0.359 NiCl2 x7 0.0012 0.0048 -6.24 -3.119 -2.57 0.124

KH2PO4 x8 0.1 1 -0.96 -0.477 -0.39 0.732 Na2HPO4 x9 0.2 2 1.62 0.81 0.67 0.57

R2=97.81%, R2(adj)=87.96% Based on the statistical analysis, the effects of soybean meal and ammonia sulfate

were (+) 17.65 and (-) 7.67, respectively, and both had confidence levels above 95%. So they were identified as influencing urease activity significantly. Others had no obvious effects and the low confidence level, so they were considered insignificant. In the result, R2 was found to 0.978, which means that model could explain 97.8% of the total variations in the system. Table 3 The Placket-Burman design variables (in coded levels) with urease activity as response.

Run Variable levels Urease activity x1 x2 x3 x4 x5 x6 x7 x8 x9 x10 x11 (mM.min-1)

1 -1 -1 1 1 1 -1 1 1 -1 -1 1 3.33 2 1 -1 -1 1 -1 1 1 -1 1 1 1 19.71 3 1 -1 1 -1 -1 -1 1 1 1 -1 -1 14.79 4 1 1 -1 1 1 -1 1 -1 -1 -1 1 22.64 5 -1 1 1 1 -1 -1 -1 -1 1 1 -1 40.89 6 -1 -1 -1 1 1 1 -1 1 1 1 -1 15.98 7 1 -1 1 -1 1 1 -1 -1 -1 1 1 7.73 8 -1 1 1 -1 1 1 1 -1 1 1 1 26.37 9 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 1 22.64 10 1 1 1 1 -1 1 -1 1 -1 -1 -1 44.36 11 -1 1 -1 -1 -1 1 1 1 -1 1 -1 31.57 12 1 1 -1 -1 1 -1 -1 1 1 -1 -1 24.24

Fig.4. Pareto chart of nine-factor standard effects on urease activity.

In the Pareto chart (Fig. 4), the maximal effect was presented in the upper portion and then progress down to the minimal effect. In addition, it directly shows that the most important factors determining urease activity were soybean meal and (NH4)2SO4.

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Table 4 Design and results of path of steepest experiment

6. Optimization by the path of steepest ascent experiment

PBD results indicated that the soybean meal was positive, whereas that of ammonia sulfate was negative. Thus increasing soybean meal and decreasing ammonia sulfate concentration should result in a higher urease activity. The center point of the PBD has been considered as the origin of the path and the urease activity was obtained. The experiments showed in Table 4 obtained when the parameters were 30g/l soybean meal and 12g/l (NH4)2SO4. It suggested that this point might be near the region of the maximum urease response. This point was chosen for further optimization. 7. Optimization by response surface methodology

The date shown in Table 5 was analyzed using Minitab 15.0 software. The t-test and P-values were used to identify the effect of each factor on urease activity. A P-value of less than 0.05 indicates that the model terms are significant. In this case, soybean meal and ammonia sulfate effect had a significant effect on urease activity (P<0.05), as quadratic terms of soybean meal and ammonia sulfate effect. The fitness of the model was examined by the coefficient of determination R2, which was found to be 0.9907, indicating that the sample variation of 99.07% was attributed to the variables and only less than 1% of the total variance could not be explained by the model. The present R2-value reflected a good fit between the observed and predicted responses, and implied that the model is reliable for urease activity in the present study. The adjusted determination coefficient (98.41%) was also satisfactory to confirm the significance of the model. The model can be shown as follows: Y=40.64+2.91X1-2.14X2-6.03X1*X1-2.90X2*X2+0.98X1*X2 Where Y is the predicted urease activity, X1 is soybean meal, and X2 is (NH4)2SO4.

Furthermore, an analysis of variance (ANOVA) for the response surface quadratic model is presented in Table 7, which also proves that this regression in statistically significant (P<0.0001) at 95% of confidence level. The model also showed statistically insignificant lack of fit (P=0.759), so the model was supposed to be adequate for prediction within the range of variables employed (Table 6). In order to gain a better understanding of the effects of the variables on the urease activity, the predicted model was presented as 3D response surface graphs as Fig. 5.

Table 5 The design and results for CCD.

Run Soybean meal (NH4)2SO4 Urease activity (mM.min-1) Code X1 X1 (g/l) Code X2 X2 (g/l)

1 -1.41421 22.4 0 12 24.86 2 0 30 0 12 40.98 3 0 30 0 12 40.04 4 0 30 0 12 41.72

Factor Urease activity (mM.min-1) Run X1 (g/l) X2 (g/l）1 18 8 25.88 2 24 10 32.84 3 30 12 37.49 4 36 14 31.24 5 42 16 24.72

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5 1 35.4 1 14.5 33.28 6 -1 24.6 1 14.5 25.12 7 0 30 -1.41421 8.46 37.68 8 -1 24.6 -1 9.5 31.82 9 0 30 0 12 40.88 10 1 35.4 -1 9.5 36.05 11 0 30 1.41421 15.5 32.28 12 0 30 0 12 39.58 13 1.41421 37.6 0 12 32.56

Table 6 Estimated Regression Coefficients for Urease activity

Term Coef. SE Coef.

T P

Constant

40.64 0.3242 125.353 0.000

X1 2.9099 0.2563 11.353 0.000 X2 -2.1383 0.2563 -8.343 0.000

X1*X1 -6.0344 0.2749 -21.955 0.000 X2*X2 -2.8994 0.2749 -10.549 0.000 X1*X2 0.9825 0.3625 2.711 0.030

S = 0.724940 PRESS = 10.4953 R2 = 99.07% R2(pred) = 97.35% R2(adj) = 98.41% Table 7 ANOVA of regression model.

Source DFa Seq SSb Adj SS Adj MSc

F P

Regression 5 393.071 393.071 78.614 149.59 0.000 Linear 2 104.322 104.322 52.161 99.25 0.000 Square 2 284.888 284.888 142.444 271.04 0.000

Interaction 1 3.861 3.861 3.861 7.35 0.03 Residual Error 7 3.679 3.679 0.526

Lack-of-Fit 3 0.856 0.856 0.285 0.4 0.759 Pure Error 4 2.823 2.823 0.706

Total 12 396.75 a DF, Degree of freedom b SS, sum of squares c S, mean square

Fig.5. 3D surface graph of soybean meal vs. (NH4)2SO4 for urease activity.

 

16

1410 12

20

30

40

102530

35

Ur ease act i v i t y

( NH4) 2SO4

Soybean meal

Sur f ace Pl ot of Ur ease act i v i t y vs ( NH4) 2SO4, Soybean meal

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8. Validation of the optimized condition On the basis of medium optimization, the quadratic model predicted that the maximum urease activity was 41.31 mM.min-1, when the soybean meal level was 31.16 g/l and (NH4)2SO4 was 11.18 g/l. To verify the predicted results, validation experiment was performed in triplicate tests. Under the optimized condition, the observed experimental urease activity was 41.01±0.14 mM.Min-1, suggesting the experimental and predicted values was in good agreement. The urease activity was 28.37 mM.min-1 in NH4-YE medium, 1.44-fold increase had been obtained. The OD600 was 3.25±0.12 and specific urease of B-20 cultured in the optimized medium was 12.62±0.48 mM.min-1.OD600, 3.78-fold than the wild-type strain. 9. S. pasteurii induced calcite carbonate production

To obtain a desired strength of sands or stones, a particular amount of calcium carbonate is required. The yield of calcium carbonate induced by S. pasteurii strains with different urease activity was measured by the concentration of Ca2+ in the solution. The yield of calcium carbonate induced by W-17 was 4.90 mM, which urease activity was 13.7 mM. And 8.49 mM calcium carbonate induced by B-20 cultured in the optimized medium with urease activity 41.10 mM. It was 1.73-fold than the wild type strain. CONCLUSION

In this study, we successfully improved the strain of S. pasteurii by mutagenesis.

The mutant (B-20) showed increased urease activity, calcite precipitation. And we found that soybean meal is a good source of nutrients that can support growth and increases urease activity also for calcite precipitation of this bacterium. This study also proved that statistical experimental designs offer an efficient and feasible approach for urease production medium optimization. A maximum urease activity of 41.31 mM.min-1 was achieved with the optimized factors of 31.16 g/l soybean meal level and 11.18 g/l (NH4)2SO4. Validation experiments were also carried out to verify the adequacy and the accuracy of the model, and results showed that the predicted value agreed with the experimental values well, and 1.44-fold increase compared to the original medium was obtain. And the yield of calcium carbonate induced by S. pasteurii was also 1.73-fold increase than the wild type strain. The results also give a basis for further study with large scale fermentation of S. pasteurii for application in civil engineering.

ACKNOWLEDEMENTS

This work was supported by National Natural Science Foundation of China (Project No. 50908122，50608041) and the State Bureau for Preservation of Cultural Relics Project (No. 2007142-12/22).

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