ارزیابی خاک پایدارشده بوسیله Bacillus sp. HAI4 در شرایط مختلف از طریق روش تاگوچی

نوع مقاله: پژوهشی- انگلیسی

نویسندگان

1 دانشجوی دکترای نانوزیست فناوری، آزمایشگاه میکروب‌شناسی، دانشگاه رازی، کرمانشاه، ایران

2 دانشیار میکروب‌شناسی، دانشگاه رازی، کرمانشاه، ایران

3 کارشناسی ارشد میکروب‌شناسی، دانشگاه رازی، کرمانشاه، ایران

چکیده

مقدمه: فرایند رسوب کلسیت، در نتیجه فعالیت‌های متابولیکی میکروارگانیسم‌ها، کلسیفیکاسیون زیستی نامیده می‌شود. در این فرایند، کلسیت (CaCO3) به وسیله فعالیت‌های میکروارگانیسم‌ها  بر روی دانه‌های خاک ته‌نشین شده و سبب افزایش سختی/ استحکام و کاهش فرسایش خاک می‌شود.
مواد و روش‏‏ها: در این مطالعه، میزان هیدرولیز اوره و ته­نشینی کلسیت در فرایند تثبیت خاک، بوسیله باکتری Bacillus sp. HAI4 از طریق روش تاگوچی مورد ارزیابی قرار گرفته است. همچنین، تونل باد برای اندازه­گیری میزان پایداری خاک، طراحی شد.
نتایج: در این پژوهش، سه فاکتور (کلرید آمونیوم، اوره و ملاس) برای بررسی تثبیت خاک، بررسی شدند. نتایج این مطالعه، نشان داد که روش تاگوچی یکی از کارامدترین روش‌ها برای بررسی فاکتورهای موثر در فرایند پایداری خاک است. در این مورد، غلظت اوره، دارای بیشترین تاثیر در رسوب‌دهی کلسیت و تثبیت خاک بوسیله این باکتری بود. همچنین، بررسی اندرکنش فاکتورها، ارتباط فاکتورها را با هم نشان داد. در مورد فاکتور کلرید آمونیوم با سه سطح (5/0، 1 و 5/1 گرم بر لیتر)، در سطح سوم، بیشترین تاثیر(353/4%) را در فرایند تثبیت خاک داشت. افزایش کلریدآمونیوم، باعث افزایش میزان پایداری خاک شد. سه سطح 20، 30 و 40 گرم بر لیتر اوره نیز مورد بررسی قرار گرفت. دراین مورد نیز، سطح سوم با میزان313/4% بیشترین تاثیر را داشت. همچنین، در این مطالعه،  طراحی تاگوچی، به صورت سه سطح ملاس (30، 40 و 50 گرم بر لیتر)، بیشینه تثبیت خاک را در سطح دوم با میزان 873/3% نشان داد. در حقیقت، ابتدا با افزایش ملاس، تثبیت خاک، افزایش و سپس کاهش یافت.
بحث و نتیجهگیری: در حالت کلی، این مطالعه نشان داد که دو فاکتور کلرید آمونیوم و اوره، تاثیر بیشتری در میزان هیدرو لیز اوره و رسوب کلسیت دارند.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Evaluation of stabilized soil by Bacillus sp. HAI4 in different conditions through Taguchi method

نویسندگان [English]

  • Mehran Alavi 1
  • Mojtaba Taran 2
  • Arezoo Berimavandi 3
1 PhD Candidate of Nano-Biotechnology, Microbiology Laboratory, Biology Department, Razi university, Kermanshah, Iran
2 Associate Professor of Microbiology, Razi University, Kermanshah, Iran
3 MSc of Microbiology, Microbiology Laboratory, Biology Department, Razi University, Kermanshah, Iran
چکیده [English]

Introduction: The process of calcite precipitation resulted from metabolic activities by microorganisms is biocalcification. In this process, calcite (CaCO3) is deposited on the soil grains by metabolic activities of microorganisms resulting in increased stiffness/strength and reduced erodibility of soil.
Materials and methods: In this study, urea hydrolysis and calcite precipitation rates in soil stabilization process were evaluated by the bacterium Bacillus sp. HAI4 through Taguchi method. Also, it was designed a wind tunnel for analysis of soil stabilization rate.
Results: In this investigation, three factors (NH4Cl, urea and molasses) were surveyed in optimization of soil stabilization. Results of this study illustrated that Taguchi method is one of effective methods for optimization of effective factors in soil stabilization process. In this case, urea concentration had higher effect on soil stabilization and calcite sedimentation by Bacillus sp. Also, evaluating factors interaction showed relationship of factors together. Three levels (0.5, 1 and 1.5 g/L), were determined for NH4Cl factor in which the highest effect (4.353%) in soil stabilization was related to level 3. Increasing of NH4Cl resulted in high soil stabilization rate. Three levels 20, 30, and 40 g/L of urea were applied. Results illustrated that effective level was 3 with 4.313%. Also, Taguchi design was in this study, as three levels of molasses (30, 40 and 50 gr/L) illustrated maximum soil stabilization in level 2 by 3.873%. In fact, by increasing of molasses, soil stabilization firstly increased and then decreased.
Discussion and conclusion: Totally, this study shows two factors (NH4Cl and urea) that have a major effect in urea hydrolysis and calcite precipitation rates.

کلیدواژه‌ها [English]

  • Biocalcification
  • Soil stabilization
  • Bacillus sp
  • Urea hydrolysis
  • Calcite precipitation
  • Ammonium chloride

Introduction

Biocalcification has illustrated some promise in soil cementation by microbially induced calcite precipitation (1, 2). In this process, calcite (CaCO3) is deposited on the soil grains by metabolic activities of microorganisms resulting in increased stiffness/strength and reduced erodibility of soil. In this case, urea hydrolysis by some bacteria such as Bacillus pasteurii or sporosarcina pasteurii can lead to calcite precipitation as crystals (3-5). 

Using this method has illustrated a promise in several fields such as strengthening of concrete and cracks remediation (6, 7), remediation of copper-contaminated soil (8), improving of strength and durability of fly ash-amended concrete (9), Biomineralization based remediation of As(III) contaminated soil (10, 11), Bioremediation of Pb-contaminated soil (12), improvement in the durability of cement mortar (13) and improving of the stiffness/strength of sandy soil (14). 

Biomineralization as production of minerals by living organisms (14) is resulted from metabolic activities of some organisms including invertebrates (carbonate), vertebrates (carbonates, phosphates and calcium), algae and diatoms (silicate) (14, 15). In the case of biologically induced mineralization (BIM), the minerals are produced extracellular by metabolic activities of the living organisms (16).

Four important factors which affect calcite precipitation include 1) concentration of the calcium, 2) dissolved inorganic carbon (DIC) concentration, 3) the pH and (4) the availability of nucleation sites (17-19). Different bacterial species can precipitate carbonates in environment with high pH and Ca2+ ions. In addition, various mechanisms can stimulate this process by bacteria (17, 20). Ability of bacteria to create environment with alkaline condition are important mechanisms. Carbonate precipitation can be developed as extracellular by successive stratification and it leads to growing carbonate crystals on the bacteria surface (21-23).

In order to calcium carbonate production, several studies applied urea and CaCl2 combinations. For example, different combinations of 0.33 and 0.67 M urea and 0.0025, 0.025 and 0.25M Ca2+ solution were used by Okwada and coworkers (2010). The results of this study, illustrate that the rate of ureolysis increases with bacterial cell concentration, and the bacterial cell concentration had a greater influence on k(urea) than initial urea concentration (24). Also, application of lower chemical amounts used over multiple injections resulted in more uniform cementation (25). In this study, urea hydrolysis and calcite precipitation rates in soil stabilization process were evaluated for the bacterium Bacillus sp. HAI4 by Taguchi method in three levels of three factors (NH4Cl, urea and molasses).

Materials and methods

Particle size analysis of soil:Sufficient aggregated samples were obtained from Wetland Hooralazim of Khuzestan province in Iran. In order to analyze soil particle size, a gradation test was performed on a soil sample in a laboratory(26). In this case, a determined weighed sample was poured into the top sieve which had a set of sieves with progressively smaller openings (Fig. 1). A mechanical shaker was used for shaking of sieve column. The results were demonstrated in a graph of percent passing versus the diameter of sieves (4.75, 2.36, 2, 1.4, 1.18, 0.85, 0.71, 0.6, 0.425, 0.3, 0.25, 0.15, 0.075) (Table 1).

 Fig. 1- Used columns of laboratory sieve in this study

Table 1- Sieve analysis results are generally expressed as the percentage of the total weight of soil that passed through different sieves

Sieve number

Diameter (mm)

Mass of soil retained on each sieve (g)

Percent retained (%)

Cumulative passed (%)

4

4.75

18.53

18.53

81.47

8

2.36

11.9

30.43

69.57

10

2

2.87

33.3

66.7

14

1.4

8.51

41.81

58.19

16

1.118

3.45

45.26

54.74

20

0.85

4.3

49.56

50.44

25

0.71

5.44

55

45

30

0.6

0.178

55.178

44.822

40

0.425

7.866

63.044

36.956

50

0.3

4.58

67.624

32.376

60

0.25

1.54

69.164

30.836

100

0.15

9.36

78.524

21.476

200

0.075

8.018

86.542

13.458

pan

pan

13.458

100

0

 

 

100

 

 

 

Bacteria preparation and treatment: Bacillus sp. HAI4 species were isolated form from Wetland Hooralazim of Khuzestan province in Iran.  Species were grown in broth nutrient at 30C for 24 hours in incubator.  In order to bacterial calcite precipitation, nutrient broth-urea containing 30 g urea, 30 g molasses, 5 g NH4Cl and 5 g milk powder per liter (10pH) was autoclaved, cooled and combined immediately with desired concentration of filtered (0.2 Mm) and sterilized CaCl2 before application onto soil. Culture medium was incubated at 30 ˚C for 48 hours. After time incubation, prepared bacteria were colored by methylene blue.

Optimization of soil stabilization by Bacteria: For obtain optimum condition of soil stabilization by bacteria, Taguchi method was applied in three levels for three factors (NH4Cl, urea and molasses) (Table 2). In this case, nine experiments were designed (Table 3). Milk powder concentration (0.1 g/L) was fixed for each experiment.

 

Table 2- Design of three factors with three levels by Taguchi method

Factors (g/L)

Level 1

Level 2

Level 3

NH4Cl

0.5

1

1.5

Urea

20

30

40

Molasses

30

40

50

 

Table 3- Nine experiments were designed by Taguchi method

Experiment number

NH4Cl (Level):(g/L)

Urea (Level):(g/L)

Molasses (Level):(g/L)

1

1= 0.5

1= 20

1= 30

2

1= 0.5

2= 30

2= 40

3

1= 0.5

3= 40

3= 50

4

2= 1

1= 20

2= 40

5

2= 1

2= 30

3= 50

6

2= 1

3= 40

1= 30

7

3= 1.5

1= 20

3= 50

8

3= 1.5

2= 30

1= 30

9

3= 1.5

3= 40

2= 40

 

In addition to nine designed experiments, two experiments were designed as control (Table 4). One experiment, number ten, contains treatment solution without bacteria and other experiment contains only bacteria without treatment solution.

 

Table 4- Control experiment designed by Taguchi Method

Experiment number

NH4Cl (g/L)

Urea (Level):(g/L)

Molasses (Level):(g/L)

10

2= 1

2= 30

2= 40

On the first day, eleven flasks contain 100 g of soil were prepared and 70 ml of bacteria with 0.5 McFarland was added to all flaks apart from control flask with number 10. Afterwards, 100 ml of treatment solutions were prepared and since the second day, 20ml of solutions were added to soils apart from controls for five days. Every day, after adding solutions, soils were incubated at 30C for 24 hours.

Biological cementation: Treatment solutions (100 mm) were prepared with adding 2.2g calcium chloride (CaCl2). Afterwards, 20 mm of treatment solution were added to each soil for 5 days (apart from control without bacteria). Due to complete drying, after five days, all soils were incubated for one mount at 30 C.

Wind tunnel testing: To analyze soil fixation, wind velocity was measured by design wind tunnel (27). Wind velocity was evaluated by the following formula:

Where ν as velocity

m=7.5×10-3 kg (mass of bullet)

g=9.8 m/s2

tanθ as angle of deviation

C as constant of integration equals 0/6

ρ as air density equals 1.3kg/m3

A as area of bullet.

In this case, two experiments at two different angles (8˚ and 12˚) were designed.

After this stage, each of weighted soil samples was exposed to wind velocity by 14.56 km/h. Then, reduced weight of soil samples was measured through weigh again of samples (Table 5). This process was applied for wind velocity by 18km/h.

 

Results

Soil fixation: Interaction of factors and optimum conditions in soil stabilization was evaluated by Qualitek-4 software. Soil stabilization rate in 11 experiments illustrates those bacteria in medium by 1g/L ammonium Chloride, 40 g/L urea and 30 g/L molasses, had maximum of soil stabilization in experiment number 6.

Table 6, demonstrates effect of each factors and their levels on soil fixation. In this case, best level is for ammonium chloride at third level (1.5 g/L), Urea at third level (40g/L), molasses at second level (40g/L).

 

Table 5- Amount of reduced weight of soil samples in ν=18km/h

Sample number

Weight of sample before wind tunnel testing

Weight of sample after wind tunnel testing

Reduction percent

1

23.5

22.85

2.77

2

15

14.7

2

3

35

34

1.45

4

31

30.7

0.97

5

27

26.2

2.96

6

25

24.7

3.61

7

18

16.5

12.5

8

16.3

15.7

3.68

9

20.3

18.7

7.88

10 (first control)

29.2

28.4

2.74

11(second control)

33

30.3

8.18

 

 

Table 6- Effect of different level of factors on soil stabilization by Bacillus sp.

Factors

Level 1

Level 2

Level 3

NH4Cl

2.523

2.513

4.353

Urea

1.49

3.136

4.313

Molasses

3.096

3.873

1.969

 

Figure 3a shows effect of ammonium chloride (nitrogen source) on soil fixation. There was a low amount of soil stabilization (2.073) in level 1, and this amount raised (2.513) in level 2, and there was a maximum amount by 4.353 in level 3.

 

 

Fig. 3- Effect of different levels of NH4Cl, urea and molasses on soil stabilization by Bacillus sp.

 

Effect of urea factor on soil stabilization is illustrated in figure 3b. Amount of soil stabilization increased form level 1by 1.49 to level 3 by 4.313. This result indicates that urea has direct effect on soil fixation. Also, effect of molasses on soil stabilization is demonstrated in figure 3c. In this case, there was increasing of soil stabilization form level 1 (3.096) to level 2 (3.873), but in level 3 (1.969), reduction of this process was observed.

Interaction of factors on soil stabilization by Bacillus sp. HAI4: Table 7 shows factors interaction on soil stabilization. Interactions of paired factors were evaluated based on amount of selected two factors which was changeable form 0.86 to 24.67. Maximum interaction was 24.67 for NH4Cl×molasses that NH4Cl was in level 3 and molasses was in level 2. Also, Interaction of urea×molasses had minimum intensity of interaction (0.86) as urea in level 3 and molasses in level 2. NH4Cl and urea in level 3 had 8.82 of interaction.

Variance analysis (ANOVA) for soil stabilization by Bacillus sp. HAI4: Variance analysis (ANOVA) illustrates effective parameters on soil stabilization by Bacillus sp HAI4. Table 8 demonstrates molasses factor had not effect but NH4Cl and urea factors had higher effect by 1.869 and 11.412 respectively. Maximum effect is related to urea which shows a major role of nitrogen in soil fixation.

Estimation of optimum conditions for soil stabilization: Table 9 illustrates share of each factor in soil stabilization. Based on these results, urea and NH4Cl factors have higher and lower shares. Expected result in optimum conditions of soil stabilization was -0.428%. NH4Cl and urea in level 1 and molasses in level 3 had suitable levels.

 

Table 7- Interaction effect of paired factors on soil stabilization by Bacillus sp.

Number

Effect of paired factors

Interaction of factors

Intensity of interaction factors

Column

Optimum conditions

NH4Cl×molasses

1

1×3

24.67

2

2.2

NH4Cl×urea

2

1×2

8.82

3

2.1

Urea×Molasses

3

2×3

0.86

1

1.2

 

Table 8- Variance analysis in soil stabilization by Bacillus sp.

Factors

DOF (f)

Sum of squares (S)

Variance (V)

F-Ratio (F)

Pure Sum (Ś)

Percent (%)

NH4Cl

2

8.777

4.388

1.076

0.644

1.869

Urea

2

12.067

6.033

1.483

3.934

11.412

Molasses

2

5.495

2.747

0.675

0

0

Total

8

34.473

 

 

 

100%

 

Table 9- Optimum conditions for soil stabilization by Bacillus sp. HAI4

Column/Factor

Level description

Level

Contribution

NH4Cl

Factor A-Level 1

1

-0.907

Urea

Factor B-Level 1

1

-1.49

Molasses

Factor C-Level3

3

-1.011

Total contribution from all factors

 

 

-3.409

Current grand average of performance

 

 

2.98

Expected result at optimum condition

 

 

-0.428

 


Discussion and conclusion

Today, stabilization of soil and dust by microorganisms is important and because of climate change, desertification and dust storm can effect on environment and human health (28). Stabilization of soils by microorganisms can be effected by different factors such as calcium, pH, and temperature and urea concentration. Therefore, improvement of soil stabilization by microorganisms can lead to increasing  microorganisms capacity in soil stabilization (1).

Results of this study illustrated that Taguchi method is one of the effective methods for optimization of effective factors in soil stabilization process. In this method, three factors (NH4Cl, urea and molasses) were surveyed in soil fixation optimization. In this case, urea concentration had higher effect on soil stabilization and calcite sedimentation by Bacillus sp HAI4. Also, interaction of two factors shows dependence of factors together.

Meyer and coworkers (2011) used NH4Cl with 10g/L concentration as nitrogen source for Sarcina pasteurii in soil stabilization (29). This group observed that soil samples as a treatment in wind tunnel by 40 km/h wind velocity had higher mass than controls. In this investigation, three levels (0.5, 1 and 1.5 g/L) were determined for this factor in which the highest effect (4.353) in soil stabilization was related to level 3 of NH4Cl (Table 6). Therefore, by increasing NH4Cl concentration, soil stabilization increased (Fig. 3). The reason of this change is high ammonium anion for rising of pH, urea hydrolysis and calcite sedimentation.

An important process in ecosystems ammonium oxidation is carried by autotrophic ammonia-oxidizing bacteria (AOB) and change in diversity and quantity of total bacteria is resulted from the application of urea to the soil (30). Also, Neg and coworkers (2012) applied 0.25M of urea for Bacillus megaterium in order to stabilize soil samples. In our study, three levels 20, 30, and 40 g/L of urea were utilized. Results illustrated that effective level was 3 with 4.313% (14). Therefore, it can result in a way that by increasing urea concentration, soil stabilization increases. Hereon, urea hydrolysis was augmented and leads to calcite sedimentation. Similarly, Dejong et al (2006) illustrated suitable result of soil stabilization in sandy samples by using 20 g/L of urea for Sarcina pasteurii (31). Amount of 250 mmol in groundwater samples for Sarcina pasteurii was used in order to close fracture and porosity in stones for inhibition of groundwater flows and contaminant transport.

Molasses is another factor that was evaluated in soil fixation. Molasses with 1 g/L was applied for Sarcina pasteurii in order to prevent water leakage in groundwater. Taguchi design in this study, as three levels of molasses (30, 40 and 50 g/L) illustrated maximum soil stabilization in level 2 by 3.873%. In fact, by increasing of molasses, soil stabilization firstly increased and then decreased (Figure 3). Before initiation of urea hydrolysis, molasses resulted in biomass augmentation; therefore this process causes pH decrease. Therefore, molasses increasing does not have an effective role in soil fixation.

 

Acknowledgements

The authors gratefully acknowledge the financial and logistic assistance provided by biology department of Razi University.

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