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

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

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

Table 3- Nine experiments were designed by Taguchi method

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

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

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

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.

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

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