J. Cent. South Univ. (2019) 26: 2596-2608

DOI: https://doi.org/10.1007/s11771-019-4197-1

Enhancement of resilient modulus of cohesive soil using an enzymatic preparation

Ahmed F. ZIDAN¹, Abdullah A. ABOUKHADRA¹, Yasser GABER^{2, 3}

1. Department of Civil Engineering, Faculty of Engineering, Beni-Suef University, Beni-Suef 62514, Egypt;

2. Microbiology Department, Faculty of Pharmacy,Beni-Suef University, Beni-Suef 62514, Egypt;

3. Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Mutah University, Al karak 61710, Jordan

Central South University Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract: The current study aims to evaluate the dynamic response of stabilized cohesive soil using an enzymatic preparation in terms of resilient modulus. We ran a series of resilient modulus testing according to AASHTO T307 on three types of cohesive soil treated with an enzymatic preparation to investigate its potential on roads construction. The results show significant improvement in the resilient modulus values, estimated at 1.4 to 4.4 times that observed for the untreated soil. Because of the complexity in conducting the resilient modulus measurement, we did a regression analysis to produce reliable correlation formula to predict the resilient modulus for untreated and stabilised soil samples involving stress state. The resilient modulus values for the subgrade materials at the anticipated field stresses were determined using a universal model. The enzymatic preparation was applied in pavement of a sample road and evaluated using the plate load test. SEM analysis for soil samples shows improvement in the soil compaction via reduction of voids between soil particles. XRD analysis shows no major structural changes in the treated soils. The enzymatic preparation contains 43 mg/mL of proteins. We used the SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) technique to identify the main protein components; however, the presence of interfering materials (surfactants) hinders the separation.

Key words: enzyme; resilient modulus; cyclic loading; regression analysis; plate load test

Cite this article as: Ahmed F. ZIDAN, Abdullah A. ABOUKHADRA, Yasser GABER. Enhancement of resilient modulus of cohesive soil using an enzymatic preparation [J]. Journal of Central South University, 2019, 26(9): 2596-2608. DOI: https://doi.org/10.1007/s11771-019-4197-1.

1 Introduction

Soils with poor properties impose great barriers for constructing roads at feasible costs. Therefore, new methods and new materials that can reduce the cost of road construction and ease the process of soil improvement are always sought. The idea of using biologically derived material such as enzymes for soil stabilization and in pavement construction is appealing due to its low cost and environmental safety profile and certain types of enzymes were reported to enhance the bonding between soil particles [1].

The improvement of weak soil with enzymatic preparations is revolutionary technique and becoming popular worldwide. Nowadays, there are many types of commercial enzymatic preparations available for soil stabilization, such as earthzyme, terrazyme and permazyme.

Many types of additives and matrices have been tested to improve soil strength and to reduce the cost of soil replacement in roads construction [2]. Enzymatic preparations have been described as potential soil stabilizers with proved improvements [3-6]. KHAN et al [7] reported improvement in soil confining compressive strength and good performance in freeze-thaw testing with Permazyme-treated soils. Furthermore, the existence of permazyme increased the lubricity of soil particles which led to decrease in the compaction effort.

The resilient modulus M_r is a key parameter to assess the soil stiffness and it is measured based on the recoverable strain under cyclic axial stress [8-15]. The 1986 AASHTO recommends using M_r for pavement design of both flexible and rigid pavements. The investigations of the effect of enzymatic preparations on resilient modulus are scarce. VELASQUEZ et al [16, 17] investigated the effect of two enzymatic preparations on the behaviour of two types of soils: soils I and II in terms of resilient modulus, shear strength and mode of action. They reported increase in M_r by an average of 54%-77% and the increase was proportional to the curing time.

Practically, enzymatic stabilization for soil in the field is usually done through five steps, explained here briefly as follows:1) the soil is treated with a grader in order to prohibit drainage of the enzymatic solution at the site of application; 2) a specific watercraft with dribble bar is used to adjust the pavement location to a moisture content near the optimum moisture concentration; 3) the enzymatic preparation is diluted with water at the specified concentration according to the manufacturer’s guidelines; 4) the diluted enzymatic solution is thoroughly mixed with soil materials, 5) levelling and compaction of the pavement is achieved by smooth drum roller [18].

The aim of the current paper is to investigate the improvement of resilient modulus of cohesive soil stabilized by an enzymatic preparation. The study is an extension of our recently published study by ABOUKHADRA et al [19], where the same cohesive soils in this study are examined to assess the impact of the enzymatic preparation on the soil resilient modulus under the effect of cyclic loading. Since the measuring of resilient modulus is a complex test and difficult task. The obtained data are used to introduce correlation formula involving the state of stresses. In addition, to support the experiments and assess the potential value of the enzymatic preparation for soil stabilization, field tests using plate load test are conducted to report on the soil strength behaviour.

2 Materials and experimental program

2.1 Investigated materials

In the current study, we examined the influence of an enzymatic preparation on three types of cohesive soils. Table 1 illustrates the different soil characteristics where C_u stands for uniformity coefficient, C_c stands for coefficient of curvature; LL stands for liquid limit; PL stands for plastic limit; PI stands for plasticity index,γ_dmax stands for dry unit weight, OMC stands for optimum moisture content. In our recently published study, the details of the examined soils such as physical and mechanical properties were reported [19].

Table 1 Particle size distribution and physical properties of untreated soil [19]

The enzymatic preparation used during this study was a commercial product distributed under the brand name Permazyme and purchased from Pacific Enzymes Inc., USA. Permazyme is described by the distributor as safe and naturally derived material for soil stabilization purposes. The exact chemical composition is, however, not revealed for commercial reasons. According to our previous work, the best concentration ratio of the enzymatic preparations in water is found to be 2 g/L therefore, through the current experimental study, the same concentration ratio was followed. Furthermore, the dynamic resilient modulus test was performed on the treated soil samples after curing period of 6 weeks in order to achieve fully interaction between the soil particles and the enzymatic solution.

2.2 Resilient modulus testing-test programme

Resilient modulus (RM, M_r) testing is an advanced dynamic test that measures the soil stiffness and provides a mean to analyze the soil behaviour under different conditions of cyclic vertical stress accompanied by confining pressure. RM measurement is influenced by several factors such as soil type, density and moisture content, test method, confining and deviator stress. In the current study, M_r for the investigated materials was determined according to AASHTO T307 “Determining the Resilient Modulus of Soil and Aggregate Materials” which describes the laboratory preparation, testing, and computation of test results [20]. The universal testing machine of 25 kN load, UTM 25 (Figure 1) at Mansoura University Highway and Airport Engineering Laboratory (H&AE-LAB), Mansoura City, Egypt, was used to measure M_r for the soil samples. Table 2 summarizes the tested materials and the ID of each sample. It should be noted that two replicates for clay I and loam soil were tested and all results are based on the average of the replicates.

The samples were prepared using the same procedure as for the static triaxial tests. After placing the sample in the triaxial cell, specified preconditioning confining pressure of 103.4 kPa was applied to the tested soils. Then the sample was subjected to 500 load cycles for preconditioning, then 15 sequences of cyclic stresses, each sequence for 100 cycles. Details of the loading sequences are given in Table 3. The applied load followed a haversine pulse with 0.2 s loading duration and 0.8 s dwell (rest). The rest time allowed between the load pulses was to permit full recovery of the resilient strain. A typical load-time and deformation-time relationship for the first loading cycle is given in Figure 2.

Figure 1 UTM-25 at Mansoura University Highway and Airport Engineering Laboratory (H&AE-LAB), Mansoura, Egypt

Table 2 Description of Sample ID

2.3 SEM and XRD analysis

The analyses of treated and untreated soils using scanning electron microscopy (SEM), and X-ray diffraction (XRD) were performed in the laboratories of Housing and Building National Research Center, Giza, Egypt, using SEM, Philips (XL30), equipped with an energy dispersive X-ray analyzer EDX. The examination aimed to investigate morphological and structural changes of the particles and voids found in the samples. For the XRD experiments, the identification of the most probable phases is carried out using PANalytical computer certified program X’Pert High Score Software 2006-Licensed modules: PW3209, with the aid of the International Center of Diffraction Database (ICDD) PDF-2 Database/CD-Release 2005-Type No. 943050001611. The data were collected with the X-ray diffraction equipment X’Pert Pro PANalytical–manufactured by Panalytical B.V Co., Netherlands (ISO 9001/14001 KEMA-0.75160) with scan type: continuous, anode material: copper (Cu), and general setting: 30 mA and 40 kV.

Table 3 Testing sequences for fine subgrade materials [21]

Figure 2 Typical load-time and deformation-time response during first loading cycle for subgrade material:

2.4 Investigation of protein content using SDS-PAGE

The widely used technique for protein analysis known as sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used in the current study to separate the protein content found in the enzymatic preparation used in the study. The technique is able to separate proteins with relative molecular masses above 10000 Da. The enzymatic preparation sample (1 mL) was treated with acetone/methanol (50:50, volume ratio) to precipitate the protein content. The precipitated protein was solubilized in water and applied to the gel for separation. Different sample masses were loaded on the SDS-PAGE and gel protein concentration was measured using Bradford assay [22].

3 Results and analysis

3.1 Improvement in resilient modulus after enzymatic treatment

As general observation, the obtained M_r values indicated significant improvement in the enzymatic treated soils compared to untreated soils.Figures 3(a)-(c) show the comparison between the measured resilient modulus values for the Clay I, Loam, and Clay II materials before and after enzymatic treatment, respectively. For Clay I (Figure 3(a)), steep decline in M_r can be noticed during the first five sequences at confining pressure of 41.4 kPa. The graph shows that the value of M_r decreased by about 32% at the end of Sequence 5. The rate of the decreasing of M_r decreases through the rest of sequences, this is attributed to soil hardening due to loading and unloading cycles. At the end of Sequence 15, the decreasing in M_r reached to 65% of its initial value. Slight increase in M_r was observed for the treated sample during all sequences, the overall increase represented approximately 9% at the end of the test. At the end of the test, the M_r value of the stabilized sample reached 110 MPa which is almost two-fold improvement compared to the untreated sample. Figure 3(b) compares the treated and the untreated soil for the loam material. Unlike Clay I, the stiffness of untreated sample increased during the sequences accompanied by the same confining pressure. It can be inferred from Figure 4 that the M_r values for treated sample ranges from 120 to 133 MPa. The improvement in the overall M_r for the stabilized soil was approximately 1.4 times compared to the untreated sample. For Clay II, as shown in Figure 3(c), insignificant change in M_r is observed for the control specimen during load sequence. On the other hand, for the stabilized sample, degradation in M_r is noticed during the sequences at the same level of confining pressure. At the end of the test, the stabilized sample gained enhancement about 4.4 times the M_r observed for control sample.

Figure 3 Sequence number versus measured M_r before and after stabilization:

3.2 Correlation formula

The term of resilience modulus M_r has become crucial for pavement design. Generally, the value of M_r is computed by conducting dynamic triaxial test on reconstituted and undistracted samples. Because the test is complex, expensive and time-consuming, M_r can be obtained by a correlation formula that depends on the stress state and the physical properties of soil [8, 23-26]. We utilized the 15 different sequences values obtained from the M_r experiments to predict a regression coefficient known as a universal model. We determined the nonlinear elastic coefficients and exponents (K values) of the universal constitutive model by using nonlinear regression analyses to fit the universal model to laboratory generated M_r test data. The generalized universal model obtained is described as follows:

where M_r is resilient modulus; θ is bulk stress=σ₁+σ₂+σ₃ (σ₁ is major principal stress, σ₂ is intermediate principal stress equal to σ₃ for cylindrical specimens and σ₃ is minor principal stress “confining pressure”); τ_oct is octahedral shear stress andP_a is normalizing stress (atmospheric pressure,e.g., 101.35 kPa); K₁, K₂ and K₃ are regression constants.

The measured laboratory data containing 16 sets of data points were used to estimate the K values. The model was solved by nonlinear optimization using the ‘Solver’ function in Microsoft^TM Excel. The values of the fitting parameters along with the goodness of fit parameters (S_e, S_y, S_e/S_y and R² where S_e is the standard error of estimate and S_y is the standard deviation of measured M_r values) are shown in Figures 4-6. It is worth mentioning that the loam materials did not follow the suggested universal model (R²<0.1) (data not shown).

Figure 4 Comparisons between measured and predicted modulus for control Clay I:

Figure 5 Comparisons between measured and predicted modulus for stabilized Clay I

3.3 Estimation of resilient modulus at anticipated field stresses

The resilient modulus values for the subgrade materials at the anticipated field stresses were determined using the universal model. For the subgrade soils, the anticipated field confining stress is in the range of 6.90-13.80 kPa and the anticipated field deviator stress in the range of 41.4-48.3 kPa [27]. Figure 7 shows the values of resilient moduli at the anticipated field stresses, it shows that the stabilized materials have a larger modulus compared to the control material for each type of soil. For Clay I the modulus increases by 150% (from 44.47 to 110.88 MPa), for the loam soil the increase in the modulus is about 19% (from 104.77 to 124.51 MPa) and 383% increase for Clay II (from 13.6 to 65.65 MPa).

Figure 6 Comparisons between measured and predicted modulus:

Figure 7 Estimated resilient moduli at anticipated field stresses

3.4 In-situ testing and plate load test

In order to verify the effect of the enzymatic preparation on soil strength, plate load tests were performed on a new road selected at the position of soil Type II. The testing permits were obtained from the official local municipal authorities. The in-situ segments were carried out at a village (El-Kom El-Ahmar) located near the Beni-Suef city, Egypt, in order to verify the suitability of the enzymatic preparation in increasing the strength of sub-base soil. This road is already serving both light and heavy public and agricultural traffics. Several samples were extracted from the road segment to measure the natural water content and compute the maximum dry density and optimum moisture content. To investigate the soil response with and without the enzymatic preparation treatment, we divided the road segment into two panels (5 m long×6 m width). For both regions, the compaction was processed on 30 cm depth. In addition, a compactor machine was used in several passes to achieve the maximum dry density at about optimum moisture content (Figure 8). The stabilized section was mixed with the diluted enzymatic solution at the same concentration obtained from the laboratory tests (2 g/L). On the other hand, we treated the control panel with potable water and compacted it using the same machine with the same compaction (Figure 8). In order to provide best results, before compaction process, scarifying equipment was used to mix the soil well with the diluted enzymatic solution (Figure 11). The soil stabilization behaviour of the two panels was investigated after curing period of 6 weeks by the plate load test.

We applied the plate load test in the current study to evaluate the performance of the stabilized soils under real conditions. We used a circular steel plate of d40 cm×2.5 cm, the load was applied through a system comprising hydraulic jack, reaction equipment (grader of 12 tons weight) and calibrated load cell (Figure 10). A seating load of 0.7 kN was applied and removed before applying the load test. The settlement of the plate was measured for each increment of stress (25, 50, 75, 100, 150, 250, 350, 450 kPa) by three dial gauges with division of 0.01 mm and the average settlement was recorded. Furthermore, the load increment was applied when the rate of settlement was reduced to 0.02 mm/min. For both tests, the load settlement curve was plotted, and the ultimate bearing capacity was determined at the intersection of two straight tangents [28].

Figure 8 Preparation of research road segment scarifying process (a), mixing (b) and compaction (c)

Figure 9 shows the load settlement curves for both treated and untreated soil with the data obtained from plat load tests. It can be noted from the behaviour of load-settlement curve that the rate of settlement observed for untreated soil is significantly higher compared to stabilized soil. Thus, the treatment of the soil with the enzymatic preparation increased the soil stiffness. As shown in Figure 8, the ultimate bearing stress for untreated soil was observed at 250 kN/m² and the value of ultimate bearing stress for stabilized soil is about 350 kN/m². In other words, the bearing capacity of stabilized soil increased to 1.4 times of that of untreated one. In addition, obviously, the enhancement in the settlement for treated soil was noticed whereas, the settlement of the plate at the ultimate load is equal to 0.25% of the plate diameter and it can be determined that the settlement was reduced by 41% as compared to the untreated soil. It can be concluded from this experiment that the enzymatic treatment has clearly improved the soil strength and carries great potential in road construction and other geotechnical applications.

Figure 9 Settlement vs stress curves applied for untreated and the treated enzymatic panels

Figure 10 Calibrated load cell

3.5 Investigation of treated versus untreated soils using SEM

Scanning electron microscopy (SEM) technique was used to investigate the effect of the enzyme preparations on all the soil samples tested in the current study. Clay and loam samples were used in the SEM imaging experiments. The images obtained before treatment and after treatment show formation of highly dense soil particles and decrease in the size of the voids found in the untreated soils. This was clear in the loam soil in particular compared to the fine sand soil. The use of XRD technique for characterization of soil mineralogy has proved high efficacy. XRD and SEM were conducted in the current study for the untreated and treated soils after being cured for 30 d. Figure 11 shows the SEM inages of samples. Curing time was 6 weeks. The untreated samples showed voids while the treated samples showed compact much less voids. Figure 11(d) also shows similar features.

3.6 XRD investigation of treated and untreated soil samples

The treated and the untreated soil samples were investigated with energy dispersive spectroscopy to reveal if structural changes in the soil mineralogy took place. The XRD investigations for the two different soil types i.e. Clay I and loam were performed. Figures 12 and 13 show the XRD chromatograms obtained. The clay sample shows characteristic peaks at positions 12° and 27° at the 2θ angel scale, which are corresponding to the gypsum and quartz, respectively. The treated samples show almost identical XRD chromatograms to the untreated samples. These major peaks identified according to the XRD machine linked database are: calcite, montmorillonite-15A, anorthite and illite. Almost the same peaks were identified in the treated clay sample. The loam sample investigated with XRD shows characteristic peaks of the following structures: quartz, gypsum, montmorillonite, kaolinite and calcite. The investigation of the treated load samples with the enzymatic preparation (Permazyme) shows the following characteristic peaks: quartz, gypsum, calcite, kaolinite and halite. These results indicate that the probable effect of the stabilizing agent is not via the formation of new structures of the soil mineralogical structures. The results obtained agree with the previous study conducted by KHAN et al [6], and KHAN et al [7].

Figure 11 SEM images of samples:

Figure 12 XRD spectra for untreated (a) and treated (b) Caly I soil with enzymatic preparation

Figure 13 XRD analysis of untreated (a) and treated (b) loam soil

3.7 Investigation of enzymatic preparation using SDS-PAGE

To shed light on the chemical composition of the enzymatic preparation used in the current study, we analyzed a sample from the preparation using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [29]. The SDS-PAGE is a technique used to separate proteins according to their molecular weights [29, 30]. The technique was used since the contents of the enzymatic preparations were expected to be composed of enzymes which are protein in nature. The test material was applied on the gel in comparison to a marker ladder protein acting as standard that contains a range of increasing molecular mass proteins. The results show no clear distinct protein bands in the enzymatic preparations even at increasing loading of the applied sample (1, 2, 5 and 10 mg of protein content based on the Bradford assay) instead, extended smears are seen in the gel, as shown in Figure 14. Different masses (1, 2, 5 and 10 mg) were loaded to different lanes in the gel based on the Bradford assay (M indicates the Marker protein ladder used as standard (10-250 kDa)). Estimation of protein concentration of the enzymatic preparation was found to be 43 mg/mL. These results indicate the presence of protein at considerable concentration, however, whether the type of the protein itself has clear enzymatic function is not certain. The probable presence of interfering materials in the enzymatic preparation of polymers or surfactants prevents good separation on the SDS-PAGE. The enzymatic preparation provider states that the enzymatic preparation is naturally prepared from fermentation products. The chemical composition is not clearly provided due to commercial reasons. Considering our previous study and in the light of the XRD results obtained in the current study, we can state that there are no new crystal structures have been formed in the treated soil samples. This indicates that physical changes rather than chemical changes in the particles of the soil surfaces could have taken place [19]. It has been described in literature that organic compounds carrying positive charges will exchange the cations (such as calcium and sodium ions) present on the surface of the soil particles and hence reduce the adsorbed water layer found on the particles’ surfaces. This will reduce the poor soil characteristics related to the presence of such water layer. The enzymatic preparation could act via providing such organic compounds. The exact chemical composition of the enzymatic preparation should be known for clear interpretation of the mechanism of the soil stabilization observed.

Figure 14 Investigation of protein content of enzymatic preparation using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

4 Conclusions

This paper aims to investigate the effect of an enzymatic preparation treatment on the soil performance in terms of resilient modulus. Based on a complex dynamic test on three clay samples, the results show a great enhancement in the measured resilience modulus using the enzymatic preparation for the stabilized soil compared to the that of original soils. Due to the enzymatic treatment, the ratio of resilient modulus of stabilized materials to the untreated materials ranges from 1.4 and 4.4 through the three types of soils used in the study. Furthermore, nonlinear regression analyses were performed on the results obtained from the laboratory expriments to determine the elastic coefficients and exponents (K values) of the universal constitutive model as well as estimation of the resilient modulus at the anticipated field stresses. The results of the plate load test show that the ultimate bearing stress for treated soil increases by 40% as compared with the untreated one.

Acknowledgments

Initiatives and National Campaigns, Academy of Scientific Research and Technology (ASRT), 101 Kasr El-Ainy St. Cairo, 11516, Egypt, are acknowledged for funding the project titled “Low Cost Technology for Roads Construction” Contract No. 24/2014, in collaboration with Beni-Suef University. Proteomics & Mass Spectrometry Facility at the Danforth Plant Science Center, Proteomics & Mass Spectrometry Facility, 975 N Warson Rd. St. Louis, MO 63132, USA is acknowledged for the SDS-PAGE analysis. The authors would like to thank the Laboratory of Highway and Airport Engineering-Mansoura University, Dr. Sherif Badawy, for the assistance for performing the experiments.

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(Edited by FANG Jing-hua)

中文导读

酶法提高黏性土回弹模量

摘要：本研究利用回弹模量来评价酶法制备稳定黏性土的动力响应。根据AASHTO T307标准，对三种经过酶制剂处理的黏性土进行了回弹模量测试，以探讨其在道路建设中的作用。结果表明，与未经过处理的土样相比，经过酶制剂处理的土样回弹模量明显提高，为原来的1.4~4.4倍。由于回弹模量测试的复杂性，进行了回归分析，建立了可靠的相关公式，预测了应力状态下未经处理的和稳定的土样的回弹模量。采用通用模型确定了路基材料在预期场强下的回弹模量。将酶法制备方法应用于样本路段，并通过平板载荷试验对其进行评价。土样的扫描电镜结果分析表明，减小土壤颗粒间的空隙可以改善土样的压实效果。XRD分析结果表明，处理后的土壤结构无明显变化。酶制剂含有43 mg/mL的蛋白质，采用SDS-PAGE(十二烷基硫酸钠聚丙烯酰胺凝胶电泳)技术鉴定主要的蛋白质组分，但干扰物质(表面活性剂)阻碍了分离作用。

关键词：酶；回弹模量；循环载荷；回归分析；平板载荷试验

Foundation item: Project supported by the Academy of Scientific Research and Technology, ASRT, Cairo,Egypt

Received date: 2018-05-14; Accepted date: 2019-01-03

Corresponding author: Yasser GABER, Associate Professor; Tel: +96-2791077619; E-mail: Yasser.Gaber@pharm.bsu.edu.eg, Yasser. Gaber@Mutah.edu.jo; ORCID: 0000-0003-2244-4406