Keeping balance between performance and cost of the roads is becoming a challenge for public and private sector. Traditionally used soil stabilizers (lime, cement and bitumen) are becoming costly. Bio-enzymes have emerged as a new chemical for soil stabilization.
Keeping balance between performance and cost of the roads is becoming a challenge for public and private sector. Traditionally used soil stabilizers (lime, cement and bitumen) are becoming costly. Bio-enzymes have emerged as a new chemical for soil stabilization.
Keeping balance between performance and cost of the roads is becoming a challenge for public and private sector. Traditionally used soil stabilizers (lime, cement and bitumen) are becoming costly. Bio-enzymes have emerged as a new chemical for soil stabilization.
Stabilization with Bio-EnzymesA Review Dr Mohd Raihan Taha Professor and Head Department of Civil & Structural Engineering, Universiti Kebangsaan Malaysia e-mail: profraihan@gmail.com Tanveer A. Khan Ph.D. Student Department of Civil & Structural Engineering, Universiti Kebangsaan Malaysia e-mail: takhan557@gmail.com Ibtehaj Taha Jawad Ph.D. Student Department of Civil & Structural Engineering, Universiti Kebangsaan Malaysia e-mail: ibtehaj78@yahoo.com Ali Akbar Firoozi Ph.D. Student Department of Civil & Structural Engineering, Universiti Kebangsaan Malaysia e-mail: a.firoozi@gmail.com Ali Asghar Firoozi Ph.D. Student Department of Civil & Structural Engineering, Universiti Kebangsaan Malaysia e-mail: mehran.firoozi@gmail.com
ABSTRACT Roads are vital for the economic growth of a country. Keeping balance between performance and cost of the roads and at the same time fulfilling the environmental regulations is becoming a challenge for public and private sector. Traditionally used soil stabilizers (lime, cement and bitumen) are becoming costly. Gravel and sand, which are introduced to alter the soil properties are depleting and becoming increasingly expansive. This scenario has led to an urgent need to identify and introduce new materials to improve the roads performance and to keep the cost at an affordable level. In this respect different types of stabilizers ranging from inorganic to organic have now been tried in laboratory and in field to evaluate their suitability as soil stabilizer. Recently bio-enzymes have emerged as a new chemical for soil stabilization. They are organic materials which are supplied as concentrated liquid. It is claimed by bio-enzymes manufacturers that their products are effective, environmental friendly (non-toxic), cost effective and convenient to use. They improve the compressive strength, reduce compaction effort and increase the density thus reducing the permeability as well. But the studies so far suggest that these claims must be verified through independent laboratory testing before applying these bio-enzymes in the field. Vol. 18 [2013], Bund. R 3882
KEYWORDS: Enzyme, soil, stabilization, roads INTRODUCTION Microbial geo-technology is an emerging branch of geotechnical engineering that deals with the application of microbiological methods to improve the mechanical properties of soil to make it more fitting or appropriate for construction and environmental purposes. In these regard two noteworthy applications, bio-clogging and bio-cementation have been explored. Bio-clogging is the production of pore-filling materials through microbial means so that the porosity and hydraulic conductivity of soil can be reduced where as bio-cementation is the generation of particle binding materials through microbial processes in situ so that the shear strength of soil can be increased [Ivanov & Chu 2008]. One promising application is the formation of the soil plugs by bacillus pasteurii in the medium containing urea and calcium chloride. Bacteria produce enzyme urease that hydrolyzes urea by the following reaction: (NB 2 ) 2 C0 +SB 2 0 2NB 4 + + BC0 3 - +0B -
Due to this enzymatic reaction, pH is increased and hydrocarbonate is produced. It is initiating precipitation of calcium carbonate, which clogging the pores and binding soil particles [Kucharski et al. 2005]. Enzymes are biological catalyst and are present in all living organisms. They are obtained from plants and animals including microorganisms by extraction using suitable solvent (Prescott 2001). Enzymes are large protein molecules which are more efficient than inorganic catalyst; the reaction rate is often increased by a factor of 106 to 1012. They usually catalyze one particular reaction therefore enzymes do not produce side reaction. They are temperature sensitive and work at mild temperature (35 o C), and loss their effectiveness at higher temperature. Also they are pH sensitive too and work good around pH value 7 [Norris et al. 2011]. The specific substance (metabolite) that fits on the enzyme surface and is converted to products(s) is called substrate. For example the enzyme urease catalyzes the reaction: (NB 2 ) 2 C0 + B 2 0 2NB 3 + C0 2
Urea is the substrate in this reaction as it fits onto the surface of enzyme. Water reacts with the urea only when urea is bond to the enzyme surface. The catalyst activity of enzyme is measured in enzyme activity which is the number of moles of substrate converted to product per minute. The complicated folding of the polypeptide chain of enzyme gives pocket on the surface of the enzyme known as active site. The substrate binds itself on the active site where catalysis takes place. The active site has specific amino acid side-chains which form weak bonds with substrate. The substrate in this manner is kept in place so that other molecules can react with it in the right orientation. [Norris et al. 2011]. Most clay has a molecular structure with a net negative charge. To maintain the electrical neutrality, cations (positively charged) are attracted to and held on the edges and surfaces of clay particles. These cations are called exchangeable cations because in most cases cations of one type may be exchange with cations of another type. When the cation charge in the clay structure is weak, the remaining negative charge attracts polarized water molecules, filling the spaces of the clays structure with ionized water. As a consequence a movement of moisture from areas of low cation concentration to areas of high cation concentration is produced to achieve the equilibrium of the cation concentration. Cations are unable to disperse freely in the soil structure because of the Vol. 18 [2013], Bund. R 3883
attractions of the negatively charged surface of the clay particles. This creates an osmotic pressure gradient, which tries to equalize the cation concentration. As a consequence a movement of moisture from areas of low cation concentration to areas of high cation concentration is produced to achieve the equilibrium of the cation concentration [Scholen 1992]. The flow of cations through the clay deposits gives the shrinking and swelling properties of the soils; when a stabilizer solution is added in to the soil, the magnitude of the effect depends on the characteristics of the particular cation. In general there are two main characteristics, the valence of the cation or number of positive charges and the size of the cation [Rauch et al. 2002]. The size determines the mobility of the cation; smaller ones will travel a greater distance throughout the soil structure (the hydrogen ion is the smallest one). With respect to the valence, the hydrogen ion is doubly effective affecting the clay structure because even though it has only a single charge, the hydrogen ion produces an effect of valence of two due to its high ionization energy. These hydrogen cations exert a stronger pull on the clay layers pulling the structure of the soil together and removing the trapped moisture permitted by the single sodium and potassium cations. The loss of moisture results in a strengthening of the molecular structure of the clay [Scholen 1992]. Enzymes speed up a chemical reaction, that otherwise would happen at a slower rate, without becoming a part of the end product. The enzyme combines with the large organic molecules to form a reactant intermediary, which exchange ions with the clay structure, breaking down the lattice and causing the cover-up effect, which prevents further absorption of water and the loss of density. The enzyme is regenerated by the reaction and goes to react again. The enzymes are absorbed by the clay lattice, and then released upon exchange with metals cations. They have an important effect on the clay lattice, initially causing them to expand and then to tighten. The enzymes can be absorbed also by colloids enabling them to be transported through the soil electrolyte media. The enzymes also help the soil bacteria to release hydrogen ions, resulting in pH gradients at the surfaces of the clay particles, which assist in breaking up the structure of the clay [Scholen 1992]. The idea of using enzyme stabilization for roads was developed from enzyme products used for treatment of soil to improve horticultural applications. A modification to the process produced a material, which was suitable for stabilization of poor ground for road traffic. When is added to a soil, the enzymes increase the wetting and bonding capacity of the soil particles. The enzyme allows soil materials to become more easily wet and more densely compacted. Also, it improves the chemical bonding that helps to fuse the soil particles together, creating a more permanent structure that is more resistant to weathering, wear and water penetration (Marasteanu et al. 2005). The basic effect of the action of the enzyme into the structure of the soil can be summarized as, initially, the film of absorbed water is greatly reduced and in fact entirely broken, as shown schematically in Figures 1 and 2.The film of water enveloping the particles, which ultimately governs the expansion and shrinkage of colloidal soil constituents, cannot be completely eliminated by purely mechanical methods. However, by means of temperature effects, addition or removal of water with mechanical pressure, it is possible to vary the amount of water held in this manner. Such variations are attended by swelling or shrinkage. This provides an ideal point of operation for the enzyme (The Carbon Group). Vol. 18 [2013], Bund. R 3884
Figure 1: Absorbed water in the structure of soil (The Carbon Group )
Figure 2: Elimination of absorbed water in the soil (The Carbon Group ) Vol. 18 [2013], Bund. R 3885
Lowering the dipole moment of the water molecule by the enzyme results in dissociation in a hydroxyl (-) and a hydrogen (+) ion. The hydroxyl ion in turn dissociates into oxygen and hydrogen, while the hydrogen atom of the hydroxyl is transformed into a hydronium ion. The latter can accept or reject positive or negative charges, according to circumstances. Normally the finest colloidal particles of soil are negatively charged. The enveloping film of absorbed water contains a sufficient number of positive charged metal ions - such as sodium, potassium, aluminum and magnesium, which ensure charge equalization with respect to the electrically negative soil ion. The positive charges of the hydronium ion or of the negatively charged hydroxyl ion will normally combine with the positively charged metal ions in the water adhering to the surface of the particles. Because of the effect of the enzyme formulation in reducing the electric charge of the water molecule, there is sufficient negative charge to exert adequate pressure on the positively charged metal ions in the absorbed water film. As a result of this, the existing electrostatic potential barrier is broken. When this reaction occurs, the metal ions migrate into the free water, which can be washed out or removed by evaporation. Thus the film of absorbed water enveloping the particles is reduced. The particles thereby lose their swelling capacity and the soil as a whole acquires a friable structure. The hydrogen ions, which are liberated in the dissociation of the water molecules, can once again react with free hydroxyl ions and form water along the gaseous hydrogen. It is important to note that the moisture content of the soil affects the surface tension and is thus a factor affecting compaction. The enzyme reduces surface tension making the soil compaction easier to perform. After the absorbed water is reduced the soil particles tend to agglomerate and as a result of the relative movement between particles, the surface area is reduced and less absorbed water can be held, which in turn reduces the swelling capacity ((The Carbon Group). EXPERIMENTAL STUDIES Unlike the standard stabilizers such as Portland cement, lime and bitumen, non-standard stabilizers have no laboratory tests that can be used to predict their field performance. Because of the lack of communication between the manufacturers (unfamiliar with the road design process) and the engineers the considerable benefits of the non-standard stabilizers remain undiscovered or not clear [Scholen 1992]. Some of the recent experimental studies of soil stabilization with bio-enzymes are presented here; Lacuoture & Gonzalez (1995) studied the effect of the TerraZyme (TerraZymesoil stabilizer is an enzyme produced in the United States by Nature Plus, Inc. and is available worldwide through local distributors or directly from NPI) soil stabilizer product on sub-base and sub-grade soils. Variation in properties was observed and no significant improvement was reported during the early days but progressive improvement was observed. Hitam et al. (1999) of Palm Oil Research Institute of Malaysia conducted field studies on improvement on plantation roads. Road section of 27.2 km was treated with TerraZyme and it was observed that road remained good after two monsoon seasons, which had serious problems due to monsoon before. Bergmann (2000) concluded through studies that Bio-Enzymes need some clay content to strengthen the soils. It was observed that at least 2% clay is needed for successful stabilization whereas 10 to 15% clay gave very good results. Shukla et al. (2003) used Bio-Enzymes to stabilize five different types of soil ranging from low clay content to very high clay content, engineering properties and strength characteristics were Vol. 18 [2013], Bund. R 3886
determined and it was found that there is little to high improvement in physical properties. Little improvement could be due to soil constituent, which has low reactivity with Bio-Enzymes. There was improvement in CBR and unconfined compression strength of soils like silty soil to sandy soil. Milburn & Parsons (2004) conducted different tests (freeze-thaw, wet-dry, leach testing, Atterberg limits and strength tests) on soils (classified as CH, CL, ML, SM, and SP) stabilized with lime, cement, Class C fly ash, and Permazyme 11-X. Compaction, Unconfined compression, stiffness, freeze-thaw, wet-dry and leaching tests were conducted on two silty soils (ML and SM) treated with Permazyme 11-X at a dosage recommended by the supplier. ML and SM soils had fines 88 and 30%, LL 30 and 20% and PI 7 and 3% respectively. Compaction test for treated soils was carried out at moisture content 1% less than the optimum. But only 4% and 1% increase in dry density was found for ML and SM soils respectively. The soil samples for two soils after 28 days of curing were tested for stiffness and no improvement was recorded. Similarly for freeze-thaw very modest improvement and for wet-dry and leaching tests no improvement was observed. Marasteanu et al. (2005) conducted resilient modulus and tri-axial tests on two soils which were stabilized with two different enzymes. Soil-I has 96% of fines (75% of clay) a SPG of 2.73 and plasticity index of 52%. Soil-II has 60% of fines (14.5% of clay) and plasticity index of 9.4%. Chemical analysis of only one enzyme (A) was conducted, as the supplier of the other enzyme (B) did not agree for this. The chemical analysis for the enzyme included pH, metals concentrations (e.g., Ca, Fe, and Al), total organic carbon concentration, and inorganic anion concentrations (e.g., Cl-, NO 3- and SO 4 2- ). The pH of product A was 4.77 and had very high concentration of potassium (K), and moderate to high concentrations of calcium (Ca), magnesium (Mg), and sodium (Na). The metal concentration and inorganic anion concentration are given in Tables 1 and 2. The tests were conducted on a base (Base-1) as well to compare the results. The protein concentration in the undiluted product A was 9230 mg/L. The presence of protein alone does not indicate that the solution will exhibit enzymatic activity therefore enzyme activity tests were conducted and it was found that the product A exhibited no detectable enzymatic activity for the used substrates. This indicates two possibilities: Product A is a highly purified enzyme solution that contains only a single enzyme or group of enzymes that catalyze reactions not tested for in our experiments or Product A may not stabilize soil via enzymatic activity but rather via some other mechanism, possibly due to their surfactant-like characteristics. Therefore surface tension test was carried out and it was found that enzyme A was more effective in reducing the surface tension of water than a common surfactant, sodium dodecyl sulfate (SDD). Comparison among the two enzymes and SDD is presented in Figure 3. The results from quantitative surface tension testing and qualitative observations suggest that product A behaves like a surfactant, which may play a role in its soil stabilization performance. Base-1, on the other hand, contains high concentrations of sodium and silicon, which suggests that it acts like cement by forming hydrated calcium silicate when added to soil. Resilient modulus tests were run with different confining (2, 4, 6 and 8 psi) and deviator stress (4, 7, 10 and 14 psi). It is difficult to discuss all the results but brief comparison is presented here:
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Table 1: Metal concentration in product-A and Base-1 Metal Concentration, mg/L A Base-1 Al 2.74 60.4 Ca 719 420 Fe 24.1 3.19 K 7800 1.55 Mg 337 2.13 Mn 2.11 <1 Na 169 31000 P <1 2.94 Rb 11 <1 Si 318 63000 Zn 3.05 <1
Table 2: Inorganic anions in product-A and Base-1 Metal Concentration, mg/L A Base-1 Cl- 1150 14.5 NO 3- Not detected Not detected SO 4 2- 664 27.8
Figure 3: Surface tension test results of products A, B and SDD Vol. 18 [2013], Bund. R 3888
For example with 7 psi deviator stress the increase in resilient modulus in soil-I was 3 % to 10 % for enzyme-A and 55 % to 85 % for enzyme-B. Similarly for the same deviator stress the increase was 51 % to 61 % with enzyme-I and 57 % to 137 % with enzyme-B for soil-II. It shows that enzyme-A was not very effective in highly expansive soil (PI of soil-I =52%) whereas enzyme-B produced significant improvement in both soils. Tri-axial shear tests were carried out on two confining pressures (4psi and 8psi). On average Enzyme-A increased the shear strength of soil-I by 9%, and by 23% the shear strength of soil II. On the other hand enzyme B increased the strength by 31% for soil I and 39% for soil II. It was also concluded that resilient modulus for all combinations of soil (I and II) and enzyme type (A, B) increases with curing time. They recommended that more mixtures of soils and enzymes be tested and laboratory data should be compared with field data. They also recommended 4 months curing time to achieve improvement in shear strength. Shankar et al. (2009) studied the effect of different dosages of Bio-Enzymes on Lateritic soil of Dakshina Kannada (district of India), having liquid limit and Plasticity Index more than 25% and 6% respectively. Tests were conducted on lateritic soil by adding different percentages of sand as well. They concluded that there is medium improvement in physical properties of lateritic soil. Therefore it was suggested that effect of Bio-Enzyme on soil should be examined in laboratory before actual field application. Higher dosage (200ml/2m 3 of soil) produced 300% increase in CBR, 450% in unconfined compressive strength and permeability was reduced by 42% after four weeks of curing. It was also observed that enzyme is not effective for cohesion less soil. Venkatasubramanian & Dhinakaran (2011) conducted tests on three soils with varied properties and different dosages of Bio-Enzyme. Three soils had liquid limits of 28, 30 and 46% and plasticity index of 6, 5 and 6%. Increase in unconfined compressive strength and CBR after 4 weeks of curing was reported as 152 to 200% and 157 to 673% respectively. Rauch et al. (2003), measured effects of three liquid stabilizers (Ionic Stabilizer, Enzyme Stabilizer, Polymer Stabilizer) on five different soils. Two soils were natural clays of high plasticity and three were composed of predominately one clay mineral: kaolinite, illite, and sodium montmorillonite. The enzyme was proprietary, concentrated, biodegradable, nonbacterial, multi- enzymatic formulation claimed to increase soil density, reduce compaction effort, improve bearing capacity, and lower soil permeability. The analytical tests to characterize the enzymes included pH, potentiometric titration, total organic carbon (TOC), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), high-performance liquid chromatography-mass spectroscopy (HPLC/MS), fast atom bombardment (FAB), and UV/Vis spectroscopy. The pH and conductivity for the enzyme stabilizer diluted 1:10,000 were 3.26 and 0.791 mS/m, respectively. This value of conductivity is in the range of high purity water. It was found through potentiometric titrations that the stabilizer was a weak acid with an acidity constant greater than Ka =10-5. Sample consisted of a 0.26N acid. The TOC of the concentrated enzyme stabilizer was 370,000 mg/L. This result suggests a very large organic carbon fraction in the enzyme stabilizer. FTIR was conducted to identify organic carbon functional groups present in the enzyme stabilizer and results shown in Table 3 are based on the J ournal of Optical Society (1988), which was further verified by NMR. HPLC/MS, FAB, UV/Vis spectroscopy suggested the presence of polyethylene glycol (C2H4O). The treated and untreated clay materials were characterized using BET surface area analysis, cation exchange capacity (CEC), environmental scanning electron microscopy (ESEM) and X-ray diffraction (XRD). Vol. 18 [2013], Bund. R 3889
BET surface area technique quantifies external surface area and pore size. BET nitrogen adsorption results for treated and untreated samples showed a significant reduction in surface area for all of the clay minerals and soils tested. These results suggest that the enzyme stabilizer caused a substantial amount of agglomeration of the soil particles regardless of the nature of the soil material, which again is consistent with the proposed mechanism. ESEM images for the treated and untreated samples also confirmed that the treated samples appear more aggregated than the corresponding untreated sample, and the clay features are less visible. Ionized water can then form linkages between packed particles to provide a cementation effect. In the presence of the enzyme stabilizer the d-spacing of the montmorillonite sample is similar to the d-spacing obtained for an untreated glycolated sample. This result suggests that the enzyme has penetrated the inner layer of the montmorillonite and forced it into an expanded state in accordance with the proposed mechanism. Whereas there was no change in the d-spacing for kaolinite and illite which means enzyme was not able to penetrate into the inner layer. This result suggests that upon application of the enzyme to expansive clay such as sodium montmorillonite, the clay layers fully expand.
Table 3: Interpretation of peaks in FTIR spectra for the enzyme stabilizer (J ournal of Optical Society, 1988) Wavelength (cm -1 ) Functional Group 1,120 C-O 1,220 tertiary butyls 1,350 C-OH 1,300 CH2=CH, CH-OH 1,460 C-H bend
ESEM images for the treated and untreated samples of the clay minerals showed that all of the clay and soils analyzed except possibly the kaolinite, the treated samples appear more aggregated than the corresponding untreated sample, and the clay features were less visible. Cation exchange capacity (CEC) was measured for untreated and treated samples of montmorillonite at three application mass ratios: 1:4, 1:1,000 and 1:6,000. There was no impact of the enzyme stabilizer on the CEC of the montmorillonite sample at any of the application mass ratios examined indicating that the expanded inner layer is still accessible for cation exchange. Overall (in terms of Atterberg limits, density, shear strength and swell potential) no marked improvement was observed, yet there were improvements in individual cases. It was suggested that a higher dosage than recommended by the supplier might produce significant benefits. Brandon et al. 2010_ENREF_2_ENREF_2 conducted Atterberg limits, density, strength and R-value test on a commercially available enzyme called Permazyme on six single source and three blended soils. It was suggested by the enzyme company that the optimum temperature is above 10 o F for a curing time of 72 hrs and that the temperature above 120 o F reduces the enzyme activity. Also Vol. 18 [2013], Bund. R 3890
the presence of strong bases and acids reduce the enzymes effectiveness. The gradation curves for the six single source soils are shown in Figure 4. The summary of Atterberg limits, increase in dry density, friction angle, cohesion and R-value are given in the Table 4. The liquid limit and plastic limit tests for treated and untreated soils were carried out only on three soils (soil-1, soil-2, and soil-6). There was decrease in plasticity index for soils-1 and soil-2. Whereas an increase of 44% in plasticity index of soil-6 was observed. By looking at the Table 2 it can be easily said that there is no definite trend in the treated and untreated soil properties except for cohesion. The increase in cohesion was observed between 6 to 64%. This increase in cohesion advocates the claim of the enzymes supplier that these products agglomerate the soil particles.
Figure 4: Gradation summary Peng et al. (2011) conducted unconfined compression tests on three soils; fine-grained, silty loam and coarse grained textures named as Soil I, Soil II and Soil III respectively. Three soils were stabilized with quicklime and an enzyme (Perma-zyme). The samples were cured up-to 60 days in two different conditions; air-dry and in sealed container. In air-dry curing the samples were allowed to dry at room temperature where as in sealed container the moisture was preserved in the samples during the curing time. Figures 5 and 6 summarize the results for two stabilizers for air-dry and sealed curing. The enzyme was found more effective in air-dry curing for Soil I and Soil II than quicklime where as it was not effective for Soil III in air-dry curing and for three soils in sealed curing too. In sealed containers, the quicklime was found more effective than the enzyme as the water in the specimens was not allowed to evaporate which promoted the further hydration of quicklime.
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Figure 5: Relation of unconfined compressive strength and curing time under air-dry condition
Figure 6: Relation of unconfined compressive strength and curing time in sealed glass containers
CONCLUSION The above studies show varying results from very modest improvement to significant improvement. Therefore the published claims or results by the manufacturer of these enzymes cannot be trusted without independent laboratory testing. But at the same time the laboratory tests are criticized for not replicating the field conditions. But if the product does not show significant results in the controlled laboratory conditions then it is more difficult to attain desired results in lesser favorable conditions in the field. Its only when laboratory testing shows significant results then the next question would be how much improvement is required to validate its use in the field.
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2. Brandon, F., Ding, C., Gary, H. & Charles, R. 2010. "Permazyme testing volume i: final testing summary report". California State University. 3. Hitam, A., Yusof, A. Z. & Samad, O. 1999. "Soil stabilizer for plantation road". National Seminar on Mechanization in Oil Palm Plantation, hlm. 4. Ivanov, V. & Chu, J . 2008. "Applications of microorganisms to geotechnical engineering for bioclogging and biocementation of soil in situ". Reviews in Environmental Science and Bio/Technology 7(2): 139-153. 5. Kucharski, E., Winchester, W., Leeming, W., Cord-Ruwisch, R., Muir, C., Banjup, W., Whiffin, V., Al-Thawadi, S. & Mutlaq, J . 2005. "Microbial biocementation". Patent Application WO/2006/066326. International Application No. PCT/AU2005: 6. Lacuoture, A. & Gonzalez, H. 1995. "Usage of organic enzymes for the stabilization of natural base soils and sub-bases in bagota". Pontificia Universidad J evariana, Faculty of Engineering: 7. Marasteanu, M. O., Hozalski, R., Clyne, T. R. & Velasquez, R. 2005. "Preliminary laboratory investigation of enzyme solutions as a soil stabilizer". Minnesota Department of Transportation, Research Services 8. Milburn, J . P. & Parsons, R. 2004. "Performance of Soil Stabilization Agents". Kansas Department of Transportation. 9. Norris, R., Ryan, L. & Acaster, D. 2011. Cambridge International AS And A Level Chemistry Coursebook. Cambridge University Press, UK, pp. 413-414.: 10. Peng, H. T., Su, H. T., Zhang, X. P. & Wang, J . 2011. "An experimental comparison of compressive strengths of soils stabilized with enzyme and ground quicklime". Advanced Materials Research 280: 9-12. 11. Prescott, C. N. 2001. "Chemistry: A Course for 'O' Level. Workbook". Times Media Private Ltd. Singapore, pp. 154. 12. Rauch, A. F., Harmon, J . S., Katz, L. E. & Liljestrand, H. M. 2002. "Measured effects of liquid soil stabilizers on engineering properties of clay". Transportation Research Record: J ournal of the Transportation Research Board 1787(1): 33-41. 13. Rauch, A. F., Katz, L. E. & Liljestrand, H. M. 2003. "An analysis of the mechanisms and efficacy of three liquid chemical soil stabilizers: volume. Work: 1". Center for Transportation Research, The University of Texas at Austin. 14. Scholen, D. E. 1992. "Non-standard stabilizers". Report FHWA-FLP-92-011. FHWA, U.S. Department of Transportation 15. Shankar, A. U., Rai, H. K. & Mithanthaya, R. 2009. "Bio-Enzyme stabilized Lateritic Soil as a Highway material". Indian Roads Congress J ournal: Vol. 18 [2013], Bund. R 3894
16. Shukla, M., Bose, S. & Sikdar, P. 2003. "Bio-enzyme for stabilization of soil in road construction a cost effective approach". IRC Seminar Integrated development of rural and arterial road networks for socio-economic development, New Delhi, hlm. 5-6. 17. The Carbon Group, L. "Perma-Zyme 11x soil stabilization for road construction and natural liners". 18. Venkatasubramanian, C. & Dhinakaran, G. 2011. "Effect of bio-enzymatic soil stabilization on unconfined compressive strength and california bearing ratio". J ournal of Engineering and Applied Sciences: 6(5):295-298.