Strength and microstructural behaviour of sand Kaolin mixtures stabilized with terrazyme and Xanthan gum

Abstract

This study evaluates the efficacy of TerraZyme (bioenzyme) and Xanthan Gum (biopolymer) in improving the mechanical behavior of Sand-Kaolin mixtures for sustainable soil stabilization. Sand and 15% Kaolin blend were treated with different additive dosages, and the specimens were evaluated via static triaxial testing under unconsolidated undrained conditions, the results are supported by further microstructural and chemical spectroscopy analyses. The optimum dosages found to be 0.075 mL/kg for TerraZyme and 1% (w/w) Xanthan Gum for increasing the shear strength up to 2.5 times after curing for 30 days. TerraZyme facilitated biochemical interaction, forming cemented bonds between sand particles and Xanthan Gum connected the particles via a polymeric network improving interparticle bonding. The results reveal that the use of bioenzymes and biopolymers in stabilization is an environmentally friendly, low-carbon option for ground improvement compared to that of chemical stabilizers.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-38011-x.

Introduction

Massive structures may appear impressive above ground level, but they often hide crucial geotechnical problems below them, which may lead to huge disasters until the potential trigger occurs. Natural causes of geotechnical issues include weak or fractured rocks, fluctuating groundwater levels, soil erosion, seepage, sinkholes, and unstable soils. Structural foundation instability arises from the unpredictable nature of the earth, compounded by natural disasters like landslides and earthquakes, causing liquefaction and flow failures¹. Excessive excavation for construction without proper planning and supervision also leads to soil failures. Traditional soil stabilization methods², such as mechanical mixing and chemical additives like cement and lime, have significant environmental drawbacks, including high energy use and groundwater contamination^2,3. The cement industry in India alone emitted 164 million metric tons of CO₂ in 2022, contributing 7–8% of global pollution⁴. Establishing sustainable ground improvement solutions is essential to minimize losses and protect the habitat⁴.

For a sustainable future, the geotechnical engineering field recognizes soil as the crucial medium for participating in the ongoing global green revolution. Harnessing the power of nature method. Strengthening soil by employing microorganisms is categorized as a bio-geotechnical stabilization method, among various other innovative ideas. Bioclogging and biocementation are vital terminologies in defining the possible mechanism behind modifying soil characteristics in bio-geotechnical engineering⁵. In that list, soil binding with precipitated calcite by the Microbially Induced Calcite Precipitation (MICP) method and biodegradable binding via biopolymers are effective for fine-grained soils^6–9. The Placement of high tensile materials as reinforcement in weaker soil (Geosynthetic-reinforced soil)^8,10 and reusing industrial waste such as fly ash¹¹, ground furnace slag¹², etc. as soil stabilizers with long-term benefits are few other effective, sustainable alternatives. Even the utilization of electric currents from renewable energy sources (electrokinetic stabilization)^13,14 in soils and plant roots (phytoremediation) for contaminated soils¹⁵ demonstrates the versatility of natural resources in different applications.

Biocementation is an innovative ground improvement technique that involves the precipitation of calcium carbonate to enhance the stability of the soil. Although these bio-based stabilization methods fall within the same sustainable framework, their underlying mechanisms differ fundamentally. In biocementation (MICP/EICP), microbial or enzymatic urea hydrolysis induces CaCO₃ precipitation, generating rigid mineral bridges between soil particles. Bioenzyme stabilization, such as TerraZyme (TD), does not rely on mineral precipitation; instead, enzyme activity modifies particle surface chemistry, reduces the thickness of the adsorbed water layer, and improves compaction behaviour. Under favorable conditions particularly in soils containing sufficient fines like Kaolin enzymes may further promote the formation of C-S-H like gel phases that coat particles, enhance bonding, and partially clog pore spaces. In contrast, biopolymers such as Xanthan Gum (XG) follow a physical mechanism: hydration and gelation produce a continuous hydrogel network that coats grain surfaces and creates polymeric bridges across voids, increasing apparent cohesion without altering mineralogy. These mechanistic differences justify the comparative examination of TD and XG in Sand-Kaolin mixtures presented in this study. These techniques have distinct preliminary procedures that involve either microbial activity or enzymes, including in MICP with Sporosarcina pasteurii¹⁶ and Enzyme-Induced Carbonate Precipitation (EICP) using urease enzymes¹⁷. EICP, while having higher reaction rates, has problems associated with enzymatic stability. However, not all biological stabilization relies on calcite precipitation. Some studies highlight the interparticle bonding and hydrogel network formation due to additives like XG in enhancing the mechanical properties of sand and mitigating wind erosion, with an emphasis on uniformity, efficiency, and cost-effectiveness^18,19. Based on recent research, XG improves strength and stiffness in clayey sand at low dosages, with curing-dependent bonding behaviour²⁰. Similarly, crosslinking XG with guar gum has reduced erosion by over 80% and significantly enhanced durability under wet dry cycles, due to the formation of a more stable hydrogel network across soils of varying plasticity²¹. Together, these studies reflect the increasing adoption of biopolymer-based soil improvement strategies in sustainable geotechnics. XG, a polysaccharide from Xanthomonas campestris, has a stable β-D-glucose backbone with side chains enhancing gelation and stability²². It exhibits pseudoplastic behavior, temperature and pH stability, and interacts with cations, making it useful in oil drilling, construction, and ground improvement²³.

Apart from the suitability of EICP and MICP in clays, bioenzymes derived from fermented plant extracts can decrease the thickness of the adsorbed water layer, thereby improving mechanical properties and making them a versatile additive for fine-grained soils, even under seismic loading conditions^22,23. TD is a fermented extract of vegetable peels and has wide applications in crop cultivation and pavement stability. It requires only low dosages; the optimal dosage is soil-specific but generally lies in a range. As the higher dosages create waste and slipping of particles, a trial lab test is recommended before direct field application^24–26. The surfactant Rhodasurf B1 in TD plays a role in enhancing the efficacy of the enzymes through the reduction in surface tension, improving enzyme penetration, and facilitating better interaction with substrates^27,28. While XG has been widely reported for improving the strength and cohesion of Sandy soils, studies on bioenzyme-based stabilization of sand, particularly using TD, are very limited. Furthermore, no comparative evaluation between a biopolymer (XG) and a bioenzyme (TD) is available in the literature, especially in the context of their distinct stabilization mechanisms. In this study, Kaolin is intentionally incorporated as a reactive fine fraction, as its surface hydroxyl groups provide additional bonding sites that influence both hydrogel network formation (XG) and enzyme-driven surface modification (TD). Moreover, systematic chemical and microstructural investigations supporting the mechanical behaviour of biopolymer- and bioenzyme-treated sand are limited. This study addresses that gap by providing a mechanism-based comparative evaluation. Bio-additive-induced cementation exhibits a low environmental impact and excellent in-situ applicability; however, current challenges such as non-uniform calcite precipitation, questionable long-term durability, and complexity in clay stabilization call for tailored approaches for maximum effectiveness.

Research on biologically stabilized soils demands an in-depth chemical investigation and microstructural study to understand the changes brought about in the behavior of soils after treatment. Such studies, within the realm of mineralogical changes and the development of cementitious bonds, have shed light on many alterations that happened in the soil fabric and findings about the mechanism behind strength improvement. Calcite precipitation formed in sand due to the MICP technique and the bridging effect on strength enhancement is generally studied by various chemical analyses^29,30. The need for high vacuum conditions and the drawback of providing only surface-level compositional information³¹ necessitate advanced investigations to get optimized results and better predictions on its long-term effect³².

Herein, this study investigates the effectiveness of biopolymer and bioenzyme stabilization for improving the strength of sand and understanding the role of interparticle bonding. Figure 1 shows the additives used and their structures along with the Kaolin and river sand to improve soil strength. Kaolin has a chemically stable structure from hydrous aluminum silicate (Al₂Si₂O₅(OH)₄), with repeated layers of tetrahedral silica and octahedral alumina sheets, which contributes to its low reactivity, resistance to swelling, and small particle size. The exposed hydroxyl groups of Kaolin at the edges can interact with polymers through hydrogen bonding or electrostatic interactions. This improves the reactivity of Kaolin with biopolymers, making it a suitable additive for stabilization applications. A critical, state-of-the-art, spectroscopic, chemical, and microstructural analysis that covers in this paper, explains the complex processes of Bio-additive-induced cementation in sand using natural additives that fill up gaps in existing literature and offer valuable insights for sustainable geotechnical engineering practices.

Fig. 1.

Structures of sand and various additives used for its strength improvement. The figure presents a Quartz-rich river sand, b Kaolin, a stable 1:1 aluminosilicate clay (Al₂Si₂O₅(OH)₄) with reactive hydroxyl edges, c TD, an enzymatic soil stabilizer, shown with its fermentation system and surfactant-like structure with a hydrophilic head and hydrophobic tail, and d XG, a polysaccharide with a β-D-glucose backbone and gel-forming side chains.

Materials and methods

Soil type

Malaysian river sand (commonly used construction-grade sand) is the main sand used in this work. This sand is representative of commercially available river sand supplied from major sand mining regions in Malaysia, including Perak (≈ 4.60°N, 101.07°E), Kuantan (≈ 3.81°N, 103.33°E), and Johor (≈ 1.49°N, 103.74°E), which are well-recognized sources for construction materials. The sand has a specific gravity of G = 2.63, Uniformity coefficient, C_u = 1.82, and Coefficient of curvature, C_c = 0.85. Thus, based on the Unified soil classification system (USCS), such soil is classified as poorly graded sand (SP) according to the American Society for Testing and Materials, ASTM D2487³³. The highly stable crystal lattice made of quartz (Silicon dioxide) making it chemically inert, weather resistant and less reactive to chemical interactions. To investigate the effect of fines on cementation, the additional soil material used is Kaolin powder from Purenso Global Pvt Ltd. Indore, India.

Non-traditional additives used

XG powder, the biopolymer used in the present work (manufacturer: Urban Platter), is food-grade and soluble in cold and warm water. TD, the bioenzyme, from Avjeet Agencies, Chennai, is non-toxic, semi-viscous and water-soluble. XG has a G value of 1.6 at 25 ℃, and that of TD is 1.0-1.1 with a pH of 2.8–3.5.

TD is a commercial fermented plant extract-based formulation with a proprietary biochemical composition. The manufacturer does not disclose its enzymatic profile or substrate specificity, and therefore its enzyme activity (U/mL) could not be quantified using standard assays. This constraint is consistent with previous TD-based studies, where its effectiveness is evaluated through soil level behavioral, chemical, and microstructural responses rather than direct enzyme activity measurement.

Material characterization

BET was employed for specific surface area determination using the equipment BELSORP MAX 11 (MicrotracBEL Corp., Japan) by degassing the samples for impurities. The samples were treated under a high vacuum of 4.381E-5 Pa and N₂ as the adsorptive at low temperatures. BET analysis estimates the specific surface area of materials by gas adsorption, which is crucial in comprehending soil-additive textural characteristics and the effect of additives in stabilizing applications³⁴. ESEM and EDS were performed on the XL-30 ESEM (Thermo Fisher Scientific, USA) and JEOL IT 300 SEM with EDAX^® EDS (JEOL Ltd., Japan), respectively. The two microscopes were operated up to 30 kV for secondary (SE) and backscattered (BSE) electron imaging. Specimens were gold coated for better conductivity and image quality while elemental characterization and mapping were performed via an integrated EDAX^® EDS detector. Microscopic imaging helps identify bonding and surface changes at a microscopic level, revealing mechanistic reasons for improvements. ESEM offers superior imaging for soils, stabilized soils, and biomaterials under variable pressure, preserving hydration and structure with minimal preparation. This enables real-time observation of dynamic processes, aiding in understanding the enhanced stress-strain response in treated soils³⁵. XRD analysis was carried out using the high-resolution Bruker D8 Advance powder X-ray diffractometer (Bruker AXS GmbH, Germany), equipped with a tungsten source, following standard sample preparation methods. The XRD technique is good for the analysis of soils and stabilized cases since it gives comprehensive information on mineral composition and structural changes important for engineering purposes in understanding mechanical properties and performances³⁶. TGA was carried out via STA 2500 (NETZSCH-Gerätebau GmbH, Germany) between 28 °C and 900 °C at a scan rate of 10.0 K/min in an Al₂O₃ crucible after standard calibration for sensitivity and temperature. TGA analyzes weight changes during heating, providing key insights into soil stabilization and additive interactions, including TD and organic decomposition. While it reveals thermal properties and material stability, TGA cannot assess long-term durability or practical performance in treated soils³⁷. FTIR was performed on a Thermo Scientific Nicolet iS50 spectrophotometer (Thermo Fisher Scientific, USA), scanning from 4000 to 400 cm⁻¹ with a resolution of 8 cm⁻¹, by using the XTR-KBr beam splitter, DTGS KBr detector and the Attenuated Total Reflectance (ATR) method for sample preparation ensures accurate results. FTIR identifies chemical changes in biocemented soils, detecting functional groups and minerals like calcium carbonate formed during Bio-additive-induced cementation. It reveals biochemical mechanisms linking microbial activity or cementation to soil property changes through observable spectral peaks³⁸. XPS on fine-dry-desiccated soil samples was conducted on a Thermo Scientific K-Alpha Surface Analysis instrument (Thermo Fisher Scientific, UK) with Source Gun A (X-Ray032 or X-Ray036 at a 400 μm focus) and Flood Gun FG03 Argon ion keeping the Source Gun B off to get precise data for non-conductive soil samples. XPS provides high surface sensitivity, precise elemental and chemical state identification, and excellent spatial resolution, making it ideal for studying biomaterials in soil stabilization. It reveals detailed interactions between biomaterials and soil components, surpassing other spectroscopic methods³². Solid-state NMR spectroscopy was performed via a JEOL ECX 500 FT-NMR (JEOL Ltd., Japan) spectrometer at 500 MHz on finely powdered soil samples packed in a 4 mm zirconia rotor and Magic Angle Spinning (MAS) at 10 kHz with single pulse excitation for aluminum and silicon analysis. NMR provides detailed atomic-level insights into chemical structures, dynamics, and interactions, making it valuable for characterizing solid and liquid samples. Solid-state NMR, particularly with MAS, is crucial for analyzing rigid structures in soils, offering high-resolution spectra by reducing anisotropic interactions³⁹.

For the XPS peak deconvolution, XPSPEAK41, Version 4.1 software was used which can be found at www.wsu.edu/~scudiero. For the illustrations, Adobe Illustrator 2024, 28.2 was used which can be found at https://www.adobe.com/in/products/illustrator.html#modal-hash. For other analysis, OriginPro 2024b, Learning Edition was used which can be found at https://www.originlab.com/. NMR analysis was done using the MNova, 15.0.1 which can be found at https://mestrelab.com/download.

Sample preparation and static triaxial testing

The river sand was air-dried, sieved through a 4.75 mm sieve, and uniformly mixed in its dry state. A Kaolin content of 15% (by dry mass of sand) was selected to ensure a fines fraction above the 7–12% threshold required for effective enzyme particle interaction. Although this fines content is primarily essential for enzymatic stabilization, the same 15% Kaolin level was maintained across all mix variants (including XG-treated and control specimens) to enable consistent and meaningful comparison of their behavior. For samples containing 15% Kaolin powder by dry weight of soil and XG powder, the Sand- Kaolin mixture was first mixed dry. Then, the XG powder was added and mixed dry with the Sand-Kaolin blend to ensure uniform distribution. The optimum moisture content (OMC) was then added, and the mixture was thoroughly hand-mixed⁴⁰ to create a homogeneous wet blend. The wet mixture was stored in a sealed container for one hour before specimen compaction. The corresponding maximum dry densities (MDD) and OMC values, obtained from standard Proctor tests⁴¹ are provided in Table 1 and were used for sample preparation. The increase in MDD with 15% Kaolin is due to fines filling the intergranular voids and producing a denser packing structure, whereas the OMC remains nearly unchanged because the moisture requirement is still governed predominantly by the sand fraction, and 15% Kaolin is insufficient to significantly alter the overall water demand. For samples treated with TD, the required dosage was mixed with water, stirred for five minutes, and then added to the soil mixture. Only the OMC was used to maintain the desired water content in the mixture. Unlike previous studies that used a wet mixing method, where XG powder was dissolved in water to form a gel before being added to the soil mixture often resulting in lumps and non-homogeneity^42,43 the current study adopted the dry mixing method. This approach ensured better control, consistency, and uniformity in the treated samples. Dry mixing also prevents premature gelation and lump formation of XG during hydration, ensuring uniform distribution of the polymer before water addition. Compaction tests indicated that the low dosages of TD and XG used in this study did not noticeably alter the OMC or MDD values of the Sand-Kaolin blend; therefore, the OMC and MDD reported in Table 1 for the base mixes were adopted for all treated specimens.

Table 1.

Summary of Soils, Additives, and Dosages.

Category	Material	Notation	Dosage	MDD (g/ml)	OMC (%)	Remarks	Ref.
Base soil	Sand	S	Pure sand	1.78	10	Used as the primary granular material	-
	Kaolin	K	Pure Kaolin	1.6	25	Used as the primary fine-grained material	-
	Sand + 15% Kaolin	S + 15%K	15% by dry weight of soil	1.97	10	Combination of sand and Kaolin for studying fines addition	-
Bioenzyme additives	TerraZyme	TD	0.02 ml/kg	-	-	Minimum dosage for testing	44,45
			0.04 ml/kg	-	-	-
			0.06, 0.075, 0.08 ml/kg	-	-	Closer dosages to study exact optimum- based on trial tests
			0.1 ml/kg	-	-	Maximum dosage for testing
Biopolymer additives	Xanthan Gum	XG	0.2% by dry weight of soil	-	-	Minimum dosage for testing	46,47
			0.5, 0.75, 1% by dry weight of soil	-	-	-
			1.5% by dry weight of soil	-	-	Maximum dosage for testing

Soil samples were prepared in cylindrical mould (3.8 cm diameter, 7.6 cm height) by compacting in three layers, followed by careful extrusion to minimize friction and disturbance during placement, compaction, and extraction. Each extruded specimen was individually sealed and placed in a desiccator for 30 days of curing under controlled conditions, where the setup prevented external moisture loss without inducing drying—despite common concerns about desiccator suitability for biopolymer-treated soils. While no active drying occurred, limited internal moisture redistribution likely resulted in specimens that were not fully saturated at testing. Details of the soil-additive combinations and curing are presented in Table 2. Water content and specimen dimension checks are carried out on the cured samples to allow further calculations after the mechanical test.

Table 2.

Soil-Additive combinations and testing Plan.

Combination	Details	Curing conditions & Testing details
S + TD	Sand (100%) + TD (0.02, 0.04, 0.06, 0.075, 0.08, 0.1 ml/kg)	Curing at ambient temperature (25 ℃) and humidity for 30 days; Static triaxial testing for evaluating peak deviatoric stress for optimal dosage.
K + TD	Kaolin (100%) + TD (0.02, 0.04, 0.06, 0.075, 0.08, 0.1 ml/kg)
S + K+TD (Fines content study)	Sand + Kaolin (2%, 5%, 8%, 10%, 12%, 15%) + 0.075 ml/kg TD
S + K+TD (Optimum TD study)	Sand + 15% Kaolin + TD (0.02, 0.04, 0.06, 0.075, 0.08, 0.1 ml/kg)
S + K+XG (Optimum XG study)	Sand + 15% Kaolin + XG (0.2%, 0.5%, 0.75%, 1.0%, 1.5%)

A series of static triaxial compression tests under unconsolidated undrained (UU) conditions was performed on all the samples, with and without additives. Triaxial compression test is the most widely used test to determine the strength of the soil specimens under a variety of simulated field stress conditions corresponding to the construction of foundations or rapid loading due to seismic events. Recognizing the potential of TD, the Indian Road Congress has approved its use for subgrade stabilization in road construction⁴⁸. Additionally, roads built using TD additive have been successfully implemented in two districts of Odisha under the Pradhan Mantri Gram Sadak Yojana (PMGSY) scheme⁴⁹. These examples showcase effectiveness of TD in in-situ applications. For the triaxial tests, the cured sample is placed in a rubber membrane, which is secured with O-rings directly on the bottom pedestal within the triaxial equipment base. The loading pad is placed on the top of the sample to transmit the axial load normally to the sample. A 100 kPa confining pressure is maintained inside the water-filled triaxial cell. The load and compression are monitored via load cells and Linear Variable Differential Transformers (LVDT). Shearing occurs at 1.25 mm/min until reaching maximum stress or 20% axial strain, plotting the stress-strain response. All tests follow IS 2720 (Part 11):1993 standards⁵⁰. Figure 2 shows the characteristic shear failure of a loaded specimen with an untested specimen for comparison.

Fig. 2.

Static triaxial cell setup, illustrated on the left, with a photograph on the right showing soil samples under Unconsolidated Undrained (UU) conditions before (top) and after shearing (bottom). The bottom sample displays the inclined failure zone, highlighting the effects of shear stress on the soil structure.

Results and discussions

Triaxial testing results for treated sand

The static triaxial test is a standard test to investigate stress-strain behavior and undrained shear strength of soil and the testing in UU conditions provides data on short-term stability under total stress conditions (without considering the water pressure from the soil pores onto the soil particles). It replicates the possible in-situ stress conditions that develop in the initial phases of construction and immediately after that under the foundation soil. This quick test is very useful for engineers to assess the suitability of a certain soil stabilization method to attain the right mix designs that provide maximum shear strength.

Stabilization of soil using bioenzyme

All samples were subjected to the same testing conditions in static triaxial tests to make the comparison of the stabilization effects on the stress-strain response possible. Tests were conducted using pure sand, pure Kaolin, and Sand-Kaolin blends at various dosages of TD to establish the critical Kaolin content necessary to ensure the required TD effect. The stress-strain plots in Fig. 3 represent the contribution of TD in all the investigated soil additive systems. The trend in the increase in peak deviatoric stress with the addition of TD is in good agreement with those obtained in other studies on virgin Kaolin samples^24,44.

Fig. 3.

Deviatoric stress versus axial strain curves obtained from Unconsolidated Undrained (UU) triaxial tests for different soil TD combinations after 30 days of curing a Influence of TD on pure sand, b Influence of TD on Kaolin, c Effect of Kaolin content on TD-treated sand with a constant TD dosage of 0.075 ml/kg, and d Effect of TD dosage in sand with a constant Kaolin content of 15%.

Sand samples treated with various TD dosages (0.02–0.1 ml/kg) exhibit higher peak and residual stresses, indicating enhanced strength, as shown in Fig. 3a. With its increase, the strengthening effect rises to 0.06 ml/kg TD dosage and then starts to decline. Perhaps the initial cohesiveness conferred by the TD enzyme to strengthen the sand clusters was overpowered by slippage between the excess amount of the TD enzyme. The optimal dosage of TD enhances the strength of sand by 151%. These observations certainly bring into question the minimum clay content concept generally associated with enzymatic soil stabilization (i.e., a minimum fines content of 7–12% is required for soil-enzyme reaction). Interestingly, the enzyme-treated Sand also exhibit more ductile behavior and strain hardening beyond peak stress, as interpreted from Fig. 3a. This means that enzymatically stabilized sand has some resistance against deformation, hence a lower chance of failure with enhanced load-carrying capacity and stability upon the incidence of stress. Pure Kaolin samples similarly exhibited strength gain with an increase in the TD dose up to the optimum dose of 0.08 ml/kg of soil and improved strength of 1.72 times compared with untreated Kaolin Fig. 3b. Prior to the optimum dose, the treated soil exhibited strain hardening after peak stress; beyond the optimum dose, the soil strain-softened and became more brittle.

The Kaolin content is kept constant at 15%, while the dosage of TD varies to study the effect of clay content on enzymatic sand stabilization (Fig. 3d). From the test results, strength increases with the increment in clay content (Fig. 3c), whereas 15% of Kaolin with an optimum dosage of TD equal to 0.075 ml/kg yields a peak stress of 2.43 and 1.72 times of pure sand and sand containing 15% of Kaolin, respectively. The graph indicates that the bearing capacity of soil subjected to TD increases with an increase in the dose, and maximum improvement is obtained at the optimum dose of 429.88 kPa. Beyond the optimum dose, the treated soil becomes brittle and shows strain-softening behavior. The strain softening observed beyond peak stress in TD treated soils is due to the breaking or slippage of bonds within the soil matrix after the maximum strength is reached. While the treatment enhances strength up to the peak, the internal structure may weaken under continued strain, causing a reduction in strength. Additionally, the ductile behavior and strain hardening observed initially may transition to softening beyond peak stress as the internal bonds of the soil are overpowered by further deformation. These results exhibit a linear relationship between the Kaolin content and enhancement in peak stress/strength in the sand TD samples. Beyond 8% fines, the growth rate becomes somewhat striking, as represented in Fig. 4a. Contrary to the general assumption, TD acts not only on clay soil but also on Sand-clay blends. Even in pure sand, there is a consistent and noticeable enhancement of strength that runs against the general notion of theoretical suitability limits (i.e., a minimum fines content of 7–12% is required for soil-enzyme reaction) of bioenzymes^24,44,45. Figure 4b is a comparison of the peak stresses developed with various dosages of TD within each tested soil-additive combination. This indicates that in every soil, there is an optimum dosage of TD at which the improvement in strength is maximum; beyond this, the strength decreases^23,51 While optimum dosages are within the same range for clay-containing soils, pure sand require only lower dosages of TD. This may involve greater reactivity and specific surface area of clay minerals, affecting the performance of the bioenzyme treatment. These results conflict with conventional views on the appropriateness of bioenzymes and provide a good insight into the optimization of TD applications over varied soil matrices, with important implications for geotechnical engineering practices^25,51.

Fig. 4.

Influence of biomaterial dosages and soil type on maximum deviatoric stress observed from static triaxial tests a Impact of Kaolin content on the maximum deviatoric stress in TD treated sand, emphasizing the strength enhancement associated with different Kaolin dosages, b Radar chart displaying the effects of various TD dosages across different soil types, illustrating the variability in maximum deviatoric stress to biomaterial treatment.

Stabilization of soil using biopolymer

These samples were prepared by mixing different dosages of XG in the soil, ranging from 0.2 to 1.5% by dry weight, and were tested after 30 days of curing to gauge the effect on peak stress and the improvement in strength. Sample preparation and curing methods could considerably affect the stress-strain response; thus, this study evaluates the practical performance of XG stabilization under relatable field conditions rather than aiming only at the maximum strength. This enables a valid comparison between the bioenzyme stabilization results reported in the current work and those of the earlier findings under similar sample preparation and curing conditions. The current study investigates the effects of XG-treated sand with 15% Kaolin and concentrates only on the strength tests since there have been previous studies concerning XG-treated pure sand and pure Kaolin samples^52–54.

The stress-strain response from the static triaxial tests (UU) shows that the addition of XG to the sand with 15% Kaolin significantly improved its mechanical properties (Fig. 5). The untreated sand has the lowest peak deviatoric stress, exhibiting rapid strain-softening, while the sand containing only Kaolin showed a slightly higher peak stress. The XG-treated mixtures show an increase in peak stresses, from which a peak reaches about 448.46 kPa at the optimum dosage of 1% XG. Also, the mixtures lead to more strain-hardening behavior. Overall, the failure response showed clear progression, where untreated sand exhibited strain-softening, the S + 15% Kaolin blend showed moderate peak strength with gradual softening, TD treatment increased peak strength with controlled post-peak reduction, and XG treatment promoted a smoother and more ductile strain-hardening response (Figs. 3 and 5). Improvement agrees with previous studies showing that XG could improve soil strength and ductility; hence, it is suitable for applications such as slope stabilization and road subgrade improvement^55,56.

Fig. 5.

Experimental deviatoric stress versus axial strain curves for Soil XG combinations after 30 days of curing under Unconsolidated Undrained (UU) conditions.

This figure illustrates the impact of varying XG dosages on the mechanical response of sand with 15% Kaolin content, highlighting the relationship between XG concentration and deviatoric stress performance.

Figure 6 presents the results of this study, showing the effect of various additive mixtures on peak and residual stresses. The observed trend aligns with findings reported by Latifi et al. (2017)⁵⁷ and Suzuki et al. (2014)⁵⁸. Specifically, the peak and residual stresses reached maximum values for pure Kaolin samples (258 and 252 kPa, respectively), while moist sand exhibited the lowest values (176 and 127 kPa, respectively), making it unsuitable for high- and low-strain applications^57,58. The addition of TD, at an optimal dosage of 0.06 ml/kg, significantly enhanced the peak stress of sand through cohesion and friction angle, contributing to an increase in shear strength according to soil shear strength theory. This was accompanied by minimal residual stress reduction at higher strains, due to increased stiffness and brittleness. A similar peak stress range of 430–448 kPa was recorded for TD-treated Sand with 15% Kaolin and pure Kaolin after 30 days of curing, as well as XG-treated Sand with 15% Kaolin; however, the rate of residual stress reduction was lower for mixtures that included sand and 15% of Kaolin. Pure Kaolin treated with TD demonstrated higher strength with 444 kPa, though its residual stress was lower at 319 kPa. The greater drop in residual strength for the K + 0.08 ml/kg TD mixture can be attributed to the formation of stronger enzyme-induced bonds at peak strength which, upon continued straining, progressively break down, and the Kaolin matrix does not readily reorganize to maintain load leading to a more noticeable reduction in residual strength. The untreated sand exhibits strain-softening, the S + 15% Kaolin blend shows moderate peak strength with gradual softening, TD treatment increases peak strength with controlled post-peak reduction, and XG treatment promotes a smoother and more ductile strain-hardening response. It could be stated that both XG and TD-treated soils with 15% clay are suitable for low and high-strain applications, respectively.

Fig. 6.

Comparison of peak and residual stress for various soil-additive combinations to evaluate strength enhancement and deformation characteristics. The left axis represents peak stress as bars (in grey), while the right axis displays residual stress, indicated by red points on the bar chart.

The peak deviatoric stress of untreated sand was 176.58 kPa, which increased significantly with treatment. For sand treated with TD, the peak stress was 429.88 kPa, reflecting a 143.44% improvement compared to the untreated soil. Similarly, sand treated with XG achieved a peak stress of 448.46 kPa, showing a 153.97% improvement over the untreated soil. Furthermore, the XG-treated Sand exhibited a 4.32% higher strength compared to the TD-treated Sand. This detailed comparison emphasizes the superior performance of XG over TD while highlighting the effectiveness of both treatments in enhancing soil strength. Biopolymers and their bioenzymes exhibit huge potential, which is measured by their significant benefits in many geotechnical applications such as foundation strengthening, slope stabilization, subgrade improvement, and seismic resilience^59–62. The overall increase in peak deviatoric stress is attributed to the denser packing introduced by Kaolin fines, the chemical bonding and surface modification induced by TD, and the hydrogel coating–bridging network formed by XG, which collectively enhance interparticle connectivity and load transfer.

Microstructural and chemical investigations

Brunauer-Emmett-Teller (BET) analysis

The samples were examined for surface area, pore volume, and pore size using the Barrett-Joyner-Halenda (BJH) technique for mesoporous materials and the Horvath-Kawazoe (HK) method for microporous materials. Supplementary Figures S1 to S4 present the graphical data. (Table 3) sand having a specific surface area of 0.65418 m²/g and an average pore diameter of 13.446 nm confirms its minimal microporosity^63,64. As observed in the BJH plot, 15% Kaolin increases the surface area of the Sand-Kaolin matrix to 1.0202 m²/g and the average pore diameter to 19.975 nm, indicating the rise in mesoporosity whereas its HK plot implies minor microporosity. For TD, the surface area improved up to 1.2605 m²/g, and the average pore diameter was 21.085 nm, indicating the structural changes and better packing due to Kaolin. BJH and HK plots presented a minor increase in meso- and micro-porous volumes compared to those for Sand-Kaolin samples possibly due to biochemical interactions of TD. This results in higher physisorption potential through Van der Waals forces⁶⁵, making it a perfect additive in in-situ applications that demand higher adsorption capacity. While BET analysis shows that increased porosity, especially mesoporosity, enhances adsorption, soil engineering concepts suggest that better packing and reduced porosity are associated with higher structural stability and strength⁶⁶. Here, it is important to note that BET captures characterize nano- to meso-scale pores related to gas adsorption, not the larger macro-voids between soil grains that primarily govern mechanical strength. Improvement in strength from stabilization arises mainly from enhanced interparticle bonding and reduced macro-voids, while increased BET mesoporosity may reflect new, smaller pores formed within aggregated structures or within stabilizing gels. Thus, the observed rise in mesoporosity does not conflict with the macro-scale strength improvement; these are distinct pore domains with different influences. The biochemical interactions involved in TD treatment further enhance the interfacial properties, improving adsorption capacity without compromising its structural integrity for specific applications.

Table 3.

BET analysis data of various soil and additive combinations.

Parameters	S	S + 15% K	S + 15% K + TD	S + 15% K + XG	Units
BET plot
Monolayer adsorption volume (Vm)	0.1503	0.2344	0.2896	0.2597	[cm3(STP) g− 1]
Specific surface area (as, BET)	0.65418	1.0202	1.2605	1.1303	[m2 g− 1]
Total pore volume (p/p0 = 0.9900)	0.00220	0.00510	0.00664	0.00901	[cm3 g− 1]
Average pore diameter	13.446	19.975	21.085	31.894	[nm]

Although the primary discussion focused on TD due to its more pronounced mineral-level effects, the BET results for XG-treated soils are also presented in Table 3 and show characteristic pore enlargement associated with hydrogel formation. Further analysis of the sample S + 15% K + XG revealed that the addition of XG significantly increased both the total pore volume and average pore diameter compared to the base sample S, indicating enhanced porosity and larger pore sizes. Specifically, the total pore volume increased substantially, and the average pore diameter more than doubled, suggesting that XG facilitated the formation of larger, more interconnected pores. However, in comparison with the TD addition, there was a slight decrease in the BET surface area, implying that XG may promote pore enlargement, which enhances porosity but reduces the surface area available for adsorption.

High-resolution environmental scanning electron microscopy (ESEM) imaging

The ESEM images show that the microstructural features of the TD-treated Sand (Fig. 7) differ significantly from those of XG-treated soil (Fig. 8). Sand treated with XG exhibits a gel-like matrix that covers the aggregated clusters and forms fibrous bridges, enhancing particle bonding. This fibrous network contributes to higher residual stress and lower compressibility, as observed in the strength results. In contrast, the TD-treated soil, which also contains Kaolin, reveals a flake-like structure, typically associated with Kaolin. This structure might result from the increased absorption and chemical modification of the TD-treated soil, causing the soil matrix to become drier and more brittle. The flake-like structure in TD-treated soils correlates with improved peak strength due to better particle packing and interparticle bonding, though it also leads to lower residual stress in comparison with XG-treated soils. Despite these differences, both treatments result in enhanced bonding, with XG-treated Sand displaying thin fibrous bridges even without Kaolin, indicating improved cohesion and stability. The distinction between the slimy coating and uncoated sand surfaces is based on the surface texture and grain-boundary features visible in the SEM images. In Fig. 7a (untreated sand), the grains exhibit clean and sharply defined surfaces with open pore spaces and minimal interparticle contact. In Fig. 7d (enzyme-treated sand), a thin, film-like coating appears over the sand grains, which smooths the grain edges and forms bridges between adjacent particles, indicating the presence of the enzymatic coating.

Fig. 7.

Environmental Scanning Electron Microscopy (ESEM) images of TD treated soils after 30 days of curing, illustrating microstructural modifications across different soil compositions. The images correspond to a pure sand, b pure Kaolin, c Sand with 15% Kaolin, d Sand treated with 0.06 ml/kg TD, e Kaolin treated with 0.08 ml/kg TD, and f Sand with 15% Kaolin treated with 0.075 ml/kg TD. All samples were gold-coated, imaged at 2000X magnification, with a scale of 20 μm, to highlight morphological changes induced by TD treatment.

Fig. 8.

Environmental Scanning Electron Microscopy (ESEM) images of XG-treated soils, highlighting microstructural variations due to XG treatment. The images correspond to a untreated Sand at 20 μm, b Sand with 15% Kaolin at 20 μm, c pure XG powder at 20 μm, d XG-treated Sand with 15% Kaolin at 20 μm, e XG-treated Sand at 200 μm, and f XG-treated Sand with 15% Kaolin at 200 μm. Images (a–d) were captured at 2000X magnification, while (e–f) were taken at 200X magnification to illustrate the broader structural changes.

It was found that the microstructural features of the XG-treated Sand (Fig. 8) are indeed very different from those of TD-treated soil (Fig. 7). Sand treated by XG has highly aggregated particles with a smooth and cohesive matrix having fibrous and film-like structures binding the sand grains, which is a sign of its excellent performance as a binding agent. On the other hand, the TD-treated soil shows a flake-like structure with closely packed particles; hence, TD improves stability due to increased particle cohesion and interparticle bonding through biochemical reactions. Both treatments enhance the mechanical properties of soil through distinct mechanisms. XG-treated soils develop a gel-like matrix that fills voids, creating a uniform, less porous structure with reduced pore volume and compressibility, as observed in studies on porosity and XG-treated soils^67,68. This cohesive network results in higher residual stress retention, making XG-treated soils suitable for long-term stability. In contrast, TD-treated soils improve particle packing and bonding strength through enzymatic action, which reduces pore volume and increases peak strength and stiffness, as supported by studies on the effect of TD^45,51. XG-treated soils are suitable for long-term stability applications like subgrade stabilization in roads, while TD-treated soils are more suitable for high-strain applications including foundation soils subjected to seismic or rapid loading. Overall, bio-based treatments are versatile for soil stability applicable to a wide range of problems.

Energy dispersive X-ray spectroscopy (EDS) and elemental mapping

Elemental evidence for morphological features like bridging, surface coating, and clusters observed in ESEM images should be provided. It presents the quantification of the percentage of every element within the matrix and the analysis of the spatial distribution of elements. EDS and elemental mapping are used to quantify the amount of each element present within the matrix and investigate their spatial distribution. EDS and elemental mapping are fast, non-destructive ways to analyze elements with high precision. Therefore, it is possible to trace chemicals and compositional changes in soils and treated soils, especially in heterogeneous materials.

The chemical composition from EDS is provided in Fig. 9. Sand is typical of quartz, with high silicon and oxygen and moderate aluminum^69,70. Adding 15% of Kaolin to the sand increases aluminum and oxygen levels, showing the presence of Kaolinite⁷¹. Adding TD to the Sand-Kaolin mixture increases the level of aluminum and oxygen and decreases the level of silicon slightly, and this might indicate that TD enhances the interaction between Kaolin and sand more. Figure 10 shows the elemental mapping of some key elements, such as Al, O and Si, of the three different samples. The elemental distribution of pure sand exhibits a typical quartz composition with moderately scattered Al and highly uniformly distributed Si and O elements and the absence of C. The Addition of 15% Kaolin to the Sand has changed the elemental distribution of Al, which is uniformly distributed by showing the incorporation of Kaolin. O levels are high and more uniform, with a level slightly higher than in pure sand; C content is very low, showing there was very low organic matter.

Fig. 9.

Energy Dispersive Spectroscopy (EDS) data of sand and bio-stabilized soils, identifying major elements such as C, O, Si and Al, along with minor constituents to assess chemical modifications due to bio-stabilization. The spectra correspond to a pure sand, b pure Kaolin, c Sand with 15% Kaolin, d Sand with 15% Kaolin and 0.075 ml/kg TD, and e Sand with 15% Kaolin and 1% XG.

Fig. 10.

Elemental mapping and detected trace element distribution in sand with 15% Kaolin and bio-stabilized Sand, highlighting variations in Al, O, and Si concentrations. The figure presents a Sand with 15% Kaolin, b Sand with 15% Kaolin and 0.075 ml/kg TD, and c Sand with 15% Kaolin and 1% XG. Elemental maps provide spatial distribution patterns of key elements, aiding in the assessment of microstructural changes and interactions due to bio-stabilization. A complete list of detected elements and their percentages is also included.

The most radical changes occur by adding both 15% Kaolin and TD to the sand. Accordingly, Al content rises even more and becomes uniformly distributed, showing further increased Kaolinite. Si remains high yet less uniform, pointing to major changes in the structure of the sand. Most importantly, the levels of O are notably higher (48%) and exhibit a smoother, more uniform appearance in the elemental mapping image, indicating greater binding and interaction in the composite. Moreover, the presence of C slightly increased, perhaps due to the inclusion of the TD additive.

X-Ray diffraction (XRD)

XRD data for sand, Kaolin, and different soil-additive combinations are presented in Fig. 11^72,73. In the sample S + 15% K + 0.075 ml/kg TD, peaks of Kaolinite are still visible, and thus TD acts by modifying the existing mineral matrix without incorporating new compounds. Characteristic peaks of Kaolin at about 12.2°, 20.2°, 24.9°, 35.0°, 38.4°, 45.0°, 62.5°, and 67.5° 2θ, typical of the layered silicate structure in Kaolinite minerals, are observed. The major peaks in sand appear at approximately 26.6°, 36.5°, 39.5°, 42.5°, 50.1°, 60.0°, and 68.2° 2θ, with quartz as the main mineral component. From XRD analysis, TD treatment seems to show structural changes in Kaolin; shifting in peak positions and variation in intensity is evidence of biochemical interaction. These changes can improve crystallinity and decrease defects in the Kaolinite structure. They also illustrate the potential of TD in modifying the physical and chemical properties of Kaolin with the aim of soil stabilization, among others. New peaks or shifts in the principal peak positions in TD indicate the formation of cementitious materials such as C-S-H and Calcium Alumino Silicate Hydrate (C-A-S-H) that increase the bonding and, thereby, strength, as explained earlier in the triaxial test result. In this respect, TD reveals a more significant effect on the crystallinity and stability of the soil compared with XG⁷⁴. EDS and elemental mapping indicate changes in the elemental composition of the soil matrix, which have been confirmed by the detection of probably formed minerals like C-S-H, C-A-S-H in the soil through XRD analysis. All these changes can affect the binding characteristics and strength behavior of the treated soil^72,73. Based on the above findings, additional TGA analysis are conducted to verify their thermal stability and composition. XRD analysis was carried out only on samples containing mineral phases (sand, Kaolin, and TD-treated variants), as XG is an amorphous biopolymer and does not produce characteristic diffraction peaks. Conversely, TGA was performed on both TD- and XG-treated soils to evaluate their thermal decomposition behaviour and quantify the influence of organic additives on mass loss.

Fig. 11.

X-ray Diffraction (XRD) patterns of soil stabilized with TD and XG, presented in a stepped format for clarity. Vertical dotted black lines aid in comparing shift in peak positions. Identified peaks are labeled with corresponding mineral phases, with descriptions provided on the right. From bottom to top, the patterns correspond to sand, Kaolin, Sand with 15% Kaolin, Sand with 0.06 ml/kg TD, Kaolin with 0.08 ml/kg TD, Sand with 15% Kaolin and 0.075 ml/kg TD, pure XG, and Sand with 15% Kaolin and 1% XG.

Thermogravimetric analysis (TGA)

All the TGA data provides a comprehensive understanding of the different thermal properties of various composites based on Kaolin and sand. In this view, TD-treated Kaolin exhibited a high mass loss of 10.22%, having an onset temperature of decomposition at about 541.7 °C with its major mass loss within the temperature interval from 773.8 up to 851.5 °C, which most probably is due to the decomposition of Kaolin or the burning off organic additives (Fig. 12). By contrast, TD-treated Sand has a small mass loss of 0.77%, reflecting high thermal stability with low organic content. The main mass loss is between 541.0 °C and 3055.2 °C, while that of TD-treated Sand + 15% Kaolin has a moderate mass loss of 1.17% due to the fact of its combined thermal characteristics obtained from the Kaolin and sand. The major mass loss in XG-treated Sand with 15% Kaolin is between 463.4 °C and 738.4 °C because of the decomposition of Kaolin and XG. This appears positive, as adding 15% Kaolin to the XG-treated Sand would raise its resistance to temperature degradation⁷⁵.

Fig. 12.

Thermogravimetric Analysis (TGA) curves of TD and XG-treated soils, illustrating thermal stability and weight loss patterns. The plotted curves represent Sand + TD (red), Kaolin + TD (blue), Sand + 15% Kaolin + TD (yellow), and Sand + 15% Kaolin + 1% XG (purple). The blue arrow indicates the dehydroxylation of Kaolin.

A distinguished, multi-dimensional analysis of thermal properties across a series of Kaolin and sand-based composites is presented, examining the effects of additives such as TD and XG. Such a detailed comparative analysis across different material compositions underlines the complex interplay between the constituents and their impact on the overall characteristics of thermal stability and decomposition. FTIR spectroscopy was applied to these samples to gain deeper insight into their molecular structure and functional groups, providing a better understanding of the material properties and their correlation with thermal behavior.

Fourier transform infrared spectroscopy analysis (FTIR)

By observing the shift of peaks in the FTIR spectra (Fig. 13), it is evident that the addition of TD caused significant changes in the vibrational characteristics of sand and Kaolin samples. In the sand samples, the calcite peaks (450–500 cm⁻¹) remain similar to those of the untreated sample; however, a new peak at 503.28 cm⁻¹ indicates that calcium oxide has transformed into another calcium compound. For Kaolin, while calcite peaks are stable, new peaks at 529.52 cm⁻¹, 546.20 cm⁻¹, and 694.73 cm⁻¹ indicate the formation of calcium silicate hydrate (C-S-H) gel or interaction with clay minerals²⁵. Mixtures of sand and Kaolin also show new peaks (e.g., at 517.85 cm⁻¹, 527.91 cm⁻¹) reflecting enhanced chemical interaction and structural change. Si-O stretching vibrations confirm the presence of quartz and silicate minerals, with new vibrational modes around 1431 cm⁻¹ and 1471 cm⁻¹ corresponding to calcium carbonate formation. The FTIR of pure TD is reported in the literature⁷⁶. Based on the reference peaks further analysis and comparison of treated and untreated soil samples were done. The organic components of TD and the surfactant Rhoda B1 contribute to the overall spectral profile, while water-related peaks demonstrate hydration state variations due to calcium chloride hygroscopicity. Specifically, peaks at 875.43 cm⁻¹ (C-O stretching) and 1426.56 cm⁻¹ (carbonate) confirm calcite formation, and peaks in the 500–800 cm⁻¹ region (e.g., 503.28 cm⁻¹) indicate C-S-H gel formation. Peaks within the 3000–3700 cm⁻¹ range further reflect hydration changes and moisture interactions in treated samples. XG treatment also induces calcite formation as evidenced by FTIR shifts between 450 and 500 cm⁻¹ and 1400–1600 cm⁻¹, with more pronounced effects in Sand-Kaolin mixtures. However, weaker or absent peaks in the 500–800 cm⁻¹ and 1000–1100 cm⁻¹ ranges suggest minimal C-S-H gel formation under XG treatment. Notably, the absence of ettringite-indicative peaks and diminished organic matter signals in XG-treated samples align with SEM observations of reduced bacterial degradation, which enhances soil durability^77,78. New peaks and shifts in the 3500–3600 cm⁻¹ and 1600–1700 cm⁻¹ ranges suggest that the XG treatment, along with moisture, promotes the formation of C-S-H gel and alters the hydration states of sand and Kaolin, indicating significant chemical interactions and changes during the curing process.

Fig. 13.

Fourier Transform Infrared (FTIR) spectra of TD and XG-treated soils, analyzed over the 4000 –400 cm⁻¹ range, relevant for soil characterization. The y-axis is in arbitrary units, with a stepped plot for clarity. From bottom to top, the spectra correspond to Sand, Kaolin, Sand + 15% Kaolin, Sand + TD, Kaolin + TD, Sand + 15% Kaolin + TD, Sand + 1% XG, and Sand + 15% Kaolin + 1% XG. Key wave number ranges, vibration types, chemical groups, and corresponding base materials are labeled within the spectra and summarized in the table below the plot.

In the XG-treated soils, the formation of C-S-H gel is confirmed by Ca–O stretching peaks and the presence of Kaolin indicates the enhancement of the hydration process. In the TD-treated soils, peak positions confirm the interaction of calcite with the TD treatment and the subsequent formation of C-S-H gel. The formation of C-S-H gel in both treatments enhance soil strength by binding particles together and improving the overall cohesion and stability of the soil, contributing to the observed strength improvement in soils stabilized with XG and TD⁴³.

X-ray photoelectron spectroscopy (XPS)

The XPS survey of TD and XG-treated soils is given in the supplementary file in Figure S5. Figure 14 shows the Al 2p narrow scan XPS spectra of soil-additive combinations, and the remaining plots of C 1s, Si 2p, and O 1s are given in the supplementary file from Figure S6 to Figure S8. Table S1 shows the chemical composition of soil-additive combinations obtained from XPS as atom% of each major element (Al, Si, O, and C) in each sample. Table S2 to Table S5 shows XPS major compounds that exist in the samples and show a relationship between the peak deviatoric stress and the atom%.

Fig. 14.

Al 2p narrow-scan X-ray Photoelectron Spectroscopy (XPS) spectra of soil-additive combinations, obtained after deconvolution, highlighting variations in aluminum bonding environments. The spectra correspond to a Sand, b Sand + 15% Kaolin, c Sand + 15% Kaolin + TD, and d Sand + 15% Kaolin + XG.

Figure 15 shows functionalities groups (Al 2p) versus peak deviatoric stress for stabilized soils, and the remaining plots of Si 2p, O 1s, and C 1s are given in the supplementary file as Figure S9-S11. Figure 15 demonstrates the three chemical environments observed: aluminosilicate, Al (OH)₃, and Al oxide. The atom % of Al oxide was found least in the sand, but it increased with the introduction of 15% Kaolin. This is because most aluminum in Kaolin occurs as aluminum oxide. Enzymatically treated Sand (S + TD) would have considerable influence on Al (OH)_3, while there were no significant alterations on aluminum silicate^79,80. All the specimens, including treated and untreated soil, contained both silicon types: aluminosilicate and SiO₂. However, these did not enhance strength significantly because the values were the same. This is supported by the EDS analysis shown in Fig. 9. With Kaolin, there was moderate development of C-C bonds; however, with both biomaterials, these bonds were lower, as shown in Table S3. The reduction in O-C = O bonds could be due to the complex post-treatment of soil and additive mixture. Thus, it influences variation in the chemical environment, which tentatively relates to peak deviatoric stress values. XG possesses a natural residue with more C-O-C bonds. This is an indication that it will be effective as a binder, but stability is inferior to that of TD because ether bonds can easily break. This phenomenon verifies the strength reduction under changing moisture conditions (curing) and temperature variations (as evident from Fig. 12). The oxygen bond in Al₂O₃ had minor impacts on all samples. Within sand, most of the oxygen atoms were coordinated to carbon environments, C-O and C = O. Most were less associated with SiO₂. Adding Kaolin decreased carbon-oxygen interactions, which mainly shifted into the SiO₂ environment. For TD-treated soils, this interaction further decreased, with oxygen existing mainly within the SiO₂ environment. This also happened with XG-treated soils, as shown in Table S2.

Fig. 15.

Correlation between Al 2p functional groups and peak deviatoric stress for treated soils. Bar plots (left axis) represent peak deviatoric stress, while point markers (right axis) indicate atomic percentage of Al 2p.

Nuclear magnetic resonance (NMR)

Solid-state NMR has been used in this work due to its ability to describe, in detail, the complex interaction of aluminum and silicon, which actively explains the stabilization mechanism involving biomaterials in soil. The study of these elements will provide valuable information on how amendments can act in improving the structure and stability of the soil, therefore better preparing them for construction applications. It enables the investigation, by solid-state NMR, of chemical shifts and couplings that provide three-dimensional structural and dynamic information. It is thus a very important tool in interpreting mechanisms of action in a wide range of biological and chemical systems^39,68 and ²⁹Si NMR spectroscopy have been documented, as shown in Figs. 16 and 17. It was observed from the NMR spectral data (Fig. 16) that peaks within 60–80 ppm represent the tetrahedral coordination in quartz Sand, and similarly, peaks at around 8 ppm manifested the same coordination. Treatment of the soil with TD caused slight shifts in the position of the aluminum peaks from 61.47 ppm to 60.28 ppm, while silicon peaks remained at -94.15 ppm and − 106.62 ppm in sand containing 15% Kaolin as shown in Fig. 17^81,82. Stability may mean that the silicate network within the soil matrix is strong, hence the high stability of the soil. Put differently, the negative values of chemical shift in NMR spectroscopy reflect a relatively increased electron shielding environment, associated with an electron-rich environment and thus useful as an insight into the electronic structure and bonding nature of the sample.

Fig. 16.

Solid-state ²⁷Al NMR spectra of TD and XG-treated soils, illustrating aluminum coordination changes. Spectra correspond to a Sand, b Sand + 15% Kaolin, c Kaolin, d Sand + TD, e Kaolin + TD, f Sand + 15% Kaolin + TD, g Sand + 0.5% XG, and h Sand + 15% Kaolin + 1% XG. Significant spectral changes are highlighted in yellow boxes with explanations.

Fig. 17.

Solid-state ²⁹Si NMR spectra of TD and XG-treated soils, illustrating changes in the silica environment. Spectra correspond to a Sand + 15% Kaolin, b Sand + 15% Kaolin + TD, and c Sand + 15% Kaolin + XG. Significant spectral changes, their possible causes, and their impact on the soil matrix are highlighted in yellow boxes with explanations.

With XG treatment, new peaks at 57.55 ppm and 7.92 ppm for aluminum (Fig. 16) and one peak of silicon at 84.26 ppm (Fig. 17) were introduced, indicating some chemical interactions, which strengthen the cohesion of the soil. The peak at 77.19 ppm in the mixture of sand and Kaolin indicates the formation of a specific aluminum site from interactions between the sand and the Kaolin. It is believed that a shift from 60.21 to 58.36 ppm for TD characterizes minor structural changes. Surprisingly, XG shifts the aluminum peak more from 58.73 ppm in the sand to 57.55 ppm in the Sand-Kaolin mixture with higher chemical stabilization than TD. These results have shown that while the silicon environment is almost insensitive to additives, the environment of aluminum significantly benefited from the addition of Kaolin due to the increase in bonding sites with biomaterials. Accordingly, the strength of the sand with 15% of Kaolin increases drastically regardless of other additives.

Mechanistic assumptions

The dominant constituent of sand is silica, which has a poor binding capacity. This is improved by the addition of aluminum-based Kaolin, which elevates the number of possible binding sites in the sand, resulting in improved bonding between particles as interpreted from XPS data. Chemical analysis shows that strength is highly influenced by alumina, as interpreted by EDS and elemental mapping. TD consists of two parts: a hydrophilic “head” and a hydrophobic “tail,” functioning similarly to a surfactant (Fig. 18a). While the hydrophilic head interacts with the Kaolin-Sand structure and develops strength, the silica in the sand binds with the hydrophobic tail of TD, which reflects on the strain softening behavior of the stabilized soil as observed in triaxial test results past the peak value obtained. XPS analysis from the study demonstrates a reduction in carbon oxygen interactions (such as C–O and C = O bonds) and a concurrent increase in oxygen associated with the SiO₂ environment following TD treatment; this shift substantiates the hypothesis that the hydrophobic tail of TD preferentially associates with silica surfaces, thereby diminishing the soil’s affinity for water and enhancing its hydrophobic nature. These findings interpret the decrease in the thickness of the adsorbed water layer caused by the breakdown of the electrostatic potential barrier around silica which in turn liberates previously adsorbed water, allowing it to convert to free water and subsequently evaporate or wash away²⁴.

Fig. 18.

(a) Proposed Mechanism of TD interaction with sand containing 15% Kaolin. The figure illustrates the surfactant structure, featuring a hydrophilic head and hydrophobic tail, demonstrating how TD bonds with the silica and alumina components of the sand and Kaolin soil matrix. This interaction enhances soil stabilization and strength. (b) Proposed Mechanism of XG interaction with sand containing 15% Kaolin. This figure depicts the weak hydrogen bonding present with the XG and Sand and Kaolin soil matrix.

XG is a highly hydrophilic biopolymer that primarily interacts with mineral surfaces through physical mechanisms, rather than direct chemical bonding⁷⁴. In sand-only systems, XG does not exhibit strong, specific interactions with the silica matrix due to the inert nature of quartz surfaces. XG can interact through relatively weak hydrogen bonding and electrostatic effects⁸³. As illustrated in Fig. 18b, XG forms physical networks within the Sand-Kaolin mixture, relying on hydrogen bonding with both sand particles and Kaolin. This mode of interaction is well documented showing XG establish hydrogen bonds with the mineral surfaces, especially with the gibbsite layers in Kaolin^83,84. The formation of hydrogel bridges between soil particles supports aggregation and fills pore spaces, leading to microstructural changes observable by ESEM but without a significant alteration in the silica environment of the sand^74,85,86. Therefore, only physical bonding was present in the sand treated by XG as observed in ESEM images, with no change in the silica environment, which was supported by the results of solid-NMR analysis.

Field implementation and limitations

Although long-term durability and cyclic loading effects are important considerations for field-scale soil stabilization, these aspects fall outside the scope of the present investigation, which focuses on short-term behavior and mechanistic understanding. The authors are currently conducting extended studies on durability and cyclic loading as part of ongoing research, and the outcomes will be reported separately. Nevertheless, the field cases referenced in this work such as TD-treated sections implemented in PMGSY road projects demonstrate promising long-term performance under real conditions, supporting the practical relevance of the findings presented here.

For shallow field applications, the sand can be modified by incorporating 15% Kaolin to improve its reactivity and bonding characteristics. Based on the optimal dosages identified in this study, TD can be applied at 0.075 mL/kg of soil, while XG can be added at 1% of the dry soil mass, depending on the required enhancement in strength and cohesion. These treatments can be introduced through shallow in-situ mixing, where the soil is loosened and blended using graders or tractor-mounted rotavators to achieve uniform distribution. After mixing, the treated material should be adjusted to a moisture level close to its optimum and compacted in thin layers using standard rollers. Although shallow mixing is practical and compatible with existing construction equipment, deep mixing remains challenging because hydrated biopolymers can clog grout pipes and injection nozzles, reducing their effectiveness at depth. Consequently, the proposed dosages and mixing approach are most suitable for near-surface stabilization in applications such as subgrades, embankments, and pavement foundation layers.

Although this study used standard triaxial samples (3.8 cm diameter × 7.6 cm height), it is important to recognize that laboratory results may not always fully represent field-scale behavior. In field conditions, larger soil volumes can exhibit variations in compaction, moisture distribution, and loading not present in laboratory specimens. Therefore, while the lab results indicate a clear improvement in soil strength with the stabilization methods described, further pilot studies at field scale are necessary to confirm performance and develop application guidelines for real engineering projects.

Despite their sustainability advantages, both TD and XG present certain limitations relevant to field applications. TD is highly dosage-sensitive and requires sufficient fines for effective enzyme particle interaction; its efficiency decreases in soils with very low clay content or high organic matter. Excess dosages may also cause particle slippage. XG, although improving cohesion and ductility, is susceptible to softening under prolonged saturation and wetting drying cycles unless supported by reactive fines such as Kaolin. Hydrated XG gels can clog injection systems, limiting deep mixing applications. The long-term durability of both additives may vary with environmental conditions, microbial activity, and moisture regimes. These considerations are crucial when evaluating their applicability for large-scale or long-term stabilization projects. Figure 19 shows the prospects of bio-stabilization methods using natural additives like TerraZyme and Xanthan gum for sustainable ground improvement.

Fig. 19.

The prospects of bio-stabilization methods using natural additives like TerraZyme and Xanthan gum for sustainable ground improvement.

Conclusions

This study confirms that biopolymers and bioenzymes significantly enhance the physical, chemical, and mechanical behavior of Sand–Kaolin mixtures, particularly when Kaolin is incorporated. Kaolin played a crucial role in increasing reactivity and bonding effectiveness, as reflected in the observed improvements in peak deviatoric stress. Microstructural analyses established increased micro- and mesoporosity, with TD-stabilized soils showing a more reactive structure. Spectroscopic analyses (XPS, EDS, NMR) confirmed that alumina-rich Kaolin enhances chemical interactions with both biomaterials. Both biomaterials remained thermally stable in Kaolin-containing sand at elevated temperatures; however, XG showed greater degradation without Kaolin, whereas TD was sensitive to Kaolin content. The dual mechanism of TD chemical bonding via its hydrophilic head with Kaolin and physical binding via its hydrophobic tail with silica contributed to enhanced bonding and strain-softening behavior. Small quantities of Kaolin significantly enhanced strength and bonding characteristics in Sandy soils. Both TD and XG achieved up to a 2.5-fold strength increase within 30 days. TD has demonstrated field-scale effectiveness in PMGSY road projects, reinforcing its potential for broader soil stabilization. Future work should explore additional bioenzymes, testing them under variable environmental conditions, and expand field trials.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Geethu Thomas: Data curation, Methodology, Lab Investigation, Funding acquisition, Software, Visualization, Writing – original draft. Ripsa Rani Nayak: Interpretation of data, Software, Graphical illustrations, Writing – review and editing. Navneet Kumar Gupta: Supervision, Interpretation of the mechanism, Writing – review and editing. Madhavi Latha Gali: Conceptualization, Supervision, Resources, Writing – review and editing.

Data availability

Data are provided within the manuscript or supplementary information files, and additional experimental data are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.