Facultative mycorrhization in a fern (Struthiopteris spicant L. Weiss) is bound to light intensity.

BACKGROUND: The establishment of mycorrhizal relationships between a fungus and a plant typically enhances nutrient and water uptake for the latter while securing a carbon source for the fungus. However, under a particular set of environmental conditions, such as low availability of light and abundant nutrients in the soil, the resources invested in the maintenance of the fungi surpass the benefits obtained by the host. In those cases, facultative mycorrhizal plants are capable of surviving without symbiosis. Facultative mycorrhization in ferns has been overlooked until now. The present study measured the response of Struthiopteris spicant L. Weiss, and its root-associated fungi to different levels of light and nutrient availability in terms of growth, mycorrhizal presence, and leaf nutrient content. This fern species exhibits a great tolerance to variable light, nutrient, and pH conditions, and it has been found with and without mycorrhizae. We conducted a greenhouse experiment with 80 specimens of S. spicant and three factors (Light, Phosphorus, and Nitrogen) resulting in eight treatments. RESULTS: We found a significant influence of the factor light on fungal community composition, plant biomass, and nutrient accumulation. Departing from a lack of colonization at the initial stage, plants showed a remarkable increment of more than 80% in the arbuscular mycorrhizal fungi (AMF) richness and abundance in their roots when grown under high light conditions, compared with the ones in low light. We also observed an upward trend of C:P and C:N ratios and the above- and belowground biomass production when AMF abundance increased. Furthermore, the compositional analysis of the whole fungal communities associated with S. spicant roots revealed clear differences among low-light and high-light treatments. CONCLUSIONS: This study is the first to investigate the importance of light and nutrient availability in determining fern-AMF relationships. We confirmed that Struthiopteris spicant is a facultative mycorrhizal plant. The composition and diversity of AMF found in the roots of this fern are strongly influenced by light and less by nutrient conditions. Our study shows that ferns respond very sensitively to changes in environmental factors, leading to shifts in the associated mycorrhizal communities.

Injection strategy for effective bacterial delivery in bioaugmentation scheme by controlling ionic strength and pore-water velocity.

For the in-situ remediation of the contaminated subsurface environment, the injection of nutrients and microorganisms changes chemical and physical conditions, which control the delivery and immobilization of microorganisms. We investigated the injection strategy for effective bacterial delivery in a bioaugmentation scheme by controlling ionic strength (IS) and pore-water velocity (v). A set of bacterial transport tests was conducted using the saturated sand column to mimic the saturated subsurface environment. The effectiveness of the injection strategies was evaluated by applying solutions with different ionic strengths into the sand column with different pore-water velocities. The deposition and delivery of bacteria through the sand column were analyzed using the first-order deposition model. The deposition and delivery of bacteria injected by various strategies were numerically simulated considering the variable deposition rate. The breakthrough curves from column experiments revealed that the bacterial deposition on the sand surface was increased by an increase in the ionic strength and by a decrease in the pore-water velocities. The rates of bacterial deposition (k1) on sand could be determined as a function of ionic strength and pore-water velocity, and it was applicable to simulate the delivery of bacteria under dynamic groundwater conditions. The numerical case study considering various injection strategies showed that the nutrient concentration controlled the bacterial delivery to the target area more significantly than the injection flow rate. Injection of bacterial solution with lower nutrient concentration could be increased the deposited bacterial concentration at the target point (Stp) by 6.2-7.1 times higher. Short pulse injection with a high injection rate decreased Stp by 67-78%. The efficiency of bacterial delivery (Ed) could be increased three times higher by lowering nutrient concentration in the injection solution. The process of evaluating the efficiency of bacterial delivery could be a prominent approach to determining the injection strategy for in-situ remediation considering variable conditions of a contaminated site.

Lasing Action from Anapole Metasurfaces.

We study active dielectric metasurfaces composed of two-dimensional arrays of split-nanodisk resonators fabricated in InGaAsP membranes with embedded quantum wells. Depending on the geometric parameters, such split-nanodisk resonators can operate in the optical anapole regime originating from an overlap of the electric dipole and toroidal dipole Mie-resonant optical modes, thus supporting strongly localized fields and high-Q resonances. We demonstrate room-temperature lasing from the anapole lattices of engineered active metasurfaces with low threshold and high coherence.

Polarization Control of Deterministic Single-Photon Emitters in Monolayer WSe2.

Single-photon emitters, the basic building blocks of quantum communication and information, have been developed using atomically thin transition metal dichalcogenides (TMDCs). Although the bandgap of TMDCs was spatially engineered in artificially created defects for single-photon emitters, it remains a challenge to precisely align the emitter's dipole moment to optical cavities for the Purcell enhancement. Here, we demonstrate position- and polarization-controlled single-photon emitters in monolayer WSe2. A tensile strain of ∼0.2% was applied to monolayer WSe2 by placing it onto a dielectric rod structure with a nanosized gap. Excitons were localized in the nanogap sites, resulting in the generation of linearly polarized single-photon emission with a g(2) of ∼0.1 at 4 K. Additionally, we measured the abrupt change in polarization of single photons with respect to the nanogap size. Our robust spatial and polarization control of emission provides an efficient way to demonstrate deterministic and scalable single-photon sources by integrating with nanocavities.

Generation of unequal nuclear genotype proportions in Rhizophagus irregularis progeny causes allelic imbalance in gene transcription.

Arbuscular mycorrhizal fungi (AMF) form mutualisms with most plant species. The model AMF Rhizophagus irregularis is common in many ecosystems and naturally forms homokaryons and dikaryons. Quantitative variation in allele frequencies in clonally dikaryon offspring suggests they disproportionately inherit two distinct nuclear genotypes from their parent. This is interesting, because such progeny strongly and differentially affect plant growth. Neither the frequency and magnitude of this occurrence nor its effect on gene transcription are known. Using reduced representation genome sequencing, transcriptomics, and quantitative analysis tools, we show that progeny of homokaryons and dikaryons are qualitatively genetically identical to the parent. However, dikaryon progeny differ quantitatively due to unequal inheritance of nuclear genotypes. Allele frequencies of actively transcribed biallelic genes resembled the frequencies of the two nuclear genotypes. More biallelic genes showed transcription of both alleles than monoallelic transcription, but biallelic transcription was less likely with greater allelic divergence. Monoallelic transcription levels of biallelic genes were reduced compared with biallelic gene transcription, a finding consistent with genomic conflict. Given that genetic variation in R. irregularis is associated with plant growth, our results establish quantitative genetic variation as a future consideration when selecting AMF lines to improve plant production.

Holobiont chronobiology: mycorrhiza may be a key to linking aboveground and underground rhythms.

Circadian clocks are nearly ubiquitous timing mechanisms that can orchestrate rhythmic behavior and gene expression in a wide range of organisms. Clock mechanisms are becoming well understood in fungal, animal, and plant model systems, yet many of these organisms are surrounded by a complex and diverse microbiota which should be taken into account when examining their biology. Of particular interest are the symbiotic relationships between organisms that have coevolved over time, forming a unit called a holobiont. Several studies have now shown linkages between the circadian rhythms of symbiotic partners. Interrelated regulation of holobiont circadian rhythms seems thus important to coordinate shifts in activity over the day for all the partners. Therefore, we suggest that the classical view of "chronobiological individuals" should include "a holobiont" rather than an organism. Unfortunately, mechanisms that may regulate interspecies temporal acclimation and the evolution of the circadian clock in holobionts are far from being understood. For the plant holobiont, our understanding is particularly limited. In this case, the holobiont encompasses two different ecosystems, one above and the other below the ground, with the two potentially receiving timing information from different synchronizing signals (Zeitgebers). The arbuscular mycorrhizal (AM) symbiosis, formed by plant roots and fungi, is one of the oldest and most widespread associations between organisms. By mediating the nutritional flux between the plant and the many microbes in the soil, AM symbiosis constitutes the backbone of the plant holobiont. Even though the importance of the AM symbiosis has been well recognized in agricultural and environmental sciences, its circadian chronobiology remains almost completely unknown. We have begun to study the circadian clock of arbuscular mycorrhizal fungi, and we compile and here discuss the available information on the subject. We propose that analyzing the interrelated temporal organization of the AM symbiosis and determining its underlying mechanisms will advance our understanding of the role and coordination of circadian clocks in holobionts in general.

Expression of putative circadian clock components in the arbuscular mycorrhizal fungus Rhizoglomus irregulare.

Arbuscular mycorrhizal fungi (AMF) are obligatory plant symbionts that live underground, so few studies have examined their response to light. Responses to blue light by other fungi can be mediated by White Collar-1 (WC-1) and WC-2 proteins. These wc genes, together with the frequency gene (frq), also form part of the endogenous circadian clock. The clock mechanism has never been studied in AMF, although circadian growth of their hyphae in the field has been reported. Using both genomic and transcriptomic data, we have found homologs of wc-1, wc-2, and frq and related circadian clock genes in the arbuscular mycorrhizal fungus Rhizoglomus irregulare (synonym Rhizophagus irregularis). Gene expression of wc-1, wc-2, and frq was analyzed using RT-qPCR on RNA extracted from germinating spores and from fungal material cultivated in vitro with transformed carrot roots. We found that all three core clock genes were expressed in both pre- and post-mycorrhizal stages of R. irregulare growth. Similar to the model fungus Neurospora crassa, the core circadian oscillator gene frq was induced by brief light stimulation. The presence of circadian clock and output genes in R. irregulare opens the door to the study of circadian clocks in the fungal partner of plant-AMF symbiosis. Our finding also provides new insight into the evolution of the circadian frq gene in fungi.

Application of an in-situ soil sampler for assessing subsurface biogeochemical dynamics in a diesel-contaminated coastal site during soil flushing operations.

Subsurface biogeochemistry and contaminant dynamics during the remediation of diesel-contamination by in-situ soil flushing were investigated at a site located in a coastal region. An in-situ sampler containing diesel-contaminated soils separated into two size fractions (<0.063- and <2-mm) was utilized in two monitoring wells: DH1 (located close to the injection and extraction wells for in-situ soil flushing) and DH2 (located beyond sheet piles placed to block the transport of leaked diesel). Total petroleum hydrocarbon (TPH) concentrations and biogeochemical properties were monitored both in soil and groundwater for six months. A shift occurred in the groundwater type from Ca-HCO3 to Na-Cl due to seawater intrusion during intense pumping, while the concentrations of Ni, Cu, Co, V, Cr, and Se increased substantially following surfactant (TWEEN 80) injection. The in-situ sampler with fine particles was more sensitive to variations in conditions during the remedial soil flushing process. In both wells, soil TPH concentrations in the <0.063-mm fraction were much higher than those in the <2-mm fraction. Increases in soil TPH in DH1 were consistent with the expected outcomes following well pumping and surfactant injection used to enhance TPH extraction. However, the number of diesel-degrading microorganisms decreased after surfactant injection. 16S-rRNA gene-based analysis also showed that the community composition and diversity depended on both particle size and diesel contamination. The multidisciplinary approach to the contaminated site assessments showed that soil flushing with surfactant enhanced diesel extraction, but negatively impacted in-situ diesel biodegradation as well as groundwater quality. The results also suggest that the in-situ sampler can be an effective monitoring tool for subsurface biogeochemistry as well as contaminant dynamics.

Hydraulic containment of TCE contaminated groundwater using pulsed pump-and-treat: Performance evaluation and vapor intrusion risk assessment.

Remedial actions for groundwater contamination such as containment, in-situ remediation, and pump-and-treat have been developed. This study investigates the hydraulic containment of Trichloroethylene (TCE) contaminated groundwater by using pulsed pump-and-treat technology. The hypothetical research site assumed the operation of pulsed pump-and-treat to manage groundwater contaminated with 0.1 mg/L of TCE. at the pump-and-treat facility. Numerical models, employing MODFLOW and MT3DMS for groundwater flow and contamination simulations, were used for case studies to evaluate the performance and risks of pump-and-treat operation strategies. Evaluation criteria included capture width, removal efficiency, and contaminant leakage. Health risks from TCE leakage were assessed using a vapor intrusion risk assessment tool in adjacent areas. In the facility-scale case study, the capture width of the pump-and-treat was controlled by pumping/injection well operations, including schedules and rates. Pumping/injection well configurations impacted facility efficiencies. Pulsed operation led to TCE leakage downstream. Site-scale case studies simulated contaminant transport through pump-and-treat considering various operation stages (continuous; pulsed), as well as various reactions of TCE in subsurface environment (non-reactive; sorption; sorption and biodegradation). Assuming non-reactive tracer, TCE in groundwater was effectively blocked during continuous operation stage but released downstream in the following pulsed operation stage. Considering chemical reactions, the influences of the pump-and-treat operation followed similar trends of the non-reactive tracer but occurred at delayed times. Groundwater contamination levels were reduced through biodegradation. Cancer and non-cancer risks could occur at points of exposure (POEs) where the contamination levels approached or fell below TCE groundwater standards.

Predictive Geogenic Radon Potential (P-GRP): A novel approach for comprehensive hazard assessment and risk modeling in subsurface environment.

Geogenic radon potential (GRP) is traditionally used for mapping radon-prone areas. However, this has challenges in the accurate assessment of radon risk because of limitations such as oversimplified soil measurements and lack of geological profiles. This study presents predictive geogenic radon potential (P-GRP), integrating geological characterization and advanced modeling for the emanation and transport of radon in the subsurface environment. Seoul, South Korea, was selected as the research area for the evaluation of hazards using P-GRP, while subway station A was selected for the assessment of indoor health risks. The geology was characterized by the layers of bedrock and soil using uranium contents and porosity. The emanation of radon was modeled considering the radioactive decay chain of uranium and the pore structures. The vertical transport of radon was modeled considering the porosity variation within geological media, which was used for the calculation of P-GRP. Without loss of continuity, the P-GRP map was constructed by calculating P-GRP at a specific depth over the Seoul area. The calculation of P-GRP in the case of subway station A demonstrates that the radon concentration in the bedrock at the platform depth was expected to be 382 million Bqm-3. The indoor radon risk was calculated using the P-GRP by coupling the vapor intrusion process. This presented a high cancer risk for the employees as well as commuters. The P-GRP map of Seoul demonstrated higher hazards in granite zones compared to banded gneiss zones. These results have demonstrated that the P-GRP could be a novel and promising approach for assessing hazard and risk by geogenic radon during subsurface development.

Laboratory Evaluation of Storage Stability for Asphalt Binder Modified with Crumb Rubber and Styrene-Isoprene-Styrene Depending on Evaluation Factors and Blending Condition.

Modified asphalt binders are still considered important in asphalt pavement. However, the comprehensive use of various modifiers is limited due to storage stability issues. Moreover, there is a scarcity of detailed analyses regarding the degree of separation for asphalt binders among each method despite the utilization of various methods to assess the storage stability of binders. Therefore, a comprehensive analysis was conducted to assess the storage stability of asphalt binder modified with a crumb rubber modifier (CRM) and styrene-isoprene-styrene (SIS), utilizing five evaluation factors following the ASTM D7173 guidelines based on four mixing methods (A: high-shear mixing method, B: low-speed agitating method, C: high-shear mixing method + low mixing method, D: low-speed agitating method + low mixing method). To produce the modified asphalt binder, the proportions of the CRM were 5% and 10% for each binder, and 10% SIS was added to all binders. The results in this study convey that (1) the addition of the modifier led to an increase in G*/sin δ with different mixing methods, but using mixing methods (C and D) for a relatively long time resulted in a lower G*/sin δ, indicating suboptimal performance; (2) through the multiple stress creep recovery (MSCR), rheological properties of Jnr and % rec exhibited trends similar to G*/sin δ evaluation, highlighting an improved elastic recovery with a higher modifier content; (3) storage stability assessment revealed consistent trends in high-shear mixing groups (A and C), while low-speed mixing groups (B and D) exhibited an elevated separation index (SI), suggesting a sensitivity to modification conditions; (4) evaluation using the MSCR method indicated that % rec with a 3.2 kPa load is effective for the sensitive assessment of binder storage stability and Jnr showed a limited sensitivity across varying loads, advocating for % rec for precise evaluation; and (5) despite permitting various tests, achieving consistent results remains challenging. Future research should explore diverse modifiers and optimal evaluation methods to enhance knowledge of binder behavior and separation dynamics.

Longevity prediction of reactive media in permeable reactive barriers considering the contamination level and groundwater velocity at the planning site, with a focus on cadmium removal by zeolite.

Zeolite is a versatile and effective reactive material used in permeable reactive barriers (PRBs) for remediating groundwater contaminated with heavy metals. In this study, we evaluated the influence of subsurface environmental conditions, namely contamination level (C0) and groundwater velocity (v), on predicting the longevity of zeolite for cadmium (Cd) removal. Batch experiments were performed to investigate the effect of C0 on Cd removal, and column experiments were performed to examine how Cd transportation through zeolite varies at different C0 and v. Breakthrough curves (BTCs) were analyzed with an advection-dispersion equation (ADE) coupled with nonequilibrium sorption rate models. The reaction parameters indicating the performance metrics of zeolite were determined using an iterative fitting approach-retardation factor (R), partitioning coefficient (ß), and mass transfer coefficient (ω). R exhibited dependence on C0, but was unrelated to v; its rapid increase at lower C0 was explained by Langmuir sorption isotherms. ß and ω, integral to sorption dynamics and mass transfer, respectively, showcased functional relationships with v. ß decreased gradually as v increased, described by the nonequilibrium sorption model, whereas ω increased steadily with v, guided by the Monod function. Using the relationship of these parameters, the fate and transport of Cd within zeolite was simulated under various subsurface environmental conditions to construct the longevity prediction function. Thus, this study introduces a method for predicting the longevity of reactive materials, which can be valuable for designing PRBs with high longevity in the future.

Spin angular momentum-encoded single-photon emitters in a chiral nanoparticle-coupled WSe2 monolayer.

Spin angular momentum (SAM)-encoded single-photon emitters, also known as circularly polarized single photons, are basic building blocks for the advancement of chiral quantum optics and cryptography. Despite substantial efforts such as coupling quantum emitters to grating-like optical metasurfaces and applying intense magnetic fields, it remains challenging to generate circularly polarized single photons from a subwavelength-scale nanostructure in the absence of a magnetic field. Here, we demonstrate single-photon emitters encoded with SAM in a strained WSe2 monolayer coupled with chiral plasmonic gold nanoparticles. Single-photon emissions were observed at the nanoparticle position, exhibiting photon antibunching behavior with a g(2)(0) value of ~0.3 and circular polarization properties with a slight preference for left-circular polarization. Specifically, the measured Stokes parameters confirmed strong circular polarization characteristics, in contrast to emitters coupled with achiral gold nanocubes. Therefore, this work provides potential insights to make SAM-encoded single-photon emitters and understand the interaction of plasmonic dipoles and single photons, facilitating the development of chiral quantum optics.

Novel method for determination of phenol degradation kinetics.

In this study, we proposed a new method for estimating biokinetic parameters in phenol degradation kinetics. The new method relies on the new formulation of q-S relation where degradation rate q is calculated from the changes of substrate concentration S for each time segment during the course of entire degradation, while in the conventional method q is obtained from the slope of the straight line that is given as substrate concentration changes with time in a semi-logarithmic scale. Thus, this new method provided more data points than the conventional method. The q-S relations obtained from the new method and the conventional method were fitted with three inhibitory kinetic models of Haldane, Yano and Edwards. Simulation of degradation profile with each kinetic model and comparison with the observed profile revealed that the new method offered a better prediction with Edwards model as the best inhibitory model.

Deposition and transport of Pseudomonas aeruginosa in porous media: lab-scale experiments and model analysis.

In this study, the deposition and transport of Pseudomonas aeruginosa on sandy porous materials have been investigated under static and dynamic flow conditions. For the static experiments, both equilibrium and kinetic batch tests were performed at a 1:3 and 3:1 soil:solution ratio. The batch data were analysed to quantify the deposition parameters under static conditions. Column tests were performed for dynamic flow experiments with KCl solution and bacteria suspended in (1) deionized water, (2) mineral salt medium (MSM) and (3) surfactant + MSM. The equilibrium distribution coefficient (K(d)) was larger at a 1:3 (2.43 mL g(-1)) than that at a 3:1 (0.28 mL g(-1)) soil:solution ratio. Kinetic batch experiments showed that the reversible deposition rate coefficient (k(att)) and the release rate coefficient (k(det)) at a soil:solution ratio of 3:1 were larger than those at a 1:3 ratio. Column experiments showed that an increase in ionic strength resulted in a decrease in peak concentration of bacteria, mass recovery and tailing of the bacterial breakthrough curve (BTC) and that the presence of surfactant enhanced the movement of bacteria through quartz sand, giving increased mass recovery and tailing. Deposition parameters under dynamic condition were determined by fitting BTCs to four different transport models, (1) kinetic reversible, (2) two-site, (3) kinetic irreversible and (4) kinetic reversible and irreversible models. Among these models, Model 4 was more suitable than the others since it includes the irreversible sorption term directly related to the mass loss of bacteria observed in the column experiment. Applicability of the parameters obtained from the batch experiments to simulate the column breakthrough data is evaluated.

Performance expectation of coal waste in permeable reactive barrier for removal of cadmium considering contamination level and pore water velocity.

The effectiveness and longevity of permeable reactive barriers (PRBs) depend on the performance of the reactive materials and the subsurface environment. The relationship of the groundwater velocity on performance of coal waste for the heavy metal removal was reported in our previous study. In this study, we investigated the performance and longevity of coal waste as a PRB material for the removal of Cd considering subsurface environmental conditions such as contamination level and groundwater velocity. The artificial groundwater contaminated by Cd were prepared with various concentrations ranging from 10 to 100 mg L-1. Lab-scale column experiments were conducted using coal waste filled columns by injecting the artificial groundwater. The breakthrough curves were analyzed advection dispersion equation coupled with equilibrium sorption model to determine the retardation factor. The Cd breakthrough curves exhibited different retardation with respect to the contamination levels. The Cd transport was more retarded as the contamination level lowered. The relationship between the retardation factor and the contamination levels could be explained with empirical equations based on non-linear sorption isotherms. By adopting the velocity dependency of sorbent performance in our previous study, transport of Cd within coal waste was simulated under various subsurface environmental conditions to construct the longevity function. The function could be used for the longevity prediction of coal waste as a PRB material considering groundwater velocity and contamination level in subsurface environment.

Longevity evaluation of non-pumping reactive wells for control of groundwater contamination: Application of upscaling methods.

Non-pumping reactive wells (NPRWs) are subsurface structures used for the passive treatment of contaminated groundwater using wells containing reactive media. In the vicinity of NPRWs, a combination of hydrogeological and chemical processes makes it difficult to predict their longevity. In this study, we evaluated the longevity of NPRWs using the upscaling methods. A horizontal two-dimensional sandbox was constructed to mimic the hydrogeological and chemical processes in a single unit of NPRW (unit NPRW). The groundwater flow and solute transport were simulated numerically to validate the processes of contaminant spreading prevention in the sandbox. Dye tracing and arsenic transport tests showed different performance of NPRW due to induced flow and uneven consumption of reactivity, which is dependent on the pathway length and residence time of the coal waste. Through numerical modeling of the experiments, the fate-related processes of contamination around NPRW were described in detail in both spatial and temporal terms. The stepwise approach of the upscaling methods was used to predict the contamination-blocking performance of the entire facility based on the reactivity of the materials and the contamination removal of the unit NPRW.

Effect of Blending and Curing Conditions on the Storage Stability of Rubberized Asphalt Binders.

Crumb rubber modifier (CRM) binders easily suffer from instability at high temperatures, with many suggestions being developed to evaluate their storage stability. However, much uncertainty around CRM binders still exists regarding the relationship between mixing methods and experiments in order to calculate the separation index. In this study, a laboratory investigation into how CRM binders behave regarding storage stability using different mixing methods and experiments was conducted. The CRM asphalt binder in this study was prepared through a wet mixing process with the addition of 5% and 10% crumb rubber modifier (by weight) at 200 °C. The three main modification methods were method A: high-shear mixing (8000 rpm) for 2 h, method B: low mixing (300 rpm) for 8 h, and method C: high-shear mixing (8000 rpm) for 2 h + low mixing (300 rpm) for 6 h. In addition, the effect of separation index (SI) on storage stability was assessed, measuring viscosity, G*/sin δ, and percentage recovery. In general, the results of this study revealed that method C appeared to have the most prominent effect on decreasing the difference between the values of the top and bottom parts; the results for 5% CRM using method C showed that there were no differences among the values for the top, middle, and bottom parts; from the MSCR test, method C was effective in decreasing the difference between the values of the top and bottom parts. It was observed that method C improved storage stability. However, the results for percentage recovery were relatively higher than the separation index when using rotational viscosity and G*/sin δ.

Effect of Kaolinite and Cloisite Na+ on Storage Stability of Rubberized Binders.

This study aimed to evaluate the impact of a two-step modification process involving kaolinite and cloisite Na+ on the storage stability of rubberized binders. The process involved the manual combination of virgin binder PG 64-22 with crumb rubber modifier (CRM), which was heated to condition it. The preconditioned rubberized binder was then modified for two hours at a high speed of 8000 rpm using wet mixing. The second stage modification was performed in two parts, with part 1 using only crumb rubber as the modifier and part 2 involving the use of kaolinite and montmorillonite nano clays at a replacement percentage of 3% to the original weight of the binder along with the crumb rubber modifier. The Superpave and multiple shear creep recovery (MSCR) test methods were used to calculate the performance characteristics and separation index % of each modified binder. The results showed that the viscosity properties of kaolinite and montmorillonite improved the performance class of the binder, with montmorillonite demonstrating greater viscosity values than kaolinite even at high temperatures. Additionally, kaolinite with rubberized binders showed higher resistance to rutting, and the % recovery value from multiple shear creep recovery testing indicated that kaolinite with rubberized binders was more effective than montmorillonite with rubberized binders, even at higher load cycles. The use of kaolinite and montmorillonite reduced phase separation between the asphaltene phase and rubber-rich phase at higher temperatures, but the performance of the rubber binder was affected by higher temperatures. Overall, kaolinite with the rubber binder generally demonstrated greater binder performance.