Design of a Clinoptilolite-Zeolite Mineral Carrier to Prolong the Shelf Life of Tea-Root Endophytic Inoculants Under Cold Storage

A prevalent issue in commercial and field applications is the swift deterioration of viable cells during storage and transportation, which impacts product consistency and reliability [1]. Because they provide chemical fertilizer substitutes and promote soil health, nutrient cycling, and plant resilience, biofertilizers and microbial inoculants are increasingly recognized as essential tools for sustainable agriculture. However, delivering the ideal quantity of living microbial cells at the time of application is crucial to its efficacy. Along with flaws in quality assurance and regulatory systems, these major issues are also affected by developments in formulation, preservation, environmental adaptability, and application techniques. To achieve consistent field performance and broader adoption in sustainable farming systems, these problems must be addressed [2]. Carrier-based formulations are essential in the design of inoculants. Conventional organic carriers, such as peat or compost, can enhance microbial viability but face challenges due to variability, moisture instability, and quality assurance [3]. A recent study underscores the significance of carriers possessing well-defined physical and chemical characteristics. In this regard, natural zeolites, particularly clinoptilolite, have emerged as promising inert, porous substrates. Their aluminosilicate structure and ion-exchange capability establish microhabitats that control moisture and ionic conditions [4]. Clinoptilolite has been thoroughly researched for agricultural applications, particularly as a stabilizing matrix for microbial inoculants. Endophytic bacteria derived from tea (Camellia sinensis) roots are increasingly recognized for their potential to improve plant health and enhance stress resilience [5]. Although these tea plant endophytic bacteria have great potential, the lack of a stable carrier formulation with clear process parameters suitable for their characteristics limits their commercialization and large-scale application [6]. Moreover, current research on zeolite-based carriers frequently omits specific processing characteristics (e.g., mesh size, heat conditioning, and moisture content), complicating comparisons of results [7]. From a formulation design standpoint, regulating these factors is crucial for consistent results. The recommended mesh size and moisture content were selected to optimize microbiological protection while ensuring practical handling: particles are tiny enough to create protective niches, yet coarse enough to allow flowability, and low moisture to reduce microbial activity during storage. This study seeks to (i) create a clinoptilolite carrier (45–80 mesh; heat-treated at 100 ℃ and 200 ℃; moisture adjusted to approximately 4% w/w) for an endophytic bacterial inoculant derived from tea roots in Gambung, West Java; and (ii) assess viability retention during cold storage at 0 ℃ for up to 150 days across various microbial loadings (12.5%, 25%, and 50% w/w) using a factorial design and total plate counts [8]. This study delineates design parameters and information on plant samples, carrier preparation, and enumeration methodologies, thereby enabling reproducible comparisons and facilitating the translation of endophyte-based inoculants into practical applications [9].

3.1 Initial bacterial counts

We counted viable cells for all treatment combinations immediately after formulation (day 0) to obtain reliable baseline values for the retention calculations that followed. Table 3 shows the first set of data, which shows the viable counts (CFU g⁻¹) for each combination of microbial loading levels (12.5%, 25%, and 50% w/w) and thermal treatment temperature (100℃ and 200℃) [28]. In this case, w/w means the ratio of microbial mass to the overall mass of the formulation. This shows the real loading percentage based on weight, not volume. These baseline measurements are important because they ensure that microbial retention measurements during the observation period are accurate and consistent [30].

Table 3. Initial viable counts (CFU g⁻¹) on day 0 for all treatment combinations

3.2 Viability retention during cold storage: Factorial analysis

Viable counts were assessed at 30, 90, and 150 days of storage at 0 ℃. Table 4 displays the comprehensive dataset for all treatment combinations, categorized by thermal treatment temperature and microbial loading [30].

Across all clinoptilolite treatments, viable counts at day 30 consistently measured approximately 10⁹ CFU g⁻¹ (Table 5), suggesting that mixing and the moisture adjustment to ~4% did not exert undue stress on the endophytic culture [2, 31-36]. The mineral carrier successfully maintained viability throughout storage. On day 90, mean counts varied from 2.85 × 10⁹ to 5.85 × 10⁹ CFU g⁻¹, contingent upon loading and temperature (Table 5). Conversely, the non-zeolite control significantly declined to roughly 5.0 × 10⁵ CFU g⁻¹ by day 90, illustrating the protective effectiveness of the clinoptilolite carrier under the same conditions. Zeolite-based carriers likely improve survival by regulating microscale water availability and offering physical protection against desiccation and osmotic stress [2, 3].

By day 150, viable counts in all clinoptilolite formulations consistently approximated 10⁹ CFU g⁻¹, with retention rates varying with initial loading (Table 6 and Figure 1). The 12.5% loading maintained 59.7% of its initial population (day 0 baseline), while the 25% and 50% loadings kept 100 °C, respectively (Table 6). This density-dependent trade-off indicates that heightened initial loads may exacerbate competition for limited pore space or nutrients within the carrier matrix, thereby hastening population decline. Similar density effects have been observed in other carrier-based inoculants [2, 36].

A two-way analysis of variance was conducted for each sampling time point to statistically assess the impacts of thermal treatment temperature, microbial loading, and their interaction on viable counts. Table 4 encapsulates the ANOVA findings.

To statistically evaluate the effects of thermal treatment temperature, microbial loading, and their interaction on viable counts, a two-way analysis of variance was performed for each sampling time point.

Table 4. Two-way analysis of variance (ANOVA) results for viable counts at days 30, 90, and 150

Table 5. Viable counts (CFU g⁻¹) at days 30, 90, and 150 for all treatment combinations

Table 6. Retention (%) relative to day-0 baseline for all treatment combinations

The ANOVA revealed that microbial loading had a highly significant effect on viable counts at all time points (p < 0.001 at days 30 and 90; p = 0.003 at day 150). In contrast, thermal treatment temperature (100 ℃ vs. 200 ℃) showed no significant effect at any sampling time (p > 0.05), and the interaction between temperature and loading was also non-significant (p > 0.05) [33]. These results indicate that the two heat treatment temperatures performed similarly in preserving viability, and their effects were consistent across all loading levels. Figure 1 presents viable counts at day 150 as bar charts with error bars (mean ± SD), with significant differences among loading levels indicated by lowercase letters (p < 0.05, Tukey’s HSD post hoc test) [34]. The non-zeolite control decreased dramatically during storage, from an initial count of approximately 1.0 × 10¹⁰ CFU per sample to 5.0 × 10⁵ CFU per sample on day 90, and became undetectable on day 150. This sharp decline underscores the protective efficacy of the clinoptilolite carrier under identical storage conditions [33].

3.3 Retention rates and density-dependent effects

Retention rates were calculated relative to the day-0 baseline (CFU₀) for each treatment combination. Table 6 presents the percent retention at days 30, 90, and 150. By day 150, retention rates showed a clear inverse relationship with initial loading: the 12.5% loading preserved approximately 59% of its initial population, whereas the 25% and 50% loadings retained only 45% and 33%, respectively (Table 6) [35]. This density-dependent decline was consistent across both temperature treatments. Several interconnected mechanisms may explain this observation:

First, spatial constraints within the carrier matrix are fundamental. The microstructural basis for this density-dependent effect becomes evident when examining the carrier architecture (Figure 2). With pore dimensions of 1–10 μm and bacterial cell diameters of 1–5 μm, each pore can accommodate only a limited number of cells. At 12.5% loading (∼3.4 × 10⁹ CFU g⁻¹), cells likely occupy pores in a dispersed pattern with adequate spacing. However, at 50% loading (∼6.7 × 10⁹ CFU g⁻¹), pore occupancy approaches or exceeds the optimal carrying capacity, forcing cells into closer proximity. This overcrowding intensifies competition for available microhabitats, leading to accelerated mortality when initial loading exceeds the carrier’s finite pore volume [36].

Second, nutrient limitations become more acute at higher cell densities. The confined pore spaces contain limited nutrients, derived either from residual organic matter in the zeolite or from carryover from the culture medium. At higher initial loadings, these nutrients are consumed more rapidly, potentially leading to early starvation stress [37].

Third, the accumulation of metabolic waste products may reach inhibitory levels sooner in densely populated microenvironments, creating localized toxicity. Comparable density-dependent effects have been reported in other carrier-based inoculants, where moderate initial loadings often yield better long-term survival than excessively high loadings. These findings suggest that optimizing microbial load rather than simply maximizing initial cell numbers is critical for formulation design.

3.4 Carrier microstructure and design implications

Scanning electron microscopy (SEM) coupled with energy-dispersive X-ray spectroscopy (EDS) was used to characterize the microstructure of the clinoptilolite carrier. Figure 2 presents representative SEM micrographs at 500× and 5000× magnification, revealing angular, porous particles with irregular surfaces [10, 18]. At higher magnification, numerous microscale voids and channels are visible, with pore sizes predominantly in the 1–10 μm range (estimated from image analysis). These dimensions are particularly significant given that bacterial cells typically measure 1–5 μm in diameter, indicating that individual pores can accommodate only a limited number of cells [2]. This microstructural observation provides direct physical evidence supporting the density-dependent mortality mechanism discussed in Section 3.3: at 50% loading (∼6.7 × 10⁹ CFU g⁻¹), pore occupancy likely exceeds optimal carrying capacity, intensifying spatial competition compared to 12.5% loading (∼3.4 × 10⁹ CFU g⁻¹).

The observed pore architecture is well-suited for bacterial colonization, as it creates protective microhabitats that shield cells from shear forces during handling while still allowing nutrient diffusion and waste exchange [14]. This pore size range is consistent with previous reports on clinoptilolite microstructure [8, 10, 16] and supports its potential as a microbial carrier matrix.

The EDS spectrum (Figure 3) confirms the expected elemental composition, showing predominant signals of silicon (Si) and aluminum (Al) characteristic of the clinoptilolite aluminosilicate framework, along with minor peaks corresponding to exchangeable cations (Ca²⁺, K⁺, Mg²⁺). This elemental profile corroborates the carrier’s moisture-buffering and ion-exchange capabilities, which are relevant to maintaining stable microenvironments for microbial survival during storage [9, 10]. The aluminosilicate framework can adsorb water and exchange cations, potentially helping to buffer against osmotic stress and maintain favorable ionic conditions within pores—factors that likely contributed to the sustained viability (> 10⁹ CFU g⁻¹ for 150 days) observed across all formulations.

From a formulation design standpoint, the selected parameters, 45–80 mesh particle size, thermal conditioning at 100 ℃–200 ℃, and approximately 4% moisture content, produced a consistent and effective carrier. Our results support the scientific basis for these choices:

• Particle size (45–80 mesh): This fraction provides an optimal balance between surface area for microbial colonization and interparticle porosity for gas exchange and flowability. The SEM images confirm the presence of pores in the 1–10 μm range, which are accessible to bacteria (typical cell size 1–5 μm) while providing physical protection from shear forces during handling [14, 21].
• Moisture content (~4%): This reduced water activity level was chosen to minimize metabolic activity during cold storage while preventing complete desiccation that could cause cell membrane damage. The successful maintenance of approximately 10⁹ CFU g⁻¹ over five months validates this selection [2, 5].
• Thermal treatment (100 ℃–200 ℃): Heat conditioning was intended to remove bound water and potentially increase pore volume [16]. However, the lack of a significant difference between 100 ℃ and 200 ℃ treatments suggests that the lower temperature (100 ℃) is sufficient to achieve the desired effects and that the additional energy input at 200 ℃ provides no further benefit for microbial preservation. This finding has practical implications for industrial scalability, as lower temperatures reduce energy consumption and processing costs without compromising product quality.

The performance of clinoptilolite-based carriers can be contextualized by comparison with other common carrier materials reported in the literature. Peat-based formulations, while widely used, often suffer from batch-to-batch variability and inconsistent moisture retention [2, 5]. Studies using peat carriers have reported declines in viability of 1–2 log units over 3–6 months at ambient temperatures (Q). In contrast, our clinoptilolite formulations maintained counts within the same log order (10⁹ CFU g⁻¹) throughout 150 days at 0 ℃. Vermiculite, another mineral carrier, has shown good moisture retention but lower mechanical stability [15]. The rigid crystalline structure of clinoptilolite offers advantages in terms of physical stability and reproducible pore architecture, as evidenced by the consistent performance across replicate samples in this study.

3.5 Limitations and future directions

This study has several limitations that should be acknowledged and addressed in future research: First, the endophytic bacterial isolates were not identified at the species or strain level. While their plant-beneficial potential is inferred from their origin (healthy tea roots), specific functional traits such as phosphate solubilization, nitrogen fixation, or phytohormone production were not verified. Future studies should include molecular identification (e.g., 16S rRNA sequencing) and functional characterization of the isolates to enable strain-specific optimization of carrier formulations [24-27]. Second, although thermal treatment was applied at two temperatures (100 ℃ and 200 ℃), the lack of a significant difference between them suggests that more extreme temperature variations or different treatment durations might be needed to observe effects. Alternatively, the benefits of heat treatment may already be saturated at 100 ℃, making 200 ℃ unnecessary. Future optimization studies could explore a wider range of temperatures and include untreated controls to quantify the baseline effect of heat conditioning. Third, the study was conducted under controlled laboratory conditions (0 ℃ storage) without field validation. While cold storage is practical for many agricultural applications, real-world performance may be affected by fluctuating temperatures and humidity, as well as interactions with soil or plant environments. Field trials are needed to evaluate whether the viability preserved during cold storage translates into effective plant colonization and growth promotion under actual growing conditions. Fourth, the absence of significant temperature × loading interactions indicates that these factors act independently, however, mechanistic studies using microscopic observation (e.g., confocal microscopy or fluorescence in situ hybridization) could provide direct evidence of how cells distribute within carrier pores and how spatial competition evolves. Fifth, the study focused solely on viability preservation and did not assess the functional integrity of the preserved cells. Future work should include bioassays to confirm that cells recovered from storage retain their plant growth-promoting activities (e.g., indole acetic acid production and phosphate solubilization) [27, 35]. Finally, while the 45–80 mesh particle size and 4% moisture content were effective, systematic optimization studies (e.g., response surface methodology) could identify the precise parameter combination that maximizes long-term survival for specific endophyte strains.

The authors thank the Tea and Quinine Research Center (Gambung, West Java) for facilitating field sampling and laboratory support. We also acknowledge the Research and Development Center for Mineral and Coal Technology (Indonesia) and the National Research and Innovation Agency (Indonesia) for providing research facilities and support.