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Characterization of mangrove-associated bacteria tolerance to stress, and their potential to increase NPK in OPMF biofertilizer, starch, and protein in duckweed
* Corresponding author: E-mail address: nazariyah@usim.edu.my (N Yahaya)
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Received: ,
Accepted: ,
Abstract
Biofertilizers offer an eco-friendly alternative to chemical fertilizers in agriculture, utilizing stress-tolerant plant growth-promoting microbes (PGPR) and sustainable carriers to enhance plant growth and yields. This study evaluates six mangrove-associated microbes (MAMs): Bacillus tropicus and Paenibacillus pasadenensis, Bacillus cereus, Bacillus thuringiensis, Acinetobacter radioresistens, and Enterobacter cloacae under environmental stressors, including extreme temperatures, salinity, and heavy metals. The findings of this study emphasize the resilience of B. cereus, B. thuringiensis, and B. tropicus under stress conditions, particularly at 37°C and in the presence of salt (NaCl) and heavy metals such as lead (Pb) and copper (Cu), as evidenced by their significant exopolysaccharide (EPS) production. Biofilm formation exhibited an inconsistent pattern among the bacterial strains; each strain responded differently to different stress levels. Flocculation activity showed insignificant formation under Pb exposure, NaCl, and 37°C, compared to non-stress conditions in B. cereus, B. thuringiensis, B. tropicus, and P. pasadenensis. These results suggest that these conditions neither enhance nor inhibit flocculation, indicating that the bacteria are resilient under moderate stress. Furthermore, analysis of the ability of these microbes to release Carbon (C), Nitrogen (N), Phosphate (P), and Potassium (K) from Oil Palm Mesocarp Fiber (OPMF) revealed that B. cereus significantly increased the P value (P < 0.05) compared to other bacteria and the control. Then, each microbe was individually inoculated into OPMF, and its effect on duckweed growth, starch, and protein content was evaluated. Duckweed growth improved significantly (P < 0.05) by day 15 when grown on OPMF inoculated with any of the microbes. A. radioresistens and E. cloacae inoculation resulted in the highest duckweed growth and protein content (P < 0.05), respectively, compared to the control. The investigation into stress-tolerant MAMs under conditions of extreme and moderate temperatures, salinity, and heavy metals was chosen to explore potential threshold effects and better understand the biological mechanisms involved.
Keywords
Biofertilizer
Duckweed
Mangrove-associated microbes (MAMs)
Oil palm mesocarp fiber (OPMF)
Stress tolerance
1. Introduction
Conventional chemical and synthetic fertilizers have raised environmental, crop quality, and human health concerns (Özkan et al., 2024). In contrast, traditional biofertilizer formulations have provided insight into the benefits of using microorganisms to support crop growth (Nur Maisarah & Yahaya, 2022). However, their effectiveness is often inconsistent compared to chemical fertilizers; hence, various biofertilizer formulations are developed to improve plant productivity (Wahab et al., 2025). Plant growth-promoting rhizobacteria (PGPR), such as nitrogen-fixing, phosphate- and potassium-solubilizing bacteria, are recognized for enhancing growth and inhibiting plant pathogens (Wahab et al., 2025).
Mangrove-associated microbes (MAMs), many of which are PGPR, contribute to nutrient cycling and plant productivity by fixing nitrogen and solubilizing phosphate and potassium, making them promising candidates for sustainable biofertilizer development (Hamdan et al., 2024; Yahaya et al., 2024). However, their survival and function may be compromised under environmental stressors such as extreme temperatures, salinity, and heavy metal contamination. To be effective, PGPR must exhibit robust growth and resilience under such conditions (Hareem et al., 2023). For instance, cold-tolerant Enterobacter cloacae improves the performance of cold-sensitive crops such as rice (Oryza sativa L.), while under heat and drought, PGPR enhance nutrient uptake and water flow (Liao et al., 2025; Udpuay et al., 2024). In saline conditions, salinity-resistant wheat varieties inoculated with PGPR strains (Bacillus safensis, Bacillus pumilus, and Zhihengliuella halotolerans), show stronger responses compared to salinity-sensitive varieties (Amini et al., 2022). Similarly, PGPR like Exiguobacterium aestuarii and Bacillus cereus can reduce Cadmium (Cd) bioaccumulation in Brassica juncea shoots grown on heavy metal-contaminated soils (Daraz et al., 2023).
PGPR functionality under stress is also supported by their ability to produce exopolysaccharides (EPS), which help maintain soil structure, limit Na⁺ uptake under salt stress, and enhance biofilm formation (El-Saadony et al., 2024; Liu et al., 2022). Under abiotic stresses such as salinity, drought, heat, and heavy metal contamination, many PGPR genera, including Acinetobacter, Azotobacter, Bacillus, Burkholderia, Klebsiella, Pantoea, Pseudomonas, and Rhizobium, form biofilms that protect cells, improve water retention, and support nutrient exchange (Brokate et al., 2024). Flocculation, the aggregation of microbial cells into clusters, also plays a crucial role in stress tolerance. Bioflocculants contain functional groups that bind heavy metals and sodium ions, neutralize charge imbalances, and reduce toxicity (Bhagat et al., 2021). EPS plays a key role in this process by facilitating the formation of larger flocs that immobilize harmful ions (Chen et al., 2022).
In biofertilizer formulation, plant or agro-industrial waste often serves as a microbial carrier (Nur Maisarah & Yahaya, 2022). Oil palm mesocarp fiber (OPMF), a lignocellulosic byproduct of palm oil processing, is an attractive option due to its availability and degradability. Despite its lower cellulose content and less research attention compared to OPEFB, OPMF is a useful biofertilizer in agriculture, though its lignocellulosic complexity can obstruct enzyme accessibility and sugar production (Gan et al., 2023).
Duckweed, a small, fast-growing aquatic plant, reproduces rapidly under favorable conditions and can double its biomass every 2-3 days (Yahaya et al., 2022). It offers multiple applications as biofuels, animal feed, phytoremediation agents, or protein-rich food source (Li et al., 2022; Raza et al., 2023; Yahaya et al., 2022). Although sometimes considered invasive, its growth is easily controlled, making it suitable for integrated systems that recycle biomass and nutrients sustainably.
Our previous study (Hamdan et al., 2024) isolated six bacterial strains from mangrove environments: Bacillus cereus and Bacillus thuringiensis (phosphate solubilizers), Acinetobacter radioresistens and Enterobacter cloacae (nitrogen fixers), and Bacillus tropicus and Paenibacillus pasadenensis (potassium solubilizers). When applied as a biofertilizer, these strains acted synergistically to promote duckweed growth and increase its protein content by providing essential N, P, and K nutrients. This present study aimed to evaluate their individual performance under environmental stress conditions, including salinity, extreme temperatures, and heavy metals, focusing on adaptive traits such as EPS production, biofilm formation, and flocculation. Each strain was inoculated into OPMF to assess its effectiveness in its carbon (C), nitrogen (N), phosphate, and potassium (K) release, and its impact on protein and starch content in duckweed. We hypothesized that MAMs tested in this study retain their adaptive mechanisms capabilities by the production of EPS, biofilm formation, as well as flocculation under stress conditions. Each of the MAMs tested in this study was hypothesized to have the potential to release one or more essential elements required for the microbes and plant growth, such as C, N, P, and K, in OPMF-based formulations. The stress-tolerant microbes identified in this study, their potential to produce C, N, P, or K, and can enhance duckweed growth and protein, demonstrating their suitability for biofertilizer development.
2. Materials and Methods
2.1 Culturing of MAMs
Six MAMs isolated from soil samples of freshwater riverine mangrove at Lukut River, Negeri Sembilan (Yahaya et al., 2024) were identified as Bacillus tropicus and Paenibacillus pasadenensis (potassium-solubilizing bacteria), Bacillus cereus and Bacillus thuringiensis (phosphate-solubilizing bacteria), and Acinetobacter radioresistens and Enterobacter cloacae (nitrogen-fixing bacteria) (Hamdan et al., 2024). Single colonies from -80°C stored microbial stocks were streaked onto nutrient agar, incubated at 30°C for 48-72 h, and transferred to 15 mL of sterile nutrient broth. Cultures were standardized to an OD range of 0.5 to 0.6 using an Eppendorf BioPhotometer Plus (Hamburg, Germany).
2.2 Characterization of bacterial stress tolerance
EPS production, biofilm formation, and bacterial flocculation were assessed under temperature, salinity, and heavy metal stress conditions (Table 1).
| Tests | Control | Treatment 1 | Treatment 2 | Treatment 3 | Treatment 4 |
|---|---|---|---|---|---|
| Temperature | 30°C | 4°C | 37°C | 50°C | 65°C |
| Salinity test | 0.0 M NaCl | 0.5 M NaCl | 1.0 M NaCl | 1.5 M NaCl | 2.0 M NaCl |
| Heavy metal | - | Ni | Cu | Pb | Fe |
2.2.1 Temperature tolerance test
Bacterial suspensions on nutrient agar (Oxoid, Basingstoke, Wade Rd, United Kingdom) were incubated at 4°C, 30°C (control), 37°C, 50°C, and 65°C.
2.2.2 Salinity tolerance test
Nutrient agar media supplemented with 10% glucose and fructose (Oxoid, Basingstoke, Hampshire, United Kingdom) were prepared with sodium chloride concentrations of 0.0 M (control), 0.5 M, 1.0 M, 1.5 M, and 2.0 M.
2.2.3 Heavy metal tolerance test
Nutrient agar media with 10% glucose and fructose were amended with 0.01 M concentrations of Nickel (Ni), Copper (Cu), Lead (Pb), and Iron (Fe). A control without heavy metals was included (Chemiz, Shah Alam, Selangor, Malaysia).
2.2.3.1 Measurement of EPS production
Six sterile filter paper discs were placed on each plate. A 5 μL aliquot of bacterial suspension was inoculated onto each disc and incubated at 30°C for 72 h. EPS production was indicated by mucoid colony formation, measured in centimeters.
2.2.3.2 Detection of biofilm formation
Single colonies were grown in 5 mL of nutrient broth and incubated at 30°C for 48 h. The inoculum was adjusted to an OD of 0.5, and 100 μL was transferred to test tubes with fresh nutrient broth, incubated at 30°C for 48 h. Tubes were washed with PBS (Oxoid, Basingstoke, Hampshire, United Kingdom), stained with crystal violet (Oxoid, Basingstoke, Hampshire, United Kingdom), and biofilm formation was quantified by measuring OD at 570 nm using a biophotometer (Eppendorf, Hamburg, Germany).
2.2.3.3 Estimation of bacterial flocculation
Single colonies were inoculated into 5 mL of nutrient broth and incubated at 30°C for 48 h. Flocculation yield was obtained by filtering the broth through Whatman No. 1 filter paper, drying at 60°C, and weighing the flocculates.
2.3 Quantification of C, N, P & K elements in OPMF
OPMF was used as a solid carrier in the formulation of biofertilizer.
2.3.1 Bacterial palletization
Bacterial cultures were centrifuged at 11,057 × g for 15 min at 4°C and then resuspended in sterile nutrient broth. Each bacterial species was mixed with pre-sterilized dry OPMF at a 1:50 ratio.
2.3.2 Biofertilizer development
OPMF was dried, ground into a fine powder, and autoclaved. Pre-sterilized OPMF packets were inoculated with bacteria at a 1:50 ratio. Dried and pre-sterilized OPMF without bacterial inoculation was used as a control. Both OPMF packets with and without bacterial inoculation were incubated at 30°C for 7 days.
2.3.3 Energy-dispersive X-ray analysis
The Carbon (C), Nitrogen (N), Phosphate (P), and Potassium (K) content in the formulated biofertilizer was examined using energy-dispersive X-ray (EDX) spectroscopy (Oxford, Abingdon, Oxfordshire, United Kingdom). Samples were placed on conductive carbon tape, and the EDX system induced emission of X-ray peaks for each element, used for quantitative analysis.
2.4 Quantification of duckweed (Wolffia globosa) growth
Duckweed (Wolffia globosa) was collected in Sepang, Selangor, Malaysia. Fronds were immersed in 1% potassium permanganate (KMnO4) with 70% ethanol for 10 min, then submerged in filtered water for seven days. The use of KMnO4 is to oxidize pollutants like organic matter and bacteria in water soaked with duckweeds (Ma et al., 2023). Ten duckweed plants were transferred to a container (20 cm × 20 cm) containing 5 g of biofertilizer and 100 mL of filtered water. Three containers for each treatment were prepared for the experiment measurement. Field and greenhouse tests were conducted at the Faculty of Science and Technology (FST) from December 10 to December 25, 2024. Plants were exposed to a 12-h light/dark cycle, at temperatures ranging from 24°C to 31°C. The pH of the water was maintained at 7. Duckweed growth was recorded every 3-4 days until Day 15.
2.5 Protein content in duckweed grown with developed biofertilizer
Fresh duckweed was ground into a fine powder and dried at 65°C for 24 h. The dried powder was immersed in sterile distilled water (1 g in 10 mL) overnight, then heated in a microwave for 15 min at 100 W. The mixture was filtered to retain the green juice. Protein content was determined using a UV spectrophotometer (Varian Cary 50, Palo Alto, California and USA) based on triplicate standard curves of bovine serum albumin (BSA) with concentrations 2, 1.5, 1, 0.75, 0.5, 0.25, and 0.125 mg/mL. A total of 2 mg/mL of bovine serum albumin (BSA) standard was diluted according to Bradford Protein Assay (Bio-Rad Laboratories, Berkeley, California) guidelines. The absorbance at 595 nm was used to calculate protein concentration based on the standard equation y = mx + c.
2.6 Starch content analysis in duckweed (W. globosa)
Duckweed biomass was collected, rinsed, and dried at 60°C before grinding into a fine powder. For starch extraction, 0.1 g of the powder was mixed with 5 mL of 80% acetone, centrifuged, and the supernatant discarded. The residue was hydrolyzed with 10 mL of 1% HCl, heated at 100°C for 30 min, cooled, and neutralized with NaOH. The solution was diluted to 25 mL with distilled water. Starch content was quantified by reacting 1 mL of the hydrolyzed solution with 2 mL of iodine-potassium iodide reagent. The starch standards were prepared before analysis by making a stock solution of 10 mg/mL starch by dissolving 1 g of starch (Sigma-Aldrich, Darmstadt, Germany) in 100 mL of distilled water. A series of dilutions was made to achieve concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL. The absorbance of the solution, either starch standard or sample, was measured at 590 nm using a UV-Vis spectrophotometer.
2.7 Statistical analysis
Measurements were done in triplicate and reported as means ± standard deviation. ANOVA was performed using MINITAB 22.2 (Pennsylvania State University, United States of America), with a significance level of P<0.05 Tukey Pairwise Comparison, and a sample size of three replicates per treatment was used for significant tests between samples by using the model Yij=μ+τi+ϵij.
Where:
Yij = observed value of the response variable for treatment i and replicate j
μ = overall mean
τi = effect of treatment i
ϵij = random error term, assumed to be normally distributed
This statistical model was used to test three hypothesis statements.
Hypothesis statement 1:
Null Hypothesis (Ho): Each of the MAMs tested in this study does not produce a significant difference in EPS, biofilm, or flocculation under varying temperature, high salinity, or metal exposure conditions.
Alternative Hypothesis (HA): At least one of the MAMs tested in this study shows a significant difference in EPS, biofilm, or flocculation under varying temperature, high salinity, or metal exposure conditions.
Hypothesis statement 2:
Null Hypothesis (Ho): OPMF-based formulation inoculated with each of the MAMs tested in this study does not show a significant difference in percentage of C, N, P, or K elements.
Alternative Hypothesis (HA): OPMF-based formulation inoculated with at least one of the MAMs tested in this study shows a significant difference in percentage of C, N, P, or K elements.
Hypothesis statement 3:
Null Hypothesis (Ho): Duckweed grows in OPMF-based formulation media inoculated with each of the MAMs tested in this study does not show a significant difference in the number of fronds and protein contents.
Alternative Hypothesis (HA): Duckweed grows in OPMF-based formulation media inoculated with at least one of the MAMs tested in this study, showing a significant difference in the number of fronds and protein contents.
The graphs for each experiment were constructed with vertical bars indicating standard deviations of the means (n=3). The statistical analysis was shown on the bars using either uppercase and lowercase letters or asterisks (*). Bars with the same uppercase letter for each bacteria species are not significantly different at P ≤ 0.05 in the same conditions, whereas bars with the same lowercase letter for each bacteria species are not significantly different at P ≤ 0.05 in different conditions.
3. Results
3.1 Exopolysaccharide production, biofilm formation, and flocculation activity of MAMs under extreme temperature conditions
MAMs used in biofertilizers are exposed to varying temperatures during production, storage, and field application. EPS production, biofilm formation, and flocculation create protective layers that retain water, forming stable microenvironments that enhance bacterial survival under extreme temperatures. This study analyzed EPS production, biofilm formation, and flocculation activity in bacterial cultures exposed to temperatures ranging from 4°C to 65°C, with 30°C as the optimal control.
In this study, the optimal temperature for EPS production in B. cereus, B. thuringiensis, and B. tropicus was observed at 37°C. No EPS was detected at extreme temperatures (4°C, 50°C, and 65°C), indicating thermal sensitivity and adaptation to its natural environments (Fig. 1a).
- Change in (a) EPSs production, (b) Biofilm formation, and (c) Flocculation in tested MAMs, in response to various temperatures. Bars with same uppercase letter for each bacteria species are not significantly different at P ≤ 0.05 in same temperature conditions, whereas bars with same lowercase letter for each bacteria species are not significantly different at P ≤ 0.05 in different temperature conditions.
Significant biofilm formation was observed in this study at 37°C in B. tropicus and P. pasadenensis, consistent with their EPS production (Fig. 1b). No biofilm formation was observed at extreme temperatures for all strains, which may suggest their limited adaptability beyond optimal conditions.
In addition, flocculation activity remained stable between 30°C and 37°C, as moderate temperatures support microbial metabolism and enzyme activity essential for flocculation. Fig. 1(c) reveals that B. cereus and B. thuringiensis exhibited the highest flocculation activity (P < 0.05) under an optimal temperature of 30°C compared to the other bacteria tested in this study (Fig. 1c). Flocculation activity declined at higher temperatures.
B. cereus emerged as the most temperature-tolerant among the MAMs studied. This was evidenced by its consistent EPS production, biofilm formation, and flocculation activity across various temperature conditions. B. thuringiensis also demonstrated some level of tolerance, particularly in biofilm formation and flocculation activity at the optimal temperature of 30°C. However, its performance significantly decreased at higher temperatures (37°C).
Conversely, E. cloacae exhibited high flocculation activity under cold conditions (4°C) but showed lower tolerance to higher temperatures. P. pasadenensis and B. tropicus displayed some tolerance under specific conditions but were generally less consistent compared to B. cereus. Overall, B. cereus was identified as the most temperature-tolerant bacterium among those tested, while E. cloacae and B. thuringiensis showed varying degrees of intolerance to temperature extremes.
3.2 Exopolysaccharide production, biofilm formation, and flocculation activity of MAMs under extreme salinity conditions
Salt levels in agricultural soil systems, including irrigation water, can cause salinity stress to crops and beneficial microbes used in biofertilizers.
Under salinity stress (0.5 M and 1.0 M NaCl), none of the bacterial strains demonstrated significant EPS production (Fig. 2a). This may be due to alternative survival adaptations, which support osmotic balance. EPS typically facilitates salinity tolerance by binding Na⁺ and maintaining ionic balance, but the lack of EPS in this study may suggest other protective mechanisms.
- Change in (a) EPSs production, (b) Biofilm formation, and (c) Flocculation in tested MAMs, in response to different salinity concentrations. Bars with same uppercase letter for each bacteria species are not significantly different at P ≤ 0.05 under the same salinity concentration, whereas bars with same lowercase letter for each bacteria species are not significantly different at P ≤ 0.05 under different salinity concentrations.
Despite low EPS yields, B. cereus, P. pasadenensis, and B. thuringiensis demonstrated optimal biofilm formation at 1.0 M NaCl, with B. thuringiensis also showing an increase at 0.5 M NaCl (Fig. 2b). This indicates that biofilm formation may be influenced by factors beyond EPS. The results for flocculation activity reveal a dramatic decrease (P < 0.05) with the presence of 1.0 M NaCl and higher, compared to 0.5 M and the control (0 M) in all bacterial species (Fig. 2c). Our findings suggest that at 0.5 M salinity, bacteria may adapt by using Na⁺ to stabilize EPS and facilitate floc formation.
Among the tested microbes, B. cereus and B. thuringiensis demonstrated the highest salinity tolerance, as evidenced by their significant biofilm formation at 1.5 M NaCl and relatively stable flocculation activity compared to the control. P. pasadenensis also showed some level of tolerance, particularly in EPS production at 0.5 M NaCl and higher flocculation yields than the control.
On the other hand, E. cloacae, B. tropicus, and A. radioresistens exhibited decreased biofilm formation and flocculation activity at higher salinity levels, indicating lower tolerance to salinity stress. Overall, B. cereus and B. thuringiensis were the most salinity-tolerant bacteria among those tested, while E. cloacae, B. tropicus, and A. radioresistens showed varying degrees of intolerance to high salinity conditions.
3.3 Exopolysaccharide production, biofilm formation, and flocculation activity of MAMs under heavy metal stress
Urbanization and industrialization near agricultural areas contribute to heavy metal contamination in soil and water. Heavy metals such as nickel (Ni), copper (Cu), iron (Fe), and lead (Pb) pose public health concerns and are associated with various diseases (Kielak et al., 2017). Microbial resistance to metal stress can be assessed by their production of EPS, which is linked to biofilm formation and flocculation activity.
Under heavy metal stress, B. cereus, B. thuringiensis, and B. tropicus showed increased EPS production in the presence of Cu and Pb (Fig. 3a), indicating their adaptability. EPS, composed of homo- or heteropolysaccharides with various functional groups, facilitates heavy metal adsorption. Similarly, Fig. 3(b) shows a significant decrease (P < 0.05) in biofilm formation compared to the control and other heavy metals, indicating that all tested bacteria were intolerant to Ni. In contrast, EPS production significantly increased with the presence of Cu in B. cereus, B. thuringiensis, B. tropicus, and A. radioresistens compared to the control.
- Change in (a) EPSs production, (b) Biofilm formation, and (c) Flocculation in MAMs, in response to heavy metals. Bars with same uppercase letter for each bacteria species are not significantly different at P ≤ 0.05 in response to the same heavy metal, whereas bars with same lowercase letter for each bacteria species are not significantly different at P ≤ 0.05 in response to different heavy metals.
In this study, a relatively high Cu concentration of 1345 mg/L (0.01 M) enhanced EPS production in B. cereus, B. thuringiensis, B. tropicus, and A. radioresistens compared to the control. Only P. pasadenensis showed a decrease (P < 0.05) in EPS formation when exposed to Cu relative to the control.
Fe had no significant impact on EPS production, likely because it plays a metabolic role rather than acting as a stressor. Overall, EPS-mediated tolerance to heavy metals is strain- and metal-dependent, with B. cereus, B. thuringiensis, and B. tropicus showing the most consistent responses.
Under heavy metal stress, most bacterial strains showed poor biofilm formation, except B. thuringiensis, which exhibited a slight increase in OD under Pb exposure (Fig. 3b). This suggests that B. thuringiensis has a relatively higher tolerance, potentially due to its ability to mitigate Pb toxicity.
In contrast, biofilm formation under Ni and Cu was significantly reduced, likely due to metal-induced toxicity (Fig. 3b). This indicates that Ni and Cu may interfere with bacterial growth and biofilm regeneration by damaging essential cellular processes. It appears that all bacteria tested in this study are intolerant to heavy metals, as evidenced by the significant decrease (P < 0.05) in biofilm formation compared to the control.
Among the strains tested, B. cereus and B. thuringiensis exhibited the highest flocculation yields under Pb stress (Fig. 3c). Flocculation activity was also reduced under heavy metal exposure, likely due to direct metal toxicity in broth culture.
3.4 C, N, P & K contents in OPMF inoculated with individual MAMs
The analysis of carbon (C), nitrogen (N), and potassium (K) elements in OPMF inoculated with each MAMs reveals that P content was significantly higher (P < 0.05) in OPMF inoculated with B. cereus compared to the other bacterial treatments and the uninoculated control (OPMF), which is consistent with its identification as a phosphate-solubilizing bacterium (Hamdan et al., 2024). In the meantime, the levels of C, N, and K remained statistically similar across treatments (Fig. 4).
- Carbon (C), Nitrogen (N), Phosphate (P), and Potassium (K) contents in OPMF inoculated with B. cereus, B. thuringiensis, B. tropicus, A. radioresistens, E. cloacae, and P. pasadenensis. Bars with * show a significant difference at P ≤ 0.05 for the element in OPMF inoculated with bacterial species.
3.5 Performance of OPMF inoculated with individual MAMs on duckweed growth
The biofertilizer potential of OPMF inoculated with MAMs was evaluated by tracking the progression of duckweed growth over 15 days (Fig. 5). Statistical analysis based on duckweed growth area on days 0 and 15 showed that all inoculated treatments significantly increased (P < 0.05) duckweed growth on day 15 compared to the uninoculated OPMF control (Fig. 6). Among the treatments, OPMF inoculated with A. radioresistens showed the highest (P < 0.05) growth of duckweed, compared to that of B. cereus, P. pasadenensis, and the control. This improvement in the duckweed growth area can be attributed to the activity of the MAMs when used in the biofertilizer. In contrast, uninoculated OPMF showed a decline in duckweed growth, likely due to insufficient nutrient release in the absence of the MAMs.
- Duckweed growth from Day-0 to Day-15 in OPMF inoculated with: (a) B. cereus, A. radioresistens, or B. thuringiensis; (b) E. cloacae, B. tropicus, or P. pasadenensis. OPMF without bacterial inoculation was used as a control.
- Duckweed growth from Day-0 to Day-15 in OPMF inoculated with: (a) B. cereus, A. radioresistens, or B. thuringiensis; (b) E. cloacae, B. tropicus, or P. pasadenensis. OPMF without bacterial inoculation was used as a control.
- Changes in duckweed (W. globosa) growth area after 15 days of growth on OPMF inoculated with B. cereus, B. thuringiensis, B. tropicus, A. radioresistens, E. cloacae, or P. pasadenensis. OPMF without bacterial inoculation was used as a control. Bars with the same letter for OPMF + each bacteria species are not significantly different at P ≤ 0.05.
3.6 Starch and protein production in duckweed grown on OPMF inoculated with individual MAMs.
Duckweed is a promising renewable resource due to its high starch content, making it valuable for industrial applications such as bioplastic and bioethanol production (Guo et al., 2023). Additionally, due to its high protein content, duckweed holds potential as a future plant-based food source (Yahaya et al., 2022). In this study, starch and protein levels were measured in duckweed grown on OPMF inoculated with various MAMs to evaluate the effectiveness of each strain in enhancing these biomolecules.
The results showed no significant differences in starch content between samples. However, duckweed grown on OPMF inoculated with E. cloacae exhibited significantly higher protein content (P < 0.05) compared to the uninoculated control (OPMF). This suggests that E. cloacae may be a promising candidate for biofertilizer formulations to increase the protein levels in duckweed. This finding aligns with our previous study that identified E. cloacae as a nitrogen-fixing bacterium, potentially contributing to increased nitrogen availability, an essential element for protein biosynthesis in duckweed and other biological organisms (Hamdan et al., 2024) (Fig. 7).
- Changes in starch and protein concentrations (mg/mL) extracted from duckweed (W. globosa) plants after 15 days of growth on OPMF inoculated with B. cereus, B. thuringiensis, B. tropicus, A. radioresistens, E. cloacae, or P. pasadenensis. OPMF without bacterial inoculation was used as a control. Bars with the same letter for OPMF + each bacteria species are not significantly different at P ≤ 0.05.
4. Discussion
Rapid population growth, industrialization, and various human activities have significantly affected the biodiversity, chemical, and physical environment (Islam & Sandhi., 2022). The combined pressures of heavy metal contamination, increased salinity, and climate-driven temperature changes pose severe threats to global agriculture, reducing crop yields and endangering food security. These environmental shifts also strain soil quality and create stressful conditions for plants, limiting their growth and productivity. Innovative biological solutions are being explored to address the pressing challenges of soil degradation and environmental stress, enhancing plant resilience under adverse conditions. Unlike chemical fertilizers, which can harm the environment and degrade soil health over time by altering soil pH, fostering pest resistance, promoting soil acidification, reducing organic matter, and impeding plant growth (Chittora, 2023), biological alternatives present a more sustainable option. Specifically, PGPR have emerged as a promising approach to improve plant stress tolerance. Our focus is now on incorporating MAMs as PGPR into biofertilizer formulations designed to enhance plant growth and provide a sustainable alternative to chemical fertilizers.
MAMs, including nitrogen-fixing and nutrient-solubilizing bacteria, are promising candidates for sustainable biofertilizers (Hamdan et al., 2024; Yahaya et al., 2024). However, their effectiveness depends on their ability to withstand environmental stressors such as salinity, extreme temperatures, and heavy metals. Adaptive traits like EPS production, biofilm formation, and flocculation are key microbial mechanisms for stress tolerance (Morcillo & Manzanera, 2021).
The findings of this study emphasize the resilience of B. cereus, B. thuringiensis, and B. tropicus under moderate stress conditions, particularly at 37°C, 0.5 M NaCl, and exposure to heavy metals such as lead (Pb) and copper (Cu), as evidenced by their significant EPS production. While biofilm formation exhibited an inconsistent pattern among the bacterial strains, each strain responded differently to different stress levels. In the meantime, flocculation activity showed insignificant under Pb exposure, 0.5M NaCl, and 37°C, compared to non-stress conditions in B. cereus, B. thuringiensis, B. tropicus, and P. pasadenensis. These results suggest that these conditions neither enhance nor inhibit flocculation, indicating that the bacteria are resilient under moderate stress. While other stress conditions significantly reduced flocculation activity. Interestingly, the conditions under which flocculation persisted align with the EPS test results, where significant EPS production was observed, reinforcing the connection between EPS synthesis and flocculation. Except for Cu exposure, where flocculation activity was notably reduced, likely due to the toxic effects of high Cu concentrations (0.01 M) in the nutrient broth. Unlike the EPS test, where Cu was incorporated into agar and had limited direct contact with bacterial cells, the direct exposure in broth likely caused toxicity that impaired flocculation.
EPS acts as a “biological glue” that holds bacterial cells together, forming a cross-linked polymer network with pores and channels (Melo et al., 2022). Its sticky nature facilitates bacterial aggregation, promoting the formation of flocs while maintaining their structural integrity and stability. The negatively charged functional groups within EPS interact with positively charged ions like sodium (Na⁺) and lead (Pb2⁺), resulting in charge neutralization, which reduces electrostatic repulsion and enhances flocculation. The efficiency of this process is influenced by the specific binding sites and functional groups present in each bacterial strain’s EPS, which determine their varying effectiveness in interacting with different ions and influence the flocculation outcomes.
The maintained flocculation yield at 0.5 M salinity in this study suggests that the bacteria have adapted to moderate salinity by utilizing sodium ions (Na⁺) to stabilize EPS and facilitate floc formation. Studies by Subramanian et al. (2010) highlighted that divalent cations such as calcium (Ca2⁺) are more effective than monovalent cations (Na⁺) in promoting flocculation. Divalent cations can bridge two negatively charged molecules, reducing repulsive forces and forming more stable flocs. In contrast, monovalent ions like Na⁺ form weaker bonds, leading to less stable flocs that are prone to shearing. This may explain the lack of a significant difference in flocculation yield compared to control conditions. At 37°C, flocculation yields also show insignificant differences from those observed under control conditions (30°C). This consistency indicates that flocculation remains stable within the optimal temperature range of the bacterial strains. Moderate temperatures are known to influence microbial metabolism and enzyme activity, both of which are critical to flocculation. However, elevated temperatures beyond the optimal range can negatively impact flocculation, as seen in past studies where wheat distillery wastewater flocculation peaked at 30°C and declined above 37°C (Diao et al., 2018).
The investigation into stress-tolerant MAMs under conditions of extreme and moderate temperatures, salinity, and heavy metals provides valuable insights into microbial resilience and adaptability. These findings have broader implications for enhancing bioremediation strategies, improving soil health in degraded or contaminated environments, and supporting sustainable agricultural practices, particularly in regions facing climate stress and industrial pollution. Furthermore, the use of MAMs offers promising potential for developing robust microbial formulations in challenging ecological conditions.
However, in this study, the concentration selected for the temperature, heavy metal, and salinity was based on preliminary estimations intended to ensure observable biological responses under controlled conditions. The concentration level may exceed typical environmental levels; however, this approach was chosen to explore potential threshold effects and better understand the biological mechanisms involved. The future studies would benefit from incorporating a range of concentrations that represent real environmental conditions to enhance ecological relevance and applicability. This will allow for a more comprehensive understanding of the system under study.
Following stress characterization, the MAMs were evaluated for nutrient release from OPMF. OPMF is a biomass abundant in oil palm cultivation areas and was used as the inoculum source in biofertilizer formulation. While the current results indicate that CNK release from OPMF is not significantly different under the tested conditions, we acknowledge the potential for further optimization. Future research will focus on improving the formulation and refining the pre-treatment and fermentation conditions of OPMF using stress-tolerant, MAMs to enhance CNPK release and overall process efficiency.
5. Conclusions
Overall, the limited ability of individual microbial strains to release C, N, P, and K from OPMF and to produce high protein and starch contents suggests that future studies must be conducted using a consortium of microbial communities, which may be more effective for biofertilizer development. However, this study highlights the potential of using MAMs, such as A. radioresistens, B. cereus, and E. cloacae, in the development of biofertilizers. However, the formulated biofertilizer must be tested beyond duckweed plants and extended to commercial crops to demonstrate its effectiveness in enhancing nutrient availability and resilience to environmental stresses, making it suitable for sustainable agriculture and improved crop productivity.
Acknowledgement
This research is funded by the Ministry of Higher Education Malaysia under the Fundamental Research Grant Scheme (FRGS/1/2024/STG03/USIM/02/2) with the project titled “Oil Palm Mesocarp Fibre as Bio-Fertilizer: Action of Selected MAMs in Enhancing Nutrient Availability.” We thank Nur Shamsinar Abdul Kadir, Siti Nur Hasyiqin Hadzri, Nur Ain Nabilah Adib, Nur Ain Muhamed Sadura, Nur Athirah Mohd Khairi, Nurul Bazilah Aminuddin, Nuralya Balqis Mohd Hamizi, and Nur Ain Syafiqah Abdul Mutalib for their contributions in conducting the experiment and collecting the data. We also thank the Faculty of Science and Technology, Universiti Sains Islam Malaysia (USIM) for the research facilities and support.
CRediT authorship contribution statement
Nazariyah Yahaya: Designed the project, supervised this study, and reviewed the manuscript. Nur Sobiha Burhan: Performed the study, collected the data, and drafted the manuscript. Shinjiro Ogita, Maryam Mohamed Rehan: Reviewed the manuscript. All the authors have read the manuscript.
Declaration of competing interest
The authors declare that they have no competing financial interests or personal relationships that could have influenced the work presented in this paper.
Declaration of Generative AI and AI-assisted technologies in the writing process
The authors confirm that there was no use of Artificial Intelligence (AI)-Assisted Technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Funding
Ministry of Higher Education Malaysia under the Fundamental Research Grant Scheme (FRGS/1/2024/STG03/USIM/02/2)
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