The growth-promoting effects of sodium alginate-montmorillonite immobilized phosphate-solubilizing bacteria on bermudagrass under salt stress

The growth-promoting effects of sodium alginate-montmorillonite immobilized phosphate-solubilizing bacteria on bermudagrass under salt stress | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 31 March 2026 V1 Latest version Share on The growth-promoting effects of sodium alginate-montmorillonite immobilized phosphate-solubilizing bacteria on bermudagrass under salt stress Authors : linqi Li 0009-0007-4757-6196 , Wenjuan Li , Yuchun li , liping Zhao , hongguo Wang , xingguo Sun , xu Zhang , Shuai Shang 0000-0003-4465-9758 [email protected] , and Jun Wang Authors Info & Affiliations https://doi.org/10.22541/au.177497865.50821560/v1 130 views 85 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract This field study evaluated the ecological effects of immobilized phosphate solubilizing bacteria (PSB) on the remediation of saline-alkali soil. Using Staphylococcus succinus YJ, we developed a sodium alginate-montmorillonite immobilized agent and applied it to bermudagrass grown on saline soil in Dongying City, Shandong Province. Compared to free bacterial liquid and control treatments, both PSB applications significantly altered the structure of the rhizosphere microbial community without reducing species richness. In particular, the immobilized agent outperformed free bacteria by specifically enhancing soil moisture and total nitrogen content, while enriching functional taxa (eg, Pseudomonas, Shewanellaceae) and metabolic pathways related to nitrogen cycling. Although the prediction of FAPROTAX indicated a potential risk of enriching pathogenic functions, immobilized PSB proved more effective in constructing a stable, growth-promoting microecological niche. We conclude that this immobilization technology is a promising strategy for improving nutrient cycling and microenvironment quality in the restoration of saline-alkali land. The growth-promoting effects of sodium alginate-montmorillonite immobilized phosphate-solubilizing bacteria on bermudagrass under salt stress Linqi Li 1,4 , Wenjuan Li 2 , Yuchun Li 3 , Liping Zhao 1 , Hongguo Wang 1 , Xingguo Sun 5 , Xu Zhang 5 , Shuai Shang 1* , Jun Wang 1* 1 College of Biological and Pharmaceutical Engineering, Shandong University of Aeronautics, Binzhou, Shandong, 256600, China; 2 Binzhou Natural Resources and Planning Bureau, Binzhou, Shandong, 256606, China; 3 Yantai Forest Resources Monitoring and Protection Service Center, Yantai, 264000, China; 4 Shandong Key Laboratory of Eco-Environmental Science for the Yellow River Delta, Shandong University of Aeronautics, Binzhou, Shandong, 256600, China; 5 Dongying Shengli Airport, Dongying, Shandong, 257000, China. Corresponding authors e-mails: [email protected] ; [email protected] Abstract: This field study evaluated the ecological effects of immobilized phosphate solubilizing bacteria (PSB) on the remediation of saline-alkali soil. Using Staphylococcus succinus YJ, we developed a sodium alginate-montmorillonite immobilized agent and applied it to bermudagrass grown on saline soil in Dongying City, Shandong Province. Compared to free bacterial liquid and control treatments, both PSB applications significantly altered the structure of the rhizosphere microbial community without reducing species richness. In particular, the immobilized agent outperformed free bacteria by specifically enhancing soil moisture and total nitrogen content, while enriching functional taxa (eg, Pseudomonas , Shewanellaceae) and metabolic pathways related to nitrogen cycling. Although the prediction of FAPROTAX indicated a potential risk of enriching pathogenic functions, immobilized PSB proved more effective in constructing a stable, growth-promoting microecological niche. We conclude that this immobilization technology is a promising strategy for improving nutrient cycling and microenvironment quality in the restoration of saline-alkali land. Keywords: Staphylococcus succinus ; immobilization technology; sodium alginate-montmorillonite; rhizosphere microorganisms; saline-alkali soil; growth-promoting effect; microbial community Introduction Staphylococcus succinus was first isolated and identified from Dominican amber in 1998 (Lambert et al., 1998), with early research focusing primarily on its taxonomic characteristics. In recent years, its value as a plant growth-promoting rhizobacterium (PGPR) has been increasingly recognized due to its excellent phosphate-solubilizing ability. For example, the S. succinus strain NG-9, isolated from the soil of the rhizosphere of the saline-alkali soil, can simultaneously solubilize inorganic phosphorus (up to 450.36 mg/L) and organic phosphorus (up to 333.15 mg/L) under saline-alkali conditions and possesses multiple growth promoting traits such as producing IAA, siderophores, and ACC deaminase. Genomic analysis revealed that it carries genes related to phosphatesolubilization, including the pst phosphate transport system and gdh, and solubilizes insoluble phosphorus by secreting organic acids such as gluconic acid and citric acid (Xie et al., 2025). Furthermore, S. succinus Mp1-Ha3, screened from the poplar rhizosphere, has a strong capacity to solubilize tricalcium phosphate (359.67 mg/L) and promotes the growth of rapeseed (Zeng et al., 2020). The SYF-3 strain, disclosed by Sun et al., has been verified to possess nitrogen-fixing, phosphate-solubilizing, and IAA-synthesizing capabilities, demonstrating significant salt tolerance and growth-promoting effects on crops such as maize and oil sunflower (Sun et al., 2026). This functional conservation within the taxonomy also suggests its similar application value in improving saline-alkali soils. The direct application of free functional bacterial strains in the soil environment often faces challenges such as low survival rates and weak competitiveness. Immobilization technology can effectively protect microorganisms, improving their colonization ability and functional stability in complex soil environments (Wen et al., 2023). Sodium alginate is a commonly used microbial encapsulation material; however, its application is limited by drawbacks such as insufficient mechanical strength and high swelling rate (Qu et al., 2016). Recent studies have found that introducing montmorillonite into the sodium alginate system can significantly improve the properties of the material. For example, adding montmorillonite to a polyvinyl alcohol/sodium alginate (PVA-SA) system to immobilize ammonia-oxidizing bacteria not only improved the mechanical stability and impact resistance of the microspheres but also significantly improved the bacteria tolerance to heavy metals (Zhang et al., 2025a). The structure of montmorillonite not only provides physical support, but its cation exchange sites can also enrich nutrients and promote biofilm formation, further enhancing the resistance to stress and functional stability of the microorganisms. Current research on the immobilization of phosphate-solubilizing bacteria has primarily focused on the impact of single carrier materials on bacterial survival rates. There is a lack of applied research on the ecological functions of immobilized microorganisms and the resulting plant growth responses, including root development and biomass accumulation. Therefore, this study immobilized Staphylococcus succinus using a sodium alginate-montmorillonite composite carrier to investigate its impact on soil biological activity and the stability of its ecological functions. Materials and Methods Source of Bacterial Strain The bacterial strain dominant in the rhizosphere was obtained from the rhizosphere soil of Suaeda salsa using enrichment culture and gradient dilution methods. The strain exhibits capabilities for nitrogen fixation, iron production, phosphate solubilization, and phytohormone production, with a maximum phosphate solubilization capacity of 130.83 mg/L and an IAA production of 19.25 mg/L. Based on comprehensive morphological characteristics, physiological and biochemical properties, and 16S rDNA sequence analysis, the strain was identified as Staphylococcus succinus and named Staphylococcus succinus YJ. To ensure traceability of the strain resource, it has been deposited at the China General Microbiological Culture Collection Center (CGMCC) under the accession number CGMCC No. 36148. Preparation of Bacterial Suspension and Immobilized Beads Preparation of bacterial suspension: A single colony was inoculated in 5 ml of liquid LB medium and cultured at 28 ° C with shaking at 180 r/min for 16 h to obtain the seed culture. The primary seed culture was then transferred at a 3% inoculation volume to 100 mL of fresh liquid LB medium and cultured under the same conditions until the logarithmic phase. Culture was centrifuged at 8000 r/min for 10 min at 4 ° C and the supernatant was discarded. The bacterial cells were washed twice with phosphate buffer (PBS, 0.1 M, pH=7.0) to remove residual medium. Cells were resuspended in an appropriate volume of sterile distilled water and gently pipetted to achieve homogeneity. Preparation of immobilized beads: Bacterial seed culture was inoculated at 3% volume in liquid LB medium and cultured with shaking until the logarithmic phase. Under sterile conditions, the culture was transferred to a conical flask containing 1 g of sterilized montmorillonite for adsorption. Following adsorption, 2% sodium alginate (w / v) was weighed, dissolved in a small amount of deionized water with heating, and stirred magnetically until completely dissolved to form a transparent viscous solution. After cooling, the montmorillonite-adsorbed bacterial suspension was added, and the mixture was continuously stirred for dispersion. The mixture was then extruded using a 10 mL sterile syringe into a 3% CaCl₂ solution for bead formation. The beads were cross-linked in a refrigerator at 4 ° C for 24 h, repeatedly rinsed with phosphate buffer, and stored in a refrigerator at 4 ° C for subsequent use. Experimental Design The experiment was carried out at Dongying Shengli Airport, Shangfei Avenue, Yong’an Town, Kenli District, Dongying City, Shandong Province. The soil was characterized as a typical saline-alkali, infertile soil. The basic physicochemical properties of the soil layer 0-20 cm were: pH 7.84, electrical conductivity 10.67 mS/cm (equivalent to a salt content of approximately 0.71%), organic matter content 4.78, total nitrogen 0.34 g / kg and available phosphorus 16 mg/kg. In the field experiment, a randomized block design was used, featuring three treatment groups, each with three replicate blocks: (1) a control group (A) without bacterial agent added; (2) a free bacterial liquid treatment (B), where a suspension of S. succinus YJ was irrigated at a rate of 1.5 mL/m²; and (3) an immobilized bacterial agent treatment (C), where an equivalent amount of S. succinus YJ was formulated into immobilized sodium alginate-montmorillonite beads and applied to the subsurface soil layer before planting. To compare the colonization effectiveness of the target functional strain in the field environment of saline-alkali, rhizosphere soil of the established plant, bermudagrass ( Cynodon dactylon ), was systematically sampled 15 days after application of each treatment (free bacterial liquid, immobilized bacterial agent and blank control). Sequencing and Bioinformatics Analysis Samples were sent to Biomarker Technologies Corporation (Qingdao, Shandong Province, China). After total DNA extraction from the samples, the hypervariable region V3-V4 of the 16S bacterial rRNA gene was amplified using universal bacterial primers: forward primer ACTCCTACGGGAGGCAGCA and reverse primer GGACTACHVGGGTWTCTAAT. After amplification, the products were purified, quantified, and normalized to construct a sequencing library. The constructed library was subjected to quality inspection and qualified libraries were sequenced on the Illumina NovaSeq 6000 platform.Paired end sequences were assembled and chimeras were removed (UCHIME, version 8.1) to obtain final high-quality sequences for subsequent analysis. After delineating features (ASV), taxonomic annotation and classification of the operational units were performed, and the number of tag sequences at various taxonomic levels (phylum, genus, etc.) for each sample was tallied. Simpson’s index was used to assess species diversity within samples, and box plots of the ACE index were generated to reflect differences in species richness among samples and their significance. Subsequently, nonmetric multidimensional scaling (NMDS) based on Bray-Curtis distance was employed to analyze the beta diversity of communities across different treatments, providing a visual representation of sample composition similarity. Simultaneously, a permutational multivariate analysis of variance (PERMANOVA) was conducted to statistically test for differences in beta diversity among treatment groups. To elucidate the community composition of each sample, bar charts of species distribution at different taxonomic levels (phylum, class, order, family, genus) were generated, revealing the dominant bacterial genera and their relative abundances in each treatment group. Furthermore, linear discriminant effect size analysis (LEfSe) was performed to identify biomarker bacterial taxa with significant differences in enrichment between groups. Finally, based on the taxonomic information obtained at the genus/species level, the prediction of ecological functions was carried out using the FAPROTAX database to analyze the potential ecological environmental functions of microbial communities. Results and Analysis Analysis of Shared and Unique ASVs in Rhizosphere Microbial Communities Under Different Treatments As shown in Figure 3.1, the bacterial liquid treatment group exhibited the highest number of unique ASVs, while the control group had the fewest. The control and bacterial liquid groups shared a set of common ASVs. The bacterial liquid treatment induced a restructuring of the rhizosphere microbial community, with its highest number of unique ASVs indicating that the exogenous bacterial agent and the resulting microenvironmental changes enriched specific bacterial taxa. The control group had the fewest unique ASV, reflecting the strong filtering effect of saline-alkali stress on the community structure. The substantial number of ASVs shared between the control and bacterial liquid groups suggests that treatment preserved the core indigenous microbial community adapted to local stress conditions. These results indicate that the primary ecological effect of the addition of bacterial liquid was the introduction and enrichment of taxa, rather than their replacement. Alpha Diversity Analysis of Rhizosphere Microbial Communities under Different Treatments To investigate whether there were differences in the alpha diversity of rhizosphere microorganisms under the different treatments in the experimental field, we used the Simpson index and the ACE index to assess species diversity and richness, respectively (Figure 3.2). Independent samples t-tests revealed that the Simpson index in both the bacterial liquid treatment group and the immobilized bacterial agent treatment group was significantly lower than in the control group (p0.05), suggesting that the species’ richness did not undergo significant changes. Beta Diversity Analysis of Rhizosphere Microbial Communities under Different Treatments The NMDS plot based on the Bray-Curtis distance (Figure 3.3) revealed a clear spatial separation between the control group and the two treatment groups, visually reflecting the restructuring of the microbial community induced by the addition of bacterial liquid or immobilized agents. PERMANOVA analysis further confirmed that exogenous amendments significantly altered the structure of the microbial community of the rhizosphere (R2 = 0.641, p = 0.001) (Figure 3.4). Integrating these findings with the results of Alpha diversity (decreased Simpson index but unchanged ACE index), we hypothesize that the introduction of the bacterial agents did not increase the number of new species. Instead, it strongly selected specific taxa, enabling them to become dominant groups. This process altered the composition of the community (Beta diversity) and evenness (Alpha diversity) without significantly changing the total richness of species. Analysis of Differences in Rhizosphere Microbial Community Species Composition under Different Treatments The structures of the microbial community in the bacterial liquid treatment group and the immobilized bacterial agent treatment group exhibited regular patterns of variation. At the phylum level (Figure 3.5A), the bacterial liquid group was distinguished primarily from the control group by enrichment of Desulfobacterota and Patescibacteria, with relative abundances reaching 3.16% and 3.19%, respectively, approximately 1.8 times and 2.4 times higher than those in the control group. Furthermore, this group exhibited the lowest proportion of unclassified bacteria (2.43%). In contrast, the immobilized group was characterized by the highest abundance of Proteobacteria (48.57%) and the highest proportion of unclassified bacteria (5.02%) compared to the control group. Furthermore, the abundance of Firmicutes in the immobilized group (2.63%) was significantly higher than that in the control group (1.61%). In summary, the bacterial liquid group was marked by high abundances of Desulfobacterota and Patescibacteria, whereas the immobilized group was characterized by elevated levels of Proteobacteria, Firmicutes, and unclassified bacteria. At the genus level, distinct differences in bacterial composition were observed among the three treatment groups (A, B, and C). Compared to the control group, the immobilized group was characterized by high relative abundances of Pseudomonas (4.10%) and Shewanella (4.08%), with its combined proportion approximately 5.5 times higher than that of the control group, indicating a proliferation of fast-growing taxa. In general, the immobilized group tended to favor rapidly growing eutrophic bacterial groups. On the contrary, the bacterial liquid group represented a unique type, simultaneously harboring photosynthetic bacteria, certain functional bacteria, and a high proportion of Sphingomonadaceae (3.62%). This composition reflects a specific selective effect, resulting in a bacterial community different from that produced under laboratory conditions. Biomarker Analysis of Rhizosphere Bacterial Communities Under Different Treatments LEfSe analysis conducted among the groups (Figure 3.6) revealed distinct enrichment patterns. In the bacterial liquid treatment group, taxa such as Campylobacterales, Arcobacter ellisii , and Sphingomonadaceae were significantly enriched, serving as representative microbial groups for this treatment. The core characteristic taxa of the immobilized treatment group were predominantly concentrated within Pseudomonas , Xanthomonadales and Shewanellaceae, establishing them as dominant components of the community structure in this group. In contrast, the control group exhibited specificity only within Acidobacteriales, with the corresponding taxa showing relatively limited abundance and diversity. FAPROTAX Ecological Function Prediction Analysis The The analysis of FAPROTAX functional prediction analysis (Figure 3.7) indicated that, compared to the control group, the bacterial liquid treatment group exhibited a significant increase in gene abundance associated with chemoheterotrophic functions. Specifically, the predicted abundances of aerobic chemoheterotrophy and chemoheterotrophy were significantly elevated (p < 0.05 for both). In contrast to this trend, photoautotrophic functions were inhibited, with decreased abundances observed for all functional genes related to photosynthesis. This was manifested as significantly reduced abundances of photoautotrophy, phototrophy, and oxygenic photoautotrophy compared to the control group (p<0.05). Compared to the control group, immobilized treatment induced a significant restructuring of the functions of the microbial community (Figure 3.8). This suite of changes collectively establishes a potential, multipathway microbial basis for growth promotion. Specifically, in the immobilized group, nitrogen cycling processes directly related to plant nutrition, including complete denitrification and nitrate reduction, exhibited extremely high statistical significance (p<0.001), indicating enhanced transformation and inorganic nitrogen supply pathways within the system. Simultaneously, significant changes in oxygenic photoautotrophy suggested adaptive adjustments in primary productivity structure, contributing to the maintenance of dissolved oxygen and microenvironmental stability, thereby indirectly supporting root metabolism. Furthermore, metal transformation processes such as manganese and iron respiration were highly significantly regulated, potentially participating in the passivation of heavy metal forms and thus alleviating their toxic stress on plants. Changes in functions, such as the degradation of aromatic compounds, also suggested the potential capacity of the system to mitigate organic pollution pressure. However, the analysis also revealed that the function of the plant pathogen showed an equally high level of differential significance, constituting a potential associated risk. In summary, immobilized treatment shaped a functionally more favorable microbial niche for plant growth by synergistically enhancing nutrient cycling, stabilizing photosynthetic oxygen production, and alleviating environmental stress. However, its application requires a careful trade-off considering the potential enrichment effect on pathogenic microorganisms. Effects on Main Physicochemical Properties of Soil Soil pH measurements taken 30 days after turf restoration (Table 3-1) showed that bacterial liquid treatment reduced soil pH from 7.82 to 7.63, a decrease of 2.43%, while immobilized bacterial agent treatment reduced pH from 7.83 to 7.72, a decrease of 1.40%. This indicates that both the liquid and immobilized inoculation methods improved soil pH. The superior performance of the bacterial liquid treatment may be attributed to the release of high concentrations of active metabolites, which can interact more rapidly with soil colloids to promote the neutralization of alkaline groups. In contrast, although the sustained release characteristics of the immobilized material offer advantages in terms of long-term efficacy, its short-term regulatory effect on soil pH was slightly inferior to that of the free bacterial liquid. Comparison of soil moisture content before and after planting revealed that the addition of free bacterial liquid had a minimal effect on soil moisture (P>0.05), with a change of only 0.97%, indicating that the free bacterial liquid has limited capacity to regulate the soil moisture microenvironment. However, after treatment with the immobilized bacterial agent, the mean soil moisture content increased significantly (rate of change: +15.71%), suggesting that the immobilization treatment effectively retains soil moisture. Regarding changes in soil electrical conductivity, no significant differences were observed between the two treatment methods in their effect on soil soluble salt content. After treatment, EC values in both the bacterial liquid addition group and the immobilized bacterial agent group decreased significantly (P<0.05), with reductions of 6.83% and 6.73%, respectively. This indicates that the addition of growth-promoting bacteria can consistently reduce the electrical conductivity of the soil. Effects on Soil Fertility Indicators at the Site When examining the general changes in soil nutrients, the accumulation of organic matter was relatively modest. The increase in the bacterial liquid addition group (+1.87%) was slightly higher than in the immobilized bacterial agent group (+0.84%), with final values of 4.86 and 4.83, respectively. This result suggests that within a single growth cycle, both treatments had a limited contribution to the organic carbon pool of the soil, and their enhancing effects were not yet fully manifested. On the contrary, the changes in total nitrogen and available phosphorus were more pronounced, and the performance of the two treatment groups differed. Regarding total nitrogen, the immobilized bacterial agent group showed a markedly greater increase (+11.76%) compared to the bacterial liquid addition group (+5.88%), indicating that the immobilization carrier may facilitate a more stable nitrogen fixation function of the bacterial community. The increase in available phosphorus was the most prominent among the three indicators, but the performance of the two groups differed from that observed for total nitrogen and organic matter. The bacterial liquid addition group exhibited an increase in available phosphorus of up to 31.97% (from 16.67 mg/kg to 22.00 mg / kg), exceeding the 21.59% increase observed in the immobilized bacterial agent group (from 17.00 mg/kg to 20.67 mg/kg). On the one hand, this confirms the significant role of growth-promoting bacteria in soil phosphorus activation. On the other hand, it suggests that under the conditions of this experiment, the free bacterial liquid may be more conducive to the short-term release of phosphorus, while immobilized treatment, while more advantageous for nitrogen accumulation, resulted in a relatively more gradual increase in phosphorus levels. Discussion Soil pH is a critical factor that influences nutrient availability for turfgrasses and microbial activity. The results of this study showed that after 30 days of remediation, both inoculation methods with growth-promoting bacteria significantly reduced soil pH, and the treatment with free bacterial liquid exhibited a more pronounced effect (a decrease of 2.43%). This phenomenon may be directly related to the high concentrations of active metabolites (such as organic acids) present in the liquid bacterial treatment. Relevant studies have confirmed that growth-promoting bacteria can secrete organic acids during their metabolism, which can react with alkaline substances in the soil, thus lowering soil pH. Previous research on Bacillus subtilis L5 also found that the addition of bacterial liquid reduced soil pH by 9.13% (Zhang et al., 2025b). When the free bacterial liquid is applied to the soil, the metabolites it carries can rapidly react with alkaline substances such as calcium carbonate in the soil, achieving a greater reduction in pH in the short term. Although the immobilized bacterial agent also exhibited an alkalinity-reducing effect, its sustained-release characteristic dictates that the release of microbial metabolites is gradual, resulting in a weaker short-term regulatory effect on soil pH compared to the liquid bacteria. Regarding the regulation of soil moisture, the two treatment methods displayed distinctly different effects. The immobilized bacterial agent treatment significantly increased soil moisture content by 15.71%, while the bacterial liquid treatment had minimal impact. This fully demonstrates the physical effect of the immobilization carrier. Immobilized bacterial agents prepared with materials such as sodium alginate possess good water absorption and retention properties. Under drought conditions, bacteria can remain dormant within the beads; during irrigation, the beads prevent bacterial leaching. The immobilization material can form tiny ”reservoirs” in the soil, reducing water evaporation and leakage, thus effectively retaining soil moisture. This holds significant application value for areas of ecological restoration prone to drought stress, such as airport slopes. On the contrary, the free bacterial liquid primarily relies on microbial metabolic activities to indirectly influence soil structure, making it difficult to achieve a significant water retention effect within a short period of only 30 days. Concerning the risk of soil salinization, electrical conductivity (EC) monitoring indicated that both growth-promoting bacteria treatments effectively reduced the soluble salt content in the soil (by approximately 6.8%). This finding is consistent with existing research. Previous studies have found that treatment with *Bacillus subtilis* L5 reduced soil CE by 23.64% (Zhang et al., 2025). Inoculation with silicate bacteria has also been confirmed to continuously decrease EC in the surface soil layer. Growth-promoting bacteria improve soil aggregate structure by secreting substances such as extracellular polysaccharides, which promotes the leaching of salts. Moreover, the absorption and assimilation of ions by microorganisms may also contribute to the decrease in EC values. It should be noted that the magnitude of the EC reduction was highly consistent between the two treatment methods, suggesting that the salt reduction is primarily attributable to the function of the microorganisms themselves and is less influenced by the formulation (liquid/immobilized). This field experiment investigated and verified the effects of both the addition of bacterial liquid and the immobilization of bacterial agents on the structure and function of the rhizosphere microbial community. The results showed that neither treatment significantly altered species richness, but both significantly reduced community diversity and induced regular changes in Beta diversity. This aligns with conclusions from similar field experiments, such as those conducted in previous studies inoculating growth-promoting bacteria into the tomato rhizosphere (Li et al., 2022), confirming that both treatments primarily regulated the composition of microorganisms rather than the number of species. In the bacterial liquid addition group, taxa such as Campylobacteria, Arcobacter ellisii , and Sphingomonadaceae were significantly enriched, serving as representative microbial groups for this treatment. Campylobacteria includes many sulfate-reducing bacteria; their activity likely drives the sulfur cycle in microanaerobic zones of the rhizosphere. Functionally potentially coupled with them, Arcobacter ellisii is a typical sulfur-oxidizing bacterium capable of oxidizing reduced sulfide compounds, thus detoxifying the environment and acidifying the local microenvironment (Ma et al., 2022). Pot culture experiments have shown that this acidification can simultaneously reduce alkalinity under laboratory conditions and also help activate insoluble phosphates in the soil, indirectly promoting the acquisition of phosphorus from plants (Jang et al., 2021). Therefore, the addition of bacterial liquid can exert growth-promoting potential by recruiting microbial groups involved in the regulation of the sulfur cycle, environmental detoxification, and direct growth promotion. The core characteristic taxa of the immobilized group were predominantly concentrated within Pseudomonadaceae, Xanthomonadales, and Shewanellaceae. Divergent from the regulatory pathways of liquid treatment, immobilized treatment specifically enriched a microbial community centered on Pseudomonadaceae, Xanthomonadales, and Shewanellaceae in the rhizosphere. This combination collectively shaped a metabolically efficient, functionally synergistic, ”growth-promoting” network. Pseudomonadaceae, as typical rhizosphere growth-promoting bacteria, their enrichment may directly contribute to IAA synthesis, siderophore secretion, and pathogen antagonism, providing direct growth stimulation and protection for plants (Srivastava et al., 2022). Shewanellaceae, renowned for their unique metal respiration and nitrate reduction capabilities, powerfully drive manganese/iron cycling and nitrogen transformation processes in the rhizosphere (Cruz et al., 2007; Fredrickson et al., 2008;Garrido et al., 2023）. This functionally explains the extreme activity observed of related pathways in the predictions and suggests their key role in mitigating metal ion toxicity in saline-alkali soils and improving nitrogen supply. Furthermore, the organic matter degradation ability of some members within Xanthomonadales helps maintain the supply of carbon sources for this highly metabolically active community(Niu et al., 2023; Kumar et al., 2023; Zhong et al., 2025). These groups, supported by the stable physical habitat and sustained nutrient release provided by the immobilization carrier, form mutually beneficial collaborative microconsortia. FAPROTAX functional prediction analysis indicated that both bacterial liquid addition and immobilized treatment significantly impacted the functions of the soil microbial community, but with distinct divergences in their action pathways and functional preferences. The liquid treatment was primarily characterized by a significant increase in chemoheterotrophic functions and inhibition of photoautotrophic functions, whereas the immobilized treatment triggered a more extensive and systematic functional reorganization, involving multiple pathways such as nitrogen cycling, metal respiration, photosynthetic oxygen production, and pollutant degradation. In the bacterial liquid addition group, the abundance of functional genes related to chemoheterotrophy and aerobic chemoheterotrophy increased significantly, while photosynthetic functions such as photoautotrophy and oxygenic photoautotrophy decreased significantly (p < 0.05). This change in metabolic mode reflects a rapid response of the microbial community to the input of easily utilizable carbon sources. The high concentrations of active metabolites and dead cell debris carried by the bacterial liquid itself provide abundant organic substrates for indigenous microorganisms, thus stimulating the explosive proliferation of heterotrophic microbes. Previous research on bacterial diversity in agricultural soils has also found that the carbon metabolism capacity of bacterial groups (including methanol oxidation, fermentation, etc.) was positively correlated with soil organic matter content (Liu et al., 2024). Currently, under conditions of abundant organic matter, heterotrophic microorganisms often gain a competitive advantage, leading to a dilution of the relative abundance and functional contribution of autotrophic microorganisms (such as photosynthetic microbes such as cyanobacteria). Research on mineral photocatalysis has pointed out that phototrophy and chemotrophy are two fundamental energy modes for microbial growth, and some microbes are even capable of switching between them (Lu et al., 2012). Therefore, inhibition of photosynthetic function observed in this study does not signify functional loss but rather a short-term manifestation after reorganization of the community structure. The short-term effect of the liquid bacterial agent lies primarily in activating heterotrophic metabolic pathways in the soil, accelerating the decomposition and turnover of organic matter; however, the recovery of autotrophic functions may require a longer succession time. In contrast, immobilized treatment exerted a more profound and multifaceted regulation on the functions of the microbial community. Concerning the nitrogen cycle, the complete denitrification pathways and the nitrate reduction functions showed extremely significant enrichment (p<0.001), indicating that the immobilization carrier provided a stable ecological niche for nitrogen cycling functional groups. Previous studies immobilizing aerobic denitrifying bacteria using a mixture of polyvinyl alcohol and sodium alginate found that immobilized bacteria exhibited higher total nitrogen removal rates compared to free bacteria (30.2% vs 55.5%, respectively) and maintained stable denitrification performance during multiple reuse cycles (Chen et al., 2021). This demonstrates that immobilization technology can effectively protect functional bacterial groups, enabling them to perform nitrogen transformation functions continuously in complex soil environments. Further research has also confirmed that after preparing Pseudomonas sp. into biochar-based immobilized bacterial pellets, both denitrification efficiency and stability were significantly improved, efficiently removing inorganic nitrogen under various aerobic conditions (Gao et al., 2023). This further corroborates that the microenvironment created by the immobilization carrier can accommodate facultatively anaerobic nitrogen-cycling functional groups, enhancing the overall efficiency of soil inorganic nitrogen transformation and providing plants with a more dynamic nitrogen supply. In terms of metal transformation, the manganese respiration and iron respiration processes were highly significantly regulated (p < 0.001), a finding with important environmental implications. Dissimilatory iron / manganese reducing bacteria can couple carbon oxidation with metal reduction. Fe(III) and Mn(IV), as electron acceptors, possess redox potentials comparable to those of nitrate and play a significant role in the oxidation of organic carbon under anaerobic conditions. Functional genes related to the nitrogen, manganese, iron, and chlorine cycles show the highest expression levels in paleosols during warm and humid periods, closely associated with higher levels of organic carbon in the soil, moisture content, and total nitrogen. In this study, immobilized treatment simultaneously increased soil moisture content (+15.71%) and abundance of metal respiration functions, suggesting that the immobilized bacterial agent may have the additional function of passing heavy metals and alleviating plant toxicity. While the reductive dissolution of iron-manganese oxides typically releases bound nutrients, under microbial mediation, reduced iron and manganese ions can re-form secondary minerals or complex with organic matter, thereby altering the speciation of heavy metals and reducing their bioavailability. Regarding primary production, significant changes in oxygenic photoautotrophic function may be attributed to the surface of the immobilization material that provides an attachment matrix for photosynthetic microorganisms (e.g. cyanobacteria). Research on syntrophic interspecies electron transfer has revealed that under anaerobic and dark conditions, the photosynthetic bacterium Rhodopseudomonas palustris can utilize electrons generated by Geobacter metabolism to drive CO₂ fixation and reduction, achieving electroautotrophic growth (Liu et al., 2021). This finding indicates that photosynthetic autotrophic microorganisms can switch their energy acquisition modes under specific conditions. In the present study, similar micro-environmental gradients might have formed within the immobilized particles, promoting synergy between photosynthetic microorganisms and other functional groups, helping to maintain dissolved oxygen balance in the microenvironment and indirectly supporting root metabolism. However, immobilized treatment also carries potential ecological risks. The highly significant enrichment of plant pathogen functions (p < 0.001) is a risk signal that cannot be ignored. A recent review has systematically outlined the dual nature of growth-promoting bacteria: On the one hand, they promote plant growth through mechanisms such as nitrogen fixation, phosphate solubilization, and hormone production; on the other hand, improper use can lead to plant hormone imbalance, production of phytotoxic metabolites, disruption of microbial diversity, and transmission of pathogenicity via horizontal gene transfer (Etesami, 2025). Another study that performed whole genome sequencing on endophytic bacteria with growth-promoting potential found that most strains were identified as multidrug resistant, also containing virulence factors associated with pathogenicity, biofilm formation, and root colonization (Youseif et al., 2025). The enrichment of pathogenic functions in this study could be because the protective effect of the immobilization carrier also benefits certain opportunistic pathogens, or because the substantial proliferation of growth-promoting bacteria alters the composition of root exudates, indirectly enriching some opportunistic pathogenic bacteria. In summary, liquid treatment and immobilized treatment exhibit complementary characteristics in functional regulation: the liquid treatment excels at rapidly activating heterotrophic metabolism, driving organic matter turnover; whereas the immobilized treatment shapes a functionally more comprehensive growth-promoting microecological niche by coupling various functions such as nutrient cycling, photosynthetic oxygen production, metal passivation, and pollutant degradation. In practical applications, the two inoculation methods can be selected or combined according to remediation goals. Simultaneously, long-term evaluation and monitoring of the risk of pathogen enrichment associated with immobilized bacterial agents are necessary. Conclusions This study systematically evaluated, through field experiments, the effects of free and immobilized Staphylococcus succinus YJ on the structure of the bermudagrass rhizosphere microbial community, the physicochemical properties of the soil, and the ecological functions of the soil saline-alkali. The results demonstrated that both treatments significantly altered the Beta diversity of the rhizosphere microbial community without significantly changing species richness. Specifically, bacterial liquid treatment enriched functional groups such as Campylobacteria and Sphingomonadaceae, while immobilized treatment significantly enriched ”growth-promoting” microorganisms, including Pseudomona and Shewanellaceae. Regarding soil improvement, both treatments significantly reduced soil pH and electrical conductivity. Immobilized treatment significantly increased soil moisture content (+15.71%) and total nitrogen content (+11.76%), while bacterial liquid treatment exhibited a more pronounced effect on increasing available phosphorus (+31.97%). The prediction of ecological function revealed that the bacterial liquid treatment primarily activated chemoheterotrophic metabolism, while the immobilized treatment systematically enhanced multiple ecological functional pathways, including nitrogen cycling, metal respiration, photosynthetic oxygen production, and pollutant degradation, thereby constructing a more stable growth-promoting microecological niche. However, this was also accompanied by a potential risk of pathogen function enrichment. In conclusion, immobilized phosphatesolubilizing bacteria with sodium alginate-montmorillonite offer significant advantages in improving the microenvironment of the saline-alkali soil and enhancing nutrient cycling efficiency, presenting a promising ecological restoration technology for saline-alkali lands. Data availability statement The raw sequence data was deposited in the NCBI Sequence Read Archive (SRA) database under the BioProject number PRJNA1431739 and the Biosample numbers SAMN 56320433-SAMN 56320441. Conflict of Interest The authors declare no conflict of interest. References Cruz-García, C., Murray, A. E., Klappenbach, J. A., et al. (2007). Respiratory nitrate ammonification by Shewanella oneidensis MR-1. Journal of Bacteriology, 189(2) , 656-662. Chen, J. L., Liu, F., Zhang, S. N., et al. (2021). Reduction of accumulated nitrite by immobilized Alcaligenes faecalis WT14. Technology of Water Treatment, 47 (11), 65-70. Etesami, H. (2025). The dual nature of plant growth-promoting bacteria: Benefits, risks, and pathways to sustainable deployment. Current Research in Microbial Sciences, 9 , 100421. Fredrickson, J. K., Romine, M. F., Beliaev, A. S., et al. (2008). Towards environmental systems biology of Shewanella . Nature Reviews Microbiology, 6 (8), 592-603. Garrido-Sanz, D., Čaušević, S., Vacheron, J., et al. (2023). Changes in structure and assembly of a species-rich soil natural community with contrasting nutrient availability upon establishment of a plant-beneficial Pseudomonas in the wheat rhizosphere. Microbiome, 11 (1), 214. Gao, Y., Zhu, J., Wang, K., et al. (2023). Discovery of a heterotrophic aerobic denitrification Pseudomonas sp. G16 and its unconventional nitrogen metabolic pathway. Bioresource Technology, 387 , 129670. Jiang, H. H., Li, J. Q., Chen, G., et al. (2021). Research progress on phosphate-solubilizing microorganisms and their application in saline-alkali soil. Soils, 53 (6), 1125-1131. Kumar, A., Rithesh, L., Kumar, V., et al. (2023). Stenotrophomonas in diversified cropping systems: Friend or foe? Frontiers in Microbiology, 14 , 1214680. Lambert, L. H., Cox, T., Mitchell, K. E., et al. (1998). Staphylococcus succinus sp. nov., isolated from Dominican amber. International Journal of Systematic Bacteriology, 48 (Pt 2), 511-518. Lu, A., Li, Y., Jin, S., et al. (2012). Growth of non-phototrophic microorganisms using solar energy through mineral photocatalysis. Nature Communications, 3(1) , 768. Liu, X., Huang, L., Rensing, C., et al. (2021). Syntrophic interspecies electron transfer drives carbon fixation and growth by Rhodopseudomonas palustris under dark, anoxic conditions. Science Advances, 7(27) , eabh1852. Li, F., Zhou, F. Y., Zhang, G. Z., et al. (2022). Effects of growth-promoting bacteria on the bacterial community in tomato roots under substrate cultivation. Microbiology China, 49 (2), 583-597. Liu, Y. J., Wang, C. B., Zhang, T., et al. (2024). Bacterial diversity exploring and functional prediction in ancient rice original-producing region of Wannian County, China. Chinese Journal of Eco-Agriculture, 32(3) , 380-390. Ma, Q. L., Du, H., Liu, Y., et al. (2022). Diversity of sulfate-reducing bacteria in mangrove wetlands and their roles in driving elemental coupling mechanisms. Acta Microbiologica Sinica, 62 (12), 4606-4627. Niu, H. Y., Guo, J. Y., Chen, B. Z., et al. (2023). Screening, degradation characteristics and metabolic pathway of a pyridine-degrading composite microbial system MD1. Acta Microbiologica Sinica, 63 (11), 4330-4343. Qu, F. N. (2016). Study on the protective effect of sodium alginate microcapsules on Lactobacillus plantarum ST-Ⅲ (Doctoral dissertation). Hubei University of Technology. Srivastava, P., Sahgal, M., Sharma, K., et al. (2022). Optimization and identification of siderophores produced by Pseudomonas monteilii strain MN759447 and its antagonism toward fungi associated with mortality in Dalbergia sissoo plantation forests. Frontiers in Plant Science, 13 , 984522. Sun, Y., Xie, W., Zhao, J., et al. (2026). A halotolerant and growth-promoting Staphylococcus succinus SYF-3 strain and its application (China Patent No. CN121574864A). Shihezi University. Wen, S., Liu, H., Yang, R., et al. (2023). Immobilization of Bacillus thuringiensis and applicability in removal of sulfamethazine from soil. Environmental Pollution, 333 , 122080. Xie, X., Xu, Z., Gan, L., et al. (2025). Simultaneous organic/inorganic phosphate solubilization by Staphylococcus succinus NG-9 under saline-alkali conditions: Insights into its characteristics, mechanisms, and potential applications. Microbiology Spectrum, 13 (9), e00490-25. Youseif, S. H., Abd El-Megeed, F. H., Soliman, M. S., et al. (2025). Nodules-associated Klebsiella oxytoca complex: Genomic insights into plant growth promotion and health risk assessment. BMC Microbiology, 25 (1), 294. Zeng, Q., Han, X., & Bao, J. (2020). A strain of inorganic phosphorus-solubilizing bacterium from poplar rhizosphere and its application (China Patent No. CN110938556A). Huaiyin Institute of Technology. Zhang, Z., Zhou, K., Garcia-Meza, J. V., et al. (2025a). Montmorillonite enhances the remediation of lead (II) by polyvinyl alcohol/sodium alginate/ Chlorella sorokiniana FK microcapsules. Applied Clay Science, 273 , 107847. Zhong, Y. Q., He, X. L., Li, Y. H., et al. (2025). Enhancing antibiotic removal in constructed wetlands: A Mg/Fe-LDHs-based strategy for optimizing microbial communities and metabolic functions. Journal of Hazardous Materials, 488 , 137412. Zhang, M. X., Zhao, M. M., Guo, Z. X., et al. (2025b). Study on the growth-promoting mechanism of Bacillus subtilis L5 and its effect on the remediation of saline-alkali soil. Journal of Lanzhou Jiaotong University, 44 (5), 136-148. Supplementary Material File (figure.docx) Download 1.39 MB File (table.docx) Download 16.24 KB Information & Authors Information Version history V1 Version 1 31 March 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords ecological experiment ecosystem ecosystem ecology ecosystem function sequencing Authors Affiliations linqi Li 0009-0007-4757-6196 Shandong University of Aeronautics View all articles by this author Wenjuan Li Binzhou Natural Resources and Planning Bureau View all articles by this author Yuchun li Yantai Forest Resources Monitoring and Protection Service Center View all articles by this author liping Zhao Shandong University of Aeronautics View all articles by this author hongguo Wang Shandong University of Aeronautics View all articles by this author xingguo Sun Dongying Shengli Airport View all articles by this author xu Zhang Dongying Shengli Airport View all articles by this author Shuai Shang 0000-0003-4465-9758 [email protected] Binzhou University View all articles by this author Jun Wang Binzhou University View all articles by this author Metrics & Citations Metrics Article Usage 130 views 85 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation linqi Li, Wenjuan Li, Yuchun li, et al. The growth-promoting effects of sodium alginate-montmorillonite immobilized phosphate-solubilizing bacteria on bermudagrass under salt stress. Authorea . 31 March 2026. DOI: https://doi.org/10.22541/au.177497865.50821560/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.177497865.50821560/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9fe5db5aaa30f047',t:'MTc3OTIyMjU5OA=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();