Alabama’s agricultural landscape is shaped by its diverse soil types and climatic conditions, which in turn determine its crop production and ecosystems. The state has seven main types of soil: Appalachian Plateau, Piedmont Plateau, Blackland Prairie, limestone valleys and uplands, floodplains, coastal marshes, and coastal plains (Mitchell, 2018). Among these, the Bama soil (20-35% clay, 45-80% sand, and less than 30% silt) and Decatur silt loam (35-60% clay, 15-35% sand, and 4-20% silt) moderately to highly acidic and thus limiting crop cultivation (Howe and Mitchell, 2010). Microorganisms play key roles in nutrient cycling, detoxification, and regulating soil microclimates (Cheng et al., 2021; Romero et al., 2023) and maintaining microbial diversity which is an effective soil management practice. This emphasis on soil microbial health which is an important strategy in sustainable agriculture for increasing agricultural resilience against challenges such as soil acidity (Msimbira and Smith, 2020).

Organic amendments, biofertilizers, and biopolymers can effectively reduce acidity while adjusting pH levels and enhancing crop productivity by improving soil structure, fertility, and microbial activity. Soil amendments can alter microbial communities and increase their diversity, which improves soil fertility and helps convert nutrients into forms that are readily available to plants (Dincă et al., 2022). The application of biofertilizers (compost and manure) and biopolymers (cork and Extracellular Polymeric Substances/Exopolysaccharide, EPS) has proven effective in enhancing soil fertility, nutrient retention, and promoting healthier crop growth (Lucchetta et al., 2023; Rompato et al., 2024; Redmile-Gordon et al., 2020; Zhang et al., 2024). Integrating such soil amendments with cover cropping (Carrera et al., 2007) not only contributes to sustainable agriculture and crop production but also Alabama’s rich soil tapestry.

Cork, a unique natural biopolymer, rich in suberin, derived from the bark of the cork oak (Quercus suber L.), has been significantly overlooked as a soil amendment despite its considerable potential to improve soil structure and microbial activity (Rompato et al., 2024). Cork improves water retention capabilities and helps buffer soil acidity (Msimbira and Smith, 2020) making it particularly beneficial in regions with higher soil acidity, such as North Alabama and Tennessee Valley. In addition, cork particles contribute to improved soil porosity, facilitating root development and promoting microbial colonization, which collectively improves overall soil health (Rompato et al., 2024). The hydrophobic nature of cork also enables it to improve aeration in heavier soils, such as silt and clay, thereby supporting diverse microbial populations. Consequently, cork presents a promising amendment for fostering improved soil structure and sustainability in high-value agro-horticultural systems (Cheng et al., 2021; Rompato et al., 2024). The unique physical and biological features of cork make it a valuable component in sustainable soil management practices.

The EPS produced by Rhizobium tropici enhances soil health by promoting beneficial microbial interactions, improving soil structure, increasing moisture retention, and facilitating nutrient cycling, all of which are important for maintaining soil fertility and effective agricultural practices (Tisdall and Oades, 1982; Flemming and Wingender, 2010). EPS also contributes to stabilizing soil structure, rendering it more resistant to erosion (Flemming and Wingender, 2010). Microorganisms can be favorably influenced by the synergic effects of soil amendments, such as cork and EPS, which enhance biogeochemical processes that convert organic matter into forms accessible to plants. For example, a few beneficial microbes convert organic phosphates into orthophosphates, enhancing nutrient uptake, particularly in low-pH soils where phosphorus availability is limited (Cheng et al., 2021). The synergistic effect of cork and EPS biopolymers further supports the proliferation of phosphate-solubilizing bacteria, thereby improving nutrient cycling and promoting both soil fertility and microbial diversity (Costa et al., 2018; Rompato et al., 2024; Redmile-Gordon et al., 2020).

The success of 16S rRNA sequencing and analysis is affected by the quality of input DNA, which relies on effective DNA isolation methods. Cell lysis and the removal of impurities in soil are essential for isolating quality DNA (Pu et al., 2025). Soil pH and acidity impact these methods, highlighting the need for standardized protocols. The microbial diversity in the rhizospheric region is likely to improve after soils are amended with biopolymers, which in turn promote soil structure, nutrient recycling, and beneficial interactions between plants and microbes. Using biofertilizer and biopolymer amendments helps increase soil microbial diversity, which supports crop production and reduces our dependence on chemical fertilizers. Despite the known benefits, limited studies have been conducted to assess the changes in microbial communities resulting from the use of biopolymer amendments. In the present study, we aimed to identify an effective DNA isolation method for determining microbial diversity among unamended and amended (cork and EPS) soils using 16S rRNA sequencing.

Soil amendments are important in modulating microbial communities, with their effects on specific bacterial species varying according to the amendment type. Positive impacts include enrichments in soil microbial biomass and biodiversity, which support resistance and resilience within microbial communities (Aguilar-Paredes et al., 2023). Certain amendments can improve beneficial bacterial diversity while concurrently mitigating the prevalence of harmful pathogens. For instance, increased carbon and nitrogen retention in soils has been shown to elevate populations of beneficial bacteria and a decrease in bacterial wilt across Southern China (Chen et al., 2020). A study reported that there is a significant change in rhizospheric compounds and a reduction of bacterial wilt disease when the soil is amended with biochar, suggesting the role of microbial activity in promoting plant health (Shuang et al., 2021). In contrast, organic amendments can negatively affect the resilience of soil microorganisms to temperature fluctuations, resulting in carbon and nitrogen fluxes in podzol soils, which also affected microbial DNA concentrations. These findings from the previous studies suggested that the strategic application of organic amendments is important in ameliorating environmental stress factors, which directly influence natural soil ecosystems (Kok et al., 2023). Our findings suggest that various soil amendments can interfere with DNA isolation processes, leading to low-quality DNA that requires purification. Utilizing the FastDNA Spin Kit, we consistently obtained high-quality DNA from 48 samples across diverse time points and treatments, which outperformed other DNA isolation methods. Isolating high-quality DNA is crucial for sequencing and our downstream microbial community and diversity analyses.

The advent of 16S rRNA amplicon sequencing has revolutionized our understanding of microbial communities across diverse ecosystems, shedding light into microbial biogeography and ecological functions (Arboleda-Baena et al., 2024; Pattnaik et al., 2020; Dubey et al., 2020). For thorough microbiome analysis, it is crucial to classify query sequences into taxonomic categories by comparing their similarity to sequences in established reference databases, such as SILVA, RDP, and Greengenes (Westcott and Schloss, 2017). Notably, amplicon-based sequencing has provided deeper insights into global microbial biodiversity (Schloss et al., 2016; Rideout et al., 2014) compared to whole-genome sequencing methodologies. Consequently, we employed 16S rRNA amplicon sequencing in this study to analyze the microbial community composition and diversity across various time points and treatments. The addition of cork has been shown to enhance soil structural stability by improving aggregate stability (Nwamba, D. C. 2020). EPS, a byproduct from Rhizobium tropici, facilitates soil particle aggregation, ultimately benefiting plants by retaining moisture and sequestering nutrients (Patwardhan et al., 2023; Nivedita et al., 2021). Our study revealed that the combination of cork and EPS significantly influenced soil bacterial diversity as analyzed through amplicon sequencing, which elucidated interspecies relationships and inter-population interactions are critical for plant growth and development.

Microbial diversity is important for nutrient cycling and soil fertility in agriculture. It facilitates nitrogen fixation and decomposition, thereby increasing nutrient availability for crops. Higher levels of microbial diversity are associated with better soil health, including improved respiration and enzymatic activity. Understanding this link is essential for good soil management and sustainable farming. Our study showed that bacterial diversity profiles changed significantly based on the time of sampling and the type of treatment. We measured these changes using the Shannon Diversity Index (SDI) ( Figures 2A and 2B ). Notably, microbial diversity was lowest on Day 0, as indicated by reduced SDI values, but exhibited a gradual increase, showing its maximum on Day 3 ( Figure 2A ). In treatment comparisons, soil.control demonstrated the highest microbial diversity, whereas soil.cork displayed the lowest SDI values, indicating a reduction in microbial richness ( Figure 2B ). Similarly, the addition of EPS has been shown to influence the biofilm integrity and the composition of microbial communities (Qiu et al., 2021). Recent studies have demonstrated that the application of cattle manure and biochar significantly improved bacterial alpha-diversity profiles in tea-planting soils, thereby improving the soil structure (Han et al., 2022). The addition of modified biochar to tea-planting soils resulted in an increase in the bacterial community’s SDI, ACE, and Chao1 indices by 3.05%, 5.07%, and 5.24%, respectively (Hua et al., 2021). Further, research revealed unique differences in bacterial alpha-diversity, represented by Shannon and Chao1 indices, among soils from major land use types characterized as highly contaminated (HC), low contaminated (LC), and non-contaminated (NC) (Li et al., 2022).

A comparative study across three different locations (West Sussex, UK; Lusignan, France; and Prato Sesia, Italy) revealed variations in bacterial beta diversity influenced by biochar treatments when compared with controls. Among the locations, UK and Italy, shown significant changes in bacterial composition and abundance, which attribute to changes in edaphic factors, such as soil pH (Jenkins et al., 2017). Our findings similarly indicated distinct temporal patterns in community clustering based on weighted and unweighted UniFrac distances with PERMANOVA analysis (p < 0.05) across different time points and treatment conditions ( Figure 3A, B ). Another study with the PERMANOVA analysis of weighted UniFrac distances revealed significant differences (p < 0.01) in bacterial community composition at varying levels of RN infestation, with PCoA plots illustrating clear clustering of samples sharing similar bacterial profiles, underscoring the impact of RN infestation on the structure of bacterial communities (Karapareddy et al., 2025). In sites across China (Kaihua, KH; Shenzhou, SZ; and Quzhou, QZ), a study on the temporal dynamics of beta diversity among niche soil communities demonstrated noticeable changes, when these sites have been treated with biogas slurry over three years sequentially and compared against controls (Xing et al., 2023).

Previous studies comparing the compositional and functional profiles of bacterial communities treated with compost (C+) and without compost (C-) soils using 16S rRNA sequencing revealed that Proteobacteria were predominant in both treatments, followed by Chloroflexi, Actinobacteria, Verrucomicrobia, Firmicutes, and Bacteroidetes. Specifically, Firmicutes were dominant in C+ soils, while Chloroflexi exhibited greater prevalence in C- soils (Kim et al., 2021). In this study, our time point analysis (Day 0 to Day 3) demonstrated stability in bacterial composition, with Actinobacteria consistently being the most abundant, followed by Proteobacteria and Acidobacteria ( Figure 5A ). Similarly, our treatment-based comparisons (soil.control, soil.cork, soil.cork.EPS, and soil.EPS) identified Actinobacteria as a dominant phylum ( Figure 5B ). At the class level, from Day 0 to Day 3, composition remained largely stable, with slight fluctuations in the relative abundance of Alphaproteobacteria and Bacilli ( Figure 6A ). Treatment-based analysis indicated consistent microbial class distributions, with Actinobacteria predominating across all treatments ( Figure 6B ). Similarly, another study identified Proteobacteria as the dominant phylum, followed by Actinobacteria and Bacteroidetes when the soils have been amended with Metarhizium (fungus) (Barelli et al., 2020). In watermelon cultivation, the use of composted cattle and chicken manure, combined with bioorganic fertilizers, effectively suppressed Fusarium wilt. Whereas, Proteobacteria, Actinobacteria, Chloroflexi, and Planctomycetes have been determined as dominant phyla, which is similar to findings identified in this study. Further they suggested that bio-organically treated soils may have promoted a few novel microbial communities that modified the rhizosphere composition of watermelon (Ayangbenro et al., 2022).

Furthermore, another study indicated that soil amendments, including bio-organic fertilizers and compost from cow and chicken manure, significantly altered the bacterial community structure compared to untreated control soils, subsequently enhancing watermelon quality and reducing Fusarium incidence (Zhao et al., 2018). In our investigation, results at the family level showed a consistent relative abundance across various time points and treatments, highlighting dominant taxa such as Bacillaceae_1, Gaiellaceae, Micromonosporaceae, and Streptomycetaceae. The microbial composition exhibited stability over time, as depicted in Figure 7A . Additionally, the treatments maintained a generally consistent order of core families in terms of abundance, with minor variations noted ( Figure 7B ). Another study identified profound shifts in bacterial ecology when the soil was amended with varying rates of sewage sludge (0: CK; 30: ST; 75: MT; and 150: HT t/ha) on a dry weight basis. The dominant families identified include Xanthomonadaceae, Hyphomicrobiaceae, and Flavobacteriaceae, with average relative abundances of 36.2%, 9.6%, and 3.8%, respectively (Li et al., 2021).

In our findings, the top 20 core microbiomes consistently featured were Bacillus and Gaiella across all treatments, with their prevalence ranging from 0.9 to 1.0. Other genera such as Thermoleophilum, Conexibacter, Streptomyces, and Solirubrobacter exhibited moderate prevalence (0.4 to 0.8), while taxa like Burkholderia, Bradyrhizobium, and Aquisphaera were less prevalent (0.1 to 0.3) ( Figure 8 ). Furthermore, another study reported core microbiomes across three planting systems (conventional, aerobic, and System of Rice Intensification, SRI) in saline environments identified 43 genera, including Sphingomonas, Sorangium, Nitrospira, Luteitalea, Candidatus_Solibacter, and Bacillus, which were found to have higher prevalence (Rokins et al., 2022). Similarly, research focusing on chemical and organic fertilizers revealed twelve predominant core bacterial genera: Acidobacterium, Bacillus, Bradyrhizobium, Clostridium, Gemmatimonas, Lysobacter, Massilia, Pseudomonas, Rhizobium, Sphingomonas, Stenotrophomonas, and Xanthomonas. Of which, Bacillus showed the highest relative abundance in soils supplemented with neem cake, contrasting with the lowest levels found in soils amended with castor cake (Su et al., 2022). Additional findings demonstrated a strong positive correlation between the relative abundances of Nocardioides, Ilumatobacter, and Gaiella under organic amendment (vermicompost), which effectively inhibited Fusarium oxysporum f. sp. lycopersici during tomato cultivation, compared to rice straw and chicken manure treatments (Zhao et al., 2019).

Certain bacterial genera identified in our study play a critical role in maintaining the health of both plants and soil. For example, Bacillus species aid in nitrogen cycling and breaking down organic matter, which boosts soil fertility and helps plants grow (Nishisaka et al., 2024). Similarly, Gaiella has been implicated in carbon and nitrogen cycling, increasing nutrient availability when treated with long-term organic and inorganic fertilization (Li et al., 2017). Streptomyces play a vital role in organic matter breakdown, contributing to carbon and nitrogen cycling; when introduced to soils with organic amendments like compost, they improve nutrient uptake and support the growth of crops such as cabbage (Brassica oleracea) and tomato (Hu et al., 2021). Burkholderia species are crucial for nitrogen fixation, with their inoculation, along with compost or biochar, shown to enhance nitrogen availability and yields in soybean (Glycine max) (Meier et al., 2021). Thermoleophilum and Solirubrobacter are thermophilic genera that contributes to organic matter decomposition (Schroder et al., 2021; Han et al., 2024), significantly impacting nutrient cycling, especially within thermophilic composting systems, thereby improving soil health and fertility in crops like lettuce (Lactuca sativa).

Furthermore, to understand how microbial diversity changes and its role in ecosystem functioning, it is essential to assess soil health. Key indicators used to evaluate microbial diversity and its ecological roles include microbial biomass, phospholipid fatty acid analysis (PLFA), enzyme activity, organic carbon levels, and nitrogen cycling. Microbial biomass indicates microbial presence and ecosystem resilience, whereas PLFA reveals the composition of microbial communities, which is essential for efficient nutrient cycling. Soil enzyme activities indicate functional diversity, while organic carbon content serves as an indicator for potential carbon sequestration, which is closely linked to microbial diversity. It is also essential to understand the composition and diversity of microbes in nitrogen cycling in order to maximize plant productivity and improve water retention. In this study, we determined microbial composition and diversity using amplicon sequencing and analysis. The cork and EPS amendments together have been demonstrated to aid in improving soil health and increasing agricultural productivity ( Supplementary Table 3 ). Synergistically, these amendments improve soil structure, porosity, and aeration, which contributes to improved water retention and increased nutrient availability. These edaphic changes promote plant growth and facilitate the formation of a rich and diverse microbial community, which is crucial for nutrient cycling. In addition, these amendments can sequester carbon, reduce greenhouse gas emissions, control soil erosion, and lower irrigation needs, particularly in arid and acidic soils. Collectively, these practices contribute to improved soil quality and increased crop yields, thereby supporting sustainable agricultural systems.

Soil microorganisms play a crucial role in biomass production, nutrient cycling, and the decomposition of organic matter. Biopolymer amendments, cork and EPS, enhance soil health by improving its structure and moisture retention. Together, they support microbial communities and resilient ecosystems. Microbial community characterization and the pursuit of rhizosphere ecology are primarily being pursued by next-generation sequencing (NGS) and microbiome investigations. Understanding the impact of organic additives on DNA isolation and microbial composition is imperative. Therefore, this study validated six DNA extraction protocols as a prelude to utilizing NGS to dissect soil microbial composition. After testing these methods on unamended and amended soils, the results show that the FastDNA Spin Kit is the most effective for extracting DNA from diverse microbial populations. Amplicon sequencing of the 16S rRNA gene revealed distinct shifts in the microbial community over time and across treatments. Both α-diversity and β-diversity analyses confirmed significant changes in microbial diversity over time and across treatments. Venn diagram analysis further highlighted the presence of unique, common, and shared ASVs across treatments and time points, emphasizing the dynamic nature of soil microbial communities. In total, this study identified twelve genera through core microbiome analysis across various time points and treatments, highlighting their crucial roles in maintaining soil ecosystem stability, carbon sequestration, nitrogen fixation, sulfur cycling, and phosphorus solubilization. In summary, this study provides valuable insights into the microbial dynamics of amended soils. It first highlights the need for careful selection of DNA extraction methods and then discusses the steps involved in amplicon sequencing followed by phyloseq analysis for reliable microbial profiling. These findings help us understand how soil microbes respond to biopolymers, which can lead to improved sustainable soil management and agriculture.