Bacillus-Based Biofertilizer Influences Soil Microbiome to Enhance Soil Health for Sustainable Agriculture

Abstract

Identifying natural alternatives to conventional chemical fertilizers is critical to preventing the widespread soil degradation and environmental damage caused by modern agriculture. Microbe-based biofertilizers have emerged as promising candidates due to their natural ability to improve nutrient bioavailability and promote plant growth. However, how biofertilizers affect the soil microbiome remains unclear. To investigate the impact of biofertilizer application on soil microbiome, LNP-1, a strain of Bacillus subtilis, was used as a biofertilizer in conjunction with no fertilizer, organic fertilizer, and chemical fertilizer for the cultivation of cabbage. Soil samples were collected and analyzed using next-generation sequencing to determine microbial abundance and diversity. Our results showed that LNP-1 supplementation not only improved cabbage yield significantly but also improved soil microbe diversity, a key indicator of soil health. Overall, soils treated with LNP-1 showed the enrichment of microbes involved in nutrient cycling and plant growth when compared to untreated groups. Notably, the yield of organically fertilized cabbage plants increased by 39.7% when treated with LNP-1. These results therefore demonstrate the potential for using biofertilizers to establish a more well-rounded, multifunctional soil microbiome to reduce reliance on chemical inputs and achieve high crop yield sustainably.

1. Introduction

As global population continues to grow, agricultural output must also be improved to keep up with the rising demand for food. However, there are escalating concerns over the environmental impact and long-term viability of conventional farming methods [1,2]. In particular, the over-application of agrochemicals such as fertilizers and pesticides severely degrades soil quality over time [3,4,5]. Soil plays a crucial role in plant growth by not only providing a physical interface between the plant and the environment and essential nutrients but also supporting a microbiological niche that fosters healthy plant development [6]. The excessive use of chemical fertilizers to support intensive cropping is now known to alter the physical, chemical, and biological properties of soil, causing substantial soil degradation. Chemical fertilizers have been shown to accelerate soil acidification, thereby disrupting the availability of various macro- and micronutrients and impeding plant growth [7,8]. Additionally, the usage of agrochemicals has been shown to cause the accumulation and concentration of mineral salts. This leads to soil compaction, a condition that significantly reduces soil permeability and water availability [9] and contributes heavily to soil erosion [10]. Furthermore, chemical fertilizers skew the natural distribution of soil microbiota by providing large quantities of certain elements such as nitrogen and phosphorus while adding little organic matter, limiting soil biodiversity and thus inhibiting key soil functions [11]. It is clear that the deleterious effects of over-reliance on agrochemicals will soon begin to outweigh any short-term benefits, and identifying a sustainable alternative to conventional fertilizers that does not jeopardize soil health is therefore of utmost importance.

Biofertilizers, or microbial inoculants designed to improve plant productivity, have emerged as promising natural substitutes for traditional chemical-based fertilizers. Biofertilizers harness the biological mechanisms inherent to microorganisms to improve crop productivity. In addition to solubilizing essential plant nutrients nitrogen, potassium, and phosphorus, biofertilizers provide additional benefits such as metabolite production [12] and defense against plant pathogens [13], and therefore represent an attractive option for effectively reducing the use of chemical fertilizers. Numerous studies have demonstrated the possibility of alleviating chemical fertilizer use while still achieving high crop yield by integrating biofertilizers during cultivation. For example, the joint application of Azotobacter strains with synthetic fertilizer resulted in improved yield in cucumber plants while reducing chemical use [14]. Another report achieved an increased yield in rice crops while applying only 50% of the recommended chemical fertilizer by adding Bacillus strains to the fertilizer [15]. Furthermore, biofertilizers show potential for improving the efficacy of organic fertilizers, another major pillar of sustainable farming. A combined formulation of N-, P-, and K-solubilizing bacteria including Rhizobacter, Bacillus, and Frateuria greatly augmented the efficiency of organic fertilizer, resulting in increased yield and nutrient content in dill plants when compared to plants treated with organic fertilizer alone [16]. Despite the many studies demonstrating the potential for increasing crop yield by implementing biofertilizers, the impact that microbial inoculants have on existing soil microbiomes remains unclear. Soil microorganisms play a crucial role in determining several significant factors including nutrient availability, resistance to biotic and abiotic stress [17], and susceptibility to pathogens [13], and their combined distribution and abundance can be used as a key indicator of soil health [18]. Indeed, the soil microbiome has been recognized as a critical element that must be properly maintained in order to achieve long-term viability [19]. However, investigations regarding the effects of biofertilizer application on soil, either with or without chemical fertilizer, are scarce. Therefore, the true impact of biofertilizers on sustainability has yet to be determined.

The goal of this study is to elucidate the effects of biofertilization on soil microbiome by conducting a comparative analysis of biofertilizer application across various fertilization regimens. LNP-1, a strain of Bacillus subtilis recently identified for its anti-fungal properties, was used as a biofertilizer to supplement three different fertilization methods: no fertilization, organic fertilization, and conventional chemical fertilization. The effects of LNP-1-supplemented fertilization on the soil microbiome after treatment were analyzed using next-generation sequencing (NGS). Examining the effects of LNP-1 application on soil microbial diversity reveals the interplay between different fertilization methods and biofertilizer supplementation and demonstrates the synergistic effects that might promote enhanced agricultural productivity while preserving soil health and ecosystem balance. Our results highlight the possibility of achieving a more balanced and environmentally conscious model that harmonizes yield optimization with long-term sustainability.

2. Materials and Methods

2.1. Materials and Apparatus

Organic and chemical fertilizers were purchased from Taiwan Fertilizer Co., Ltd. (Tainan, Taiwan). Organic fertilizer consisted of the following composition: total nitrogen (N)—2.3%; total potassium oxide (K₂O)—2.0%; total phosphorus pentoxide (P₂O₅)—2.7%; and organic matter (cow dung, sawdust, and bagasse)—68.7%. Chemical fertilizer consisted of the following composition: total nitrogen (N)—15.0%; total potassium oxide (K₂O)—15.0%; total phosphorus pentoxide (P₂O₅)—15.0%; magnesium oxide (MgO)—3.0%; calcium oxide (CaO)—7.5%; and organic matter—3.0%. Tryptic soy broth (TSB) was purchased from Kingfex Co., Ltd. (Taichung, Taiwan). Bacterial strain Bacillus subtilis LNP-1 was isolated from crop soil samples collected from the Taiwan Agricultural Research Institute. An RNA extraction kit was purchased from Allbio Science Inc. (Taichung, Taiwan). NGS was performed using a MiSeq sequencer (Illumina, San Diego, CA, USA) according to manufacturer protocols.

2.2. Cultivation of Brassica oleracea

The experimental field, situated at the Taiwan Agricultural Research Institute in Taichung, Taiwan (24°01′48.4″ N, 120°41′47.5″ E), was divided into nine plots dedicated to the cultivation of Brassica oleracea. The area of each plot was 270 m². Overall, 175 cabbage seedlings were planted in each plot, roughly 30 cm apart. Among the plots, three distinct fertilization methods were implemented, including no fertilization, organic fertilization, and chemical fertilization. Each plot was subdivided into two sections: one treated with LNP-1 biofertilizer and one without microbial fertilizer treatment (mock). To reduce the effect of local soil factors on any singular treatment group, replicates of each treatment category were arranged using a randomized complete block design. The layout of the groups is shown in Figure 1.

Before cultivation, in each plot where organic fertilization was applied, 125 kg of organic fertilizer was used. In plots subjected to chemical fertilization, 3.6 kg of chemical fertilizer was applied. All fertilizers were applied according to commercial instructions and in accordance with normal farming practices to determine how biofertilizer treatment would interact with commonly used fertilization strategies. Then, 22 days after the application of fertilizer, biofertilizer was applied to the LNP-1 treatment groups.

Following cultivation, 63 heads of cabbage from each treatment group were collected (some plants from the control group were obviously wilted prior to harvest, and were discarded). The average weight of each treatment group was calculated to obtain the yield.

2.3. Culture and Field Application of Bacillus subtilis LNP-1

The bacterial strain LNP-1 was cultivated under standardized laboratory conditions to ensure consistent growth and metabolic activity. The strain was inoculated into TSB. The culture was incubated at 28 °C with constant agitation at 150 revolutions per minute using a rotary shaker. The incubation period was maintained for a total of four days, during which the bacterial cells proliferated and entered a metabolically active phase suitable for downstream applications.

Following incubation, a suspension was prepared by diluting the cultured broth 400× (original concentration: 1 × 10⁹ CFU/mL), and the resulting solution was applied via soil drenching at the base of each plant. In total, 12 L total was applied to each treatment group.

2.4. Soil Sampling and Microbiome Analysis

Soil samples were collected from the soil rhizosphere once before cultivation and once right before harvesting. Soil was collected from the four corners. as well as the center of each plot. at a depth of 10 cm and homogenized for each treatment group. Then, approximately 100 g of each sample was placed in individual bags for the further 16S rDNA amplicon sequencing of microorganisms. After soil samples were collected from the plots, the total genome DNA from the samples was extracted using soil DNA kit, followed by library preparation, Illumina sequencing, and sequencing data analysis.

For library preparation, V3 and V4 hypervariable regions of 16S ribosomal DNA (rDNA) from bacteria were selected and primers were designed at relatively conserved regions bordering the V3 and V4 regions of bacteria 16S rDNA by using forward primers with the sequence “CCTACGGRRBGCASCAGKVRVGAAT” and reverse primers with the sequence “GGACTACNVGGGTWTCTAATCC”. This was followed by ligating 5′ and 3′ adapters to both ends of fragmenting DNA simultaneously. Adapter-ligated DNA fragments were then amplified with a polymerase chain reaction (PCR) in triplicate, followed by a standard Illumina sequencing protocol.

For sequencing data analysis, the VSEARCH clustering tool (v1.9.6) was used for operational taxonomic unit (OTU) clustering with sequence similarity set to 97%. Then, using the Ribosomal Database Project (RDP) classifier Bayesian algorithm to identify OTU taxonomy from domain to genus, we obtained the community abundance of each OTU.

2.5. Microbial Abundance and Diversity Analysis

Microbial alpha diversity indices, including the Shannon diversity index and observed OTUs, were calculated using custom Python scripts (Python v3.9). OTU tables were first normalized to relative abundance before diversity metrics were applied. The Shannon index was computed as the negative sum of the relative abundance of each out, multiplied by its log2-transformed value. Observed OTUs were defined as the number of non-zero OTUs per sample. Boxplots were generated using the Seaborn 0.13.2 and Matplotlib 3.8.3 libraries, and Hedges’ g was used to estimate the effect size between selected sample pairs for each alpha diversity index.

To evaluate differential microbial abundance between experimental groups, log2 fold change and p-values were calculated using two-tailed unpaired t-tests (SciPy). Taxa with an absolute log2 fold change greater than 1 and a p-value less than 0.05 were considered significantly differentially abundant. These results were visualized using volcano plots, where significantly upregulated and downregulated features were highlighted in red and blue, respectively.

All analyses were conducted in Python using the NumPy, Pandas, SciPy, Seaborn, and Matplotlib libraries. Effect size (Hedges’ g) was reported directly using alpha diversity plots for interpretability.

3. Results

3.1. Effect of Biofertilizer Treatment on Crop Yield Under Different Fertilization Strategies

Cabbage plants were organized into 3 different categories based on fertilization method: a control group received no fertilizer, an organic group received fertilizer derived from organic matter, and a chemical group received commercial synthetic fertilizer. Furthermore, each category was either additionally treated with LNP-1 or not additionally treated with anything (mock), resulting in 6 different methods of cultivation. Each method of cultivation was repeated in triplicate for a total of 18 different plots of cabbage plants, as depicted in Figure 1.

The final yield of cabbage plants grown under different treatments is shown in Figure 2. Without LNP-1 treatment, cabbage plants that received no fertilizer (plots A1, B3, and C2) showed the lowest growth of the three groups, while those treated with chemical fertilizer (plots A2, B1, and C3) showed the highest growth. The yield of plants fertilized with organic fertilizer (plots A3, B2, and C1) was significantly greater compared to non-fertilized plants and significantly smaller than that of chemical-treated plants. Notably, while supplementation with biofertilizer did not result in increased yield in non-fertilized plants, LNP-1 treatment significantly improved yield in both organic and chemical-fertilized plants.

3.2. Effect of LNP-1 Biofertilizer on Soil Microbial Diversity

To determine the effect of LNP-1 treatment on the distribution of soil microorganisms and identify possible reasons explaining the improved growth resulting from LNP-1 treatment, soil samples were collected and NGS was performed.

Changes in soil microbiome diversity are shown in Figure 3. In general, the process of cultivating cabbage resulted in significantly fewer observed OTUs when compared to soil prior to cultivation (Figure 3A). Across all three fertilization regimens, treatment with LNP-1 increased the number of observed OTUs. In chemically fertilized soil, LNP-1 treatment prevented significant reduction in species richness, while in organically fertilized soil, LNP-1 treatment resulted in a significant increase in observed OTUs. Cabbage cultivation did not cause any significant change in Shannon index (Figure 3B). However, both control and organic groups showed significantly increased diversity when treated with LNP-1, while chemical groups did not. Figure 3C,D compare species richness and diversity across different treatment regimens, respectively. Both observed OTUs and Shannon index were higher in organically fertilized soil treated with LNP-1 than those treated with any other fertilization strategy. In contrast, the use of chemical fertilizer alone resulted in significantly lower richness and diversity index. Although LNP-1 improved species richness in chemically fertilized soil, the Shannon index was slightly decreased.

3.3. Effect of LNP-1 Biofertilizer on Relative Abundance of Specific Soil Microbial Genera

To further identify specific changes in soil microbiome caused by different fertilization strategies, we performed differential abundance analysis. Changes in soil microbiome resulting from different fertilization regimens are shown in Figure 4. All significantly regulated microbial genera are listed in

Table A1. Control (−): after vs. before.

Taxa	Log2FC	p-Value	Function	Reference
Chryseolinea	3.2456	0.0032	Suppresses plant diseases such as Fusarium.	Y Ou et al. (2019) [39]
Paenibacillus	−19.8761	<0.0001	Nitrogen fixation, reduces fertilizer use and enhances growth.	Liu X et al. (2019) [32]
Minicystis	2.7806	0.0159	Degrades organic matter, enhances nutrient cycling and soil fertility.	Rayapadi et al. (2024) [40]
Panacagrimonas	4.1974	0.0052	Ables to decompose chemical pollutants such as chlorinated hydrocarbons, aides in the breakdown of harmful substances in contaminated soils.	Zhou, Y. et al. (2014) [41]
Fictibacillus	4.7720	0.0084	Degrades lignocellulosic agricultural residues.	YF Chen et al. (2020) [42]
Dongia	1.0233	0.0292	Suppresses the growth of soil-borne pathogens.	Han, L. et al. (2018) [43]
Devosia	1.0811	0.0412	Establishes nitrogen-fixing symbioses with legumes, enhances soil fertility and promotes plant growth.	Rivas et al. (2002) [44]
			Produces indole acetic acid (IAA) that stimulates root development and overall plant vigor.	Chhetri et al. (2022) [45]
			Produce siderophores which chelate iron from the environment, making iron more available to plants.	Chhetri et al. (2022) [45]
			Mitigates the adverse effects of salinity on seed germination, enhances plant resilience to abiotic stresses.	Monjezi et al. (2023) [46]

,

Table A2. Control (+): after vs. before.

Taxa	Log2FC	p-Value	Function	Reference
Chryseolinea	3.8447	0.0022	Suppresses plant diseases such as Fusarium.	Y Ou et al. (2019) [39]
Terrimonas	1.4473	0.0271	Promotes plant growth, mitigates replant disease.	Yim B. et al. (2017) [47]
Paenibacillus	−1.2907	0.0073	Nitrogen fixation, reduces fertilizer use and enhances growth.	Liu X et al. (2019) [32]
Minicystis	3.1712	0.0448	Degrades organic matter, enhances nutrient cycling and soil fertility.	Rayapadi et al. (2024) [40]
Clostridium_sensu_stricto	−19.0959	0.0077	Produces exotoxins in the environment and affect crop growth.	S Jin et al. (2023) [48]
Pseudarthrobacter	−2.2595	0.0239	Identified as aerobic auxin-producing bacterium.	Nanetti et al. (2023) [49]
			Increases the availability of nitrogen, phosphorus, and potassium in growing medium.	Issifu et al. (2022) [50]
			Protects plants from various biotic and abiotic stresses.	Ham et al. (2022) [51]
			Produces siderophores molecules that bind and solubilize iron, making it more accessible to plants.	Mghazli et al. (2022) [52]
Mycobacterium	2.9279	0.0287	Identified as pathogenic bacteria.	B Wang et al. (2023) [53]
Kribbella	−21.3685	0.0341	Plant growth promoting bacteria, as a biocontrol agent.	Mehmood et al. (2022) [54]
Flavihumibacter	−18.5318	0.0331	Growth promotion, disease control and tolerance to abiotic stress.	Seo et al. (2024) [55]
Streptomyces	−3.7314	0.0320	Solubilizes phosphate, making it more accessible to plants.	Chouyia et al. (2022) [56]
			Produces siderophore and making it available for plant growth.	Omar et al. (2022) [57]
Gordonia	20.2721	0.0268	Nitrogen fixation in saline-affected areas.	Kayasth et al. (2014) [58]

,

Table A3. Organic (−): after vs. before.

Taxa	Log2FC	p-Value	Function	Reference
Pseudarthrobacter	−2.9450	0.0094	Increases the availability of nitrogen, phosphorus, and potassium in growing medium.	Issifu et al. (2022) [50]
			Protects plants from various biotic and abiotic stresses.	Ham et al. (2022) [51]
			Produces siderophores molecules that bind and solubilize iron, making it more accessible to plants.	Mghazli et al. (2022) [52]
			Identified as aerobic auxin-producing bacterium.	Nanetti et al. (2023) [49]
Panacagrimonas	20.5296	0.0296	Ables to decompose chemical pollutants such as chlorinated hydrocarbons, aides in the breakdown of harmful substances in contaminated soils.	Zhou, Y. et al. (2014) [41]
Dongia	1.0755	0.0091	Suppresses the growth of soil-borne pathogens.	Han, L. et al. (2018) [43]
			Involves in nutrient cycling in soil.	Jia M. et al. (2022) [59]
Reyranella	2.1292	0.0473	Participates in the reduction of nitrates to nitrogen gas, thus playing a role in the nitrogen cycle within soil ecosystems.	Dhanoa et al. (2023) [60]
Chitinophaga	1.8816	0.0332	Possess a diverse array of enzymes capable of breaking down complex carbohydrates, including chitin and plant cell wall polysaccharides. Facilitates the decomposition of organic matter, enhances soil fertility and structure.	Fernandes et al. (2021) [61]
			Exhibits antifungal activity against plant pathogens like Botrytis cinerea. Potential use as biological control agents to manage plant diseases.	Kim et al. (2023) [62]
Dactylosporangium	20.0412	0.0261	Exhibits antifungal activities against various plant pathogenic fungi.	JY Lee and BK Hwang (2002) [63]
Gordonia	4.9206	0.0197	Nitrogen fixation in saline-affected areas.	Kayasth et al. (2014) [58]

,

Table A4. Organic (+): after vs. before.

Taxa	Log2FC	p-Value	Function	Reference
Noviherbaspirillum	3.7877	0.0056	Identified as denitrifying bacteria.	Peta et al. (2021) [37]
Planifilum	18.4313	<0.0001	Produces enzymes such as xylanases and proteases, break down complex organic compounds into simpler forms. Enhances soil health and promotes plant growth.	Zhang, H. et al. (2021) [36]
Pseudarthrobacter	−1.5332	0.0404	Identified as aerobic auxin-producing bacterium.	Nanetti et al. (2023) [49]
			Increases the availability of nitrogen, phosphorus, and potassium in growing medium.	Issifu et al. (2022) [50]
			Protects plants from various biotic and abiotic stresses.	Ham et al. (2022) [51]
			Produces siderophores molecules that bind and solubilize iron, making it more accessible to plants.	Mghazli et al. (2022) [52]
Rhodomicrobium	3.0364	0.0316	Fixation of atmospheric nitrogen, converting into forms usable by plants.	Eric et al. (2023) [38]

,

Table A5. Chemical (−): after vs. before.

Taxa	Log2FC	p-Value	Function	Reference
Microvirga	−2.3531	0.0399	Nitrogen fixation, solubilizes phosphate and produce siderophores, facilitating nutrient uptake and promoting plant health.	Jimnez-Gmez et al. (2019) [34]
Sphingomonas	−1.1674	0.0358	Synthesizes phytohormones like indole-3-acetic acid (IAA), which promote cell division and elongation in plants.	Lombardino et al. (2022) [35]
			Produces auxins and gibberellins, enhancing plant tolerance to salinity and cadmium stress.	Lombardino et al. (2022) [35]
			Increases number of lateral roots and root hairs of Arabidopsis thaliana, thus improving water and nutrient uptake.	Luo Y. et al. (2019) [64]
Terrimonas	1.6384	0.0393	Promotes plant growth, mitigates replant disease.	Yim B. et al. (2017) [47]
Minicystis	20.4337	0.0180	Degrades organic matter, enhances nutrient cycling and soil fertility.	Rayapadi et al. (2024) [40]
Chitinophaga	−18.8338	0.0229	Exhibits antagonistic properties against plant pathogens. Potential as a biocontrol agent in agriculture.	Kim et al. (2023) [62]

and

Table A6. Chemical (+): after vs. before.

Taxa	Log2FC	p-Value	Function	Reference
Terrimonas	1.6049	0.0050	Promotes plant growth, mitigates replant disease.	Yim B. et al. (2017) [47]
Minicystis	19.8817	<0.0001	Degrades organic matter, enhances nutrient cycling and soil fertility.	Rayapadi et al. (2024) [40]
Nannocystis	2.0670	0.0466	Produces antimicrobial secondary metabolites, suppresses soil-borne pathogens and promote plant health. Potential biocompetitive agent against aflatoxigenic Aspergillus moulds.	Visioli G. et al. (2018) [65]
Fictibacillus	4.2241	0.0011	Degrades lignocellulosic agricultural residues.	YF Chen et al. (2020) [42]
Roseomonas	−18.4296	0.0422	Produces plant-growth-promoting substances such as indole-3-acetic acid (IAA), siderophores, and phytase. Enhances nutrient availability and uptake.	HJ Lee and KS Whang (2022) [66]

.

4. Discussion

Improving the sustainability of modern agriculture is critical to minimizing environmental damage and maintaining the global food supply. The effects of LNP-1 biofertilizer under three different fertilization regimens was investigated to explore the potential of improving both the yield and sustainability of cabbage cultivation.

4.1. LNP-1 Biofertilizer Improves Cabbage Yield

The overuse of chemical fertilizers causes numerous deleterious environmental effects such as soil acidification and water pollution. Organic fertilization has been proposed as a more sustainable alternative to synthetic fertilizers [20]. Despite its lower environmental impact, however, organic fertilization alone results in a substantially lower crop yield [21]. Our results show that supplementing organic fertilizer with LNP-1 biofertilizer significantly improved cabbage yield. The improved yield may be attributed to the innate nutrient-solubilizing ability of Bacillus strains. While organic fertilizers are composed of biomass rich in organic compounds necessary for plant growth, the normally slow breakdown of organic material hinders the fertilization effect. However, bacterial strains such as Bacillus have been shown to participate in processes such as nitrogen fixation and phosphate solubilization, accelerating the introduction of soluble nutrients to soil [22,23]. The combination of organic fertilizer with Azobacter and Rhizobium strains was shown to greatly enhance growth in dill plants [16]. The use of biofertilizer in conjunction with organic fertilizer improved walnut yield [24]. According to our results, supplementing traditional synthetic fertilizers with LNP-1 also resulted in a significant increase in cabbage yield. This increase can likely be attributed to the solubilization of insoluble nitrogen and phosphorus naturally found in soil, augmenting the effects of chemical fertilizer use. Indeed, the combined application of chemical fertilizer and strains of Burkholderia and Herbaspirillum enhanced rice crop yield by improving the bioavailability of soil phosphorus [25]. Therefore, our results highlight the possibility of using biofertilizers such as LNP-1 to improve productivity in both organic and chemical fertilization regimens.

4.2. LNP-1 Biofertilizer Promotes Soil Health by Increasing Microbial Diversity

Soil microbes are critical regulators of both soil health and plant productivity. In addition to nutrient cycling and solubilization capabilities, bacteria also play vital roles in metabolite production [12] and plant defense [26], and the robust diversity of soil microbiota is a key indicator of soil health [27,28]. Traditional farming practices using synthetic fertilizers alter soil physiochemical properties and disrupt microbial distribution, as demonstrated in other studies as well as our data. As expected, after fertilization with only chemical fertilizers, both observed OTUs as well as Shannon index dropped significantly. Notably, this decrease in species richness was prevented when chemical fertilizers were supplemented with LNP-1. Moreover, while organic fertilization alone improved diversity of soil bacteria, combining organic fertilizer with LNP-1 further strengthened microbial distribution, resulting in the highest Shannon index of all nine treatment groups. Similar results were noted in a study on maize rhizosphere, where results exhibited higher richness and diversity following treatment with B. subtilis biofertilizer [29]. Biofertilizers may alter soil community structure in many ways. Nutrient-fixing bacteria such as Bacillus increase the available nutrients in soil, which not only benefits plant growth but may also recruit other nutrient-responsive bacteria not present in soil. For example, B. amyloliquefaciens was found to recruit both Pseudomonadacea and Flavobacteriaceae in rapeseed plants by increasing the available nitrogen in soil environment [30]. Additionally, many soil bacteria demonstrate symbiotic relationships that allow for successful colonization. Pseudomonas are well-known plant growth-promoting bacteria often found in legume nodules, but many species are unable to induce nodule formation on their own and rely on rhizobia to enable colonization [31]. Our data therefore suggests that the use of biofertilizers such as LNP-1 may assist in mitigating the polarizing effect of synthetic fertilizers by improving microbial diversity to maintain soil health, a cornerstone of sustainable agriculture.

4.3. LNP-1 Biofertilizer Modulates Different Bacterial Genera to Improve Soil Function

In addition to improving overall soil diversity, biofertilizers may modulate different types of soil bacteria to enhance specific functions. Following cabbage cultivation, Paenibacillus was reduced drastically (nearly 20 Log2FC) in unfertilized soil not treated with LNP-1. However, treatment with LNP-1 in the same soil largely prevented this decrease. Notably, multiple species of Paenibacillus were found to cycle atmospheric nitrogen into forms accessible to plants [32]. Furthermore, some members of Paenibacillus improve plant growth by producing phytohormones including indole acetic acid [33]. Likewise, in chemically fertilized soil, LNP-1 prevented the significant reduction of Microvirga and Sphingomonas, both of which are involved in nutrient cycling and uptake [34,35]. In addition to mitigating the loss of certain bacteria due to the cultivation process, treatment with biofertilizers can also enrich beneficial bacteria to improve plant growth. In organically fertilized soil, LNP-1 treatment resulted in significant increases in Planifilum, Noviherbaspirillum, and Rhodomicrobium that were not observed in soil not treated with biofertilizer. Planifilum has been shown to accelerate the composting process by releasing enzymes such as xylanases and proteases [36], while both Noviherbaspirillum and Rhodomicrobium are involved in nitrogen cycling and fixation [37,38]. These shifts suggest that LNP-1 does not act merely as a microbial supplement, but as a selective agent that reshapes microbial communities to favor beneficial interactions in soil.

4.4. Limitations of the Study

Although this research highlights the potential for improving the practicality of sustainable agriculture, the number of replicates was limited, and further repeats are needed to fully validate correlation between increased diversity and improved yield. In addition, differences in soil properties could alter the interaction between biofertilizers and existing soil microbiomes. Finally, while we observed significant increases in several important genera, we did not evaluate improvements in soil conditions such as nutrient cycling, etc. Measuring metabolic activity and functional gene expression could further reveal key mechanisms behind soil dynamics in future studies.

5. Conclusions

In this research, we investigated the effects of biofertilizer application on the soil microbiome by using LNP-1 biofertilizer to supplement three different fertilization regimens. LNP-1 significantly improved the yield of cabbage when used in conjunction with both organic and chemical fertilizers. Analysis of soil samples showed that both abundance and diversity of soil microbiota were improved following treatment with LNP-1. Notably, decreased microbial abundance resulting from chemical fertilization was mitigated, while both abundance and Shannon diversity index were substantially increased in organically fertilized soil. Furthermore, beneficial bacteria such as Paenibacillus and Rhodomicrobium were enriched in soil treated with LNP-1, but not in untreated soil. Our results demonstrate the potential to organically establish a robust, multifunctional soil microbiome that promotes plant growth sustainably.