Recovery, Identification, and Presumptive Agricultural Application of Soil Bacteria

Abstract

Conventional and organic agriculture can both cause soil microbial community structure (SMCS) destruction, infertility, and abandonment. The application of soil productivity-improving biofertilizers is a sustainable practice that requires holistic knowledge, including complex biointeractions, diverse microbial metabolism, and culture requirements, the last of which rely on methodology and technology. In this study, a holistic culture-based and meta-analysis approach was employed to explore pristine and domesticated soils for presumptive plant growth-promoting (PGP) bacteria. Various soil samples were logistically acquired and processed using enrichment and heat alternatives. Morphologically diverse isolates were streak-purified and analyzed for 16S rRNA bacterial identification. Meta-analysis of PGP bacteria in domesticated environments was conducted using Google Search and NCBI PubMed. Soil fertility was analyzed for the pH and nitrogen/phosphorus/potassium (NPK) contents using biochemical tests. Notably, 7 genera and 15 species were differentially recovered, with Bacillus being the most prevalent and diverse in species. Conversely, Aeromonas, Lactobacillus, Lelliottia, Pseudomonas, and Staphylococcus were found only in pristine soil. While soil pH was consistent in all pristine soil samples, NPK contents ranged widely across the pristine (i.e., P/K) and domesticated samples (i.e., N/P/K). These findings could enhance biofertilizer SMCS, function, and effectiveness in the agricultural productivity needed to feed the expanding population.

1. Introduction

Microorganisms, including biofertilizers, are ubiquitous in agricultural environments, contributing to symbiotic roles in their ecosystems, including the host plants [1,2,3]. Through various sites of interactions, including root, phyllosphere, and endosphere [4], beneficial bacteria may contribute to the host’s biotic resistance [5], abiotic resistance [2], nutrient acquisition [6,7,8,9,10,11], growth-promoting soil structure (i.e., improve water- and nutrient-holding capacity) [2], and enhanced growth [12] directly [1,12]. These plant growth-promoting soil bacteria consist of diverse genera and species [2,4] that coexist [2,4] and can synergistically contribute to the health and yield of plants [2,4,13]. Additionally, their availability is environment-dependent and dictated by temperature [14], synthetic fertilizer and pesticide manipulations [15], and the sites of a plant [4]. Both bacterial and fungal genera and species, including the endospore-forming bacteria Bacillus subtilis [16], B. amyloliquefaciens [17], Burkholderia [18], Azotobacter [19], Azospirillum, Paenibacillus, and Pseudomonas [2], as well as arbuscular mycorrhizal fungi [20], are well-reported biofertilizers, in which the combined bacterial-fungal synergistic growth-promoting effect was reported [1]. Among them, Azospirillum spp. [21], Azotobacter [19], and Paenibacillus [22,23] are nitrogen-fixing and nutrient-uptaking beneficial bacteria. Note that artificially formulated microbial formulations (i.e., biofertilizer) that already exist [17,18,19,24,25] in the market could be reformulated per this holistic study.

Soil particles are composites of organic and inorganic (i.e., mineral) compounds, formulated by biological dissimilation (i.e., decomposition) of dead materials, including carcasses, plant components, and microorganisms [1,11,26,27], and biological assimilation of carbon [1] and nitrogen [1,2,6,21], which contribute to an agriculturally fit soil ecosystem and plant growth promotion. The domain of bacteria includes structurally and physiologically variant microorganisms [14], including Gram-positive/-negative [28] and non-endospore-forming/endospore-forming bacteria [29], respectively. Physiological variation (i.e., endospore formation) enables the bacteria to persist in harsh environmental conditions, including drought [30] and heat [31,32], and increases their environmental availability [29]. Separately or synergistically, they serve multiple functions to ensure soil productivity, including stabilizing soil structure [2,27], cycling nutrients [15], holding water [2,33,34], decomposing organic [15] and inorganic (i.e., heavy metal) compounds [2,15], maintaining the availability of limiting nutrients [15], and producing antimicrobial compounds to protect plants [13], thereby enhancing healthy plant growth [2,15]. Healthy plants could be determined by their environmentally dictated capacity to uptake environmental nutrients, including N [2,35,36,37], P [2,38], K [2,39], and water [1,11], and withstand stresses, including biotic and abiotic challenges [1].

U.S. agriculture has a long history of producing major cash crop commodity products, including corn, soybean, wheat, cotton, grain products, and peanuts [40], generating an annual revenue of ~USD 300 billion in 2023 [40], of which corn and soybeans combined contributed more than half of the total crop revenues [41]. The United States, which contributed nearly a tenth of the total annual revenue, is the world’s leading exporter of cotton [42]. According to an agriculture productivity growth study reported by the USDA, U.S. agricultural practices, including dryland (i.e., primary practice) and irrigated [43], have evolved from input-focused to output-focused technologies, including genetically modified, stress-resistant crops, equipment, chemicals, and educated farm organization, which reduced the size of land and labor needed while tripling the total agricultural output [41] between 1948 and 2021 [41]. Necessarily, agricultural innovations have gradually accrued operation/production costs, including increasing differential fertilizer expenses per planted acre (~0.4 hectare) for different crops (i.e., including corn, cotton, soybean, rice, peanuts; USD 7.97–156.51, 2003–2013; USD 25.06–243.28, 2014–2023) [43], totaling USD 300–400 from 2004 to 2010 and USD 400–500 billion from 2011 to 2023 as the technology has progressively evolved [41], thereby increasing the market cost of products, and burdening the small family farm operation financially [44]. Hence, biofertilizers are particularly essential in meeting the need for agricultural innovations to feed a population of nearly 10 billion in 2050 [45].

Cultivation practices, including tilling [46], burning [47], crop rotation [48,49], and nutrient application [15], could lead to erosion-attributed nutrient loss, edible nutrient availability disruption, topsoil removal, and an imbalanced functional ecosystem (i.e., microbial nutrient regeneration), respectively, thereby negatively impacting bacterial community functional structure [50] and plant health [49,50,51]. It is worth mentioning that nutrient applications (i.e., synthetic fertilizer) could cause intensified biodecomposition of organic matter by microorganisms, poor water-holding capacity of soil, and lead to plant health issues [15,26]. In contrast, in pristine (i.e., undisturbed) environments, biological formulations are naturally actively performed by microbial decomposers, including bacteria and fungi [2]. These negative impacts caused by agricultural practices are evident in the state of Alabama, which was renowned for its rich, fertile, dark soil [52,53], distributed in hills and grassland areas [54,55]. This soil enabled the flourishing of the cotton industry and a booming population [56]. As a result of overuse and the introduction of invasive species, the land has reverted to non-agricultural purposes [57,58,59].

Biological promotion of plant growth is a longstanding, continuously investigated topic due to inconsistent findings among reports, which could be attributed to unstandardized management practices and methodology. In particular, this could be attributed to missing microbial symbionts that are needed to improve plant production in a coordinated way, leading to a reduced functional microbial community. This study employed a holistic approach and metadata analyses with literature information from 1981 to 2025 to determine the prevalence of bacteria, including previously reported plant growth-promoting bacteria and a group of three bacterial genera found exclusively in pristine soil and the presumptive members of the plant-productivity-enhancing biofertilizer community. The benchwork comparatively analyzed total bacteria acquired from disturbed (i.e., domesticated) and undisturbed (i.e., pristine) soil particles to anticipate developing a bacterial community structure that is conducive to soil fertility and sustainable agriculture. Using non-pre-enrichment alone (i.e., regular), pre-enrichment alone, heat process alone, and subsequent pre-enrichment and heat processes, endospore-forming and endosporeless bacteria were recovered from domesticated and pristine soil samples collected from multiple sites in Alabama, an agricultural state of the U.S., owing to its logistics, including proximity to the research laboratory and ease of access. Further analyses of individual bacterial isolates, including 16S rRNA bacterial identification and in silico research of reported agriculture-benefiting biofertilizers and other functional bacteria, revealed a presumptive enhanced community of bacterial biofertilizers that could synergistically enhance the regeneration of soil productivity biologically, economically, and effectively.

2. Materials and Methods

2.1. Soil Collection

Domesticated and pristine soil samples were collected (~120 g each, with a depth span of 0–0.1 m) in Sumter County, Alabama (GPS coordinates: disturbed soil, 32.5843° N, 88.1872° W; pristine soil, 32.525671, −88.139611), in the Fall (early October 2017) using sterile tubes and kept at ambient temperature (25 °C) prior to experimental analyses (Figure 1). Briefly, under the environments’ humid condition, various soil types determined by visible differential colors, including black, light black, and white (Figure 2), were separately acquired from various sites of land that were not disturbed by human activity according to locals, whereas red soil was acquired from a domesticated area whose soil was reportedly fit for cultivating plants. Subsequently, black, light black, red, and white color soil samples were determined as 1.7/1 (black), 5/2 (grayish brown), 7/8 (yellow orange), and 8/1 (light gray), respectively, with reference to the revised Munsell soil color charts (i.e., Hue 7.5 YR) [60,61].

Figure 1. Soil microbiology experimental scheme.

Figure 2. Various types of soil. Pristine ((A) black; (B) light black; (D) white) and domesticated ((C) red) soil samples were collected.

2.2. Bacterial Holistic Enrichment, Isolation, Storage, and Culture Conditions

Bagged bacterial dilution solutions (250 g; 1:10 dilution) (Filtra-bag^®, VWR, Pittsburgh, PA, USA) containing soil samples (25 g) and buffered peptone water (BPW, 0.1%, w/v) (Difco, Detroit, MI, USA) were homogenized (Stomacher^® 400 Circulator lab blender, Seward, Weber Scientific, Hamilton, NJ, USA) before being subject to various processes, including suspension only (i.e., non-enrichment alone) in BPW, suspension and enrichment (35 °C, 16–20 h) (i.e., enrichment alone), suspension (BPW) and heated (80 °C, 20 min) (i.e., heated alone), and suspension, enrichment (BPW, 35 °C, 16–20 h) and heated (i.e., enrichment and heated), were inoculated into Brain Heart infusion (BHI, Difco, Detroit, MI, USA) agar plates containing amphotericin B fungicide (0.25 μg/mL, per the manufacturer instructions) (Thermo Fisher Scientific, Waltham, MA, USA), incubated (35 °C) until visible bacterial colonies formed (i.e., 24–72 h), and bacterial colonies (i.e., in each sample) with different morphologies (i.e., shape, size, and color) (Figure 3) were streaked for purification on BHI agar plates. Pure bacterial cultures were suspended in sterile glycerol-supplemented BHI broth and kept at −72 °C storage temperature. Prior to experimental investigation, thawed bacterial cultures were sub-cultured (1:10 bacterial dilution) twice in BHI at 35 °C for ~20 h.

Figure 3. Partial representation (of four technical replicates) of morphologically variant bacterial colonies acquired in this study. Soil samples (i.e., black, light black, red, and white color soil samples) were processed with enrichment alone (A) and enrichment followed by heat treatment (B) prior to inoculation of bacterial dilutions on BHI agar media and incubation at 35 °C until visible bacterial colonies formed.

2.3. Bacterial DNA Extraction, Purification, and Analysis

The bead-collision method of Tiong and Muriana [62] was employed to extract bacterial chromosomal DNA. Briefly, sub-cultured bacteria (i.e., fresh cultures) were spun in a high-speed microcentrifuge (12,000 RPM, 2 min) (VWR, Suwanee, GA, USA) and resuspended in sterile distilled water (DI water) twice before resuspended in solution containing sterile glass beads (0.5 mm) (VWR) and Tris buffer (10 mM, pH 7.4), followed by sequential bead-collision processing, including step 1 (i.e., 1 min vortex and 2 min refrigeration) and step 2 (i.e., 1 min vortex and 4 min refrigeration) processes, to shear bacterial cell envelope and release intracellular chromosomal DNA. Bacterial lysates containing chromosomal DNA were spun (12,000 RPM, 2 min), and the pure DNA-containing supernatants were transferred into sterile Eppendorf tubes and kept at −20 °C until further analyses. DNA purity and concentration were analyzed and determined with a spectrophotometer (Thermo Scientific, South San Francisco, CA, USA) at OD₂₆₀/OD₂₈₀ and OD₂₆₀ wavelengths, respectively. Agarose gel electrophoresis (i.e., 1.6%, w/v) was used to confirm the DNA quality and quantity visually.

2.4. PCR Conditions, Purification, and Bacterial Identification

16S rRNA PCR reactions were prepared according to the GoTaq Flexi DNA Polymerase’s instructions (Promega, Madison, WI, USA). Briefly, PCR mixtures, containing MgCl₂ (1.5 mM, Promega), dNTPs (0.2 mM, Fisher Scientific, Fair Lawn, NJ, USA), PCR buffer (5×, Promega), primers (0.4 µM, (16S 515F, GTGCCAGCMGCCGCGGTAA; 16S 1391R, GACGGGCGGTGTGTRCA) [63] (IDT, Coralville, IA, USA), DNA template (<0.5 µg/50 µL), and DNA polymerase (1.25 U, Promega), were subject to PCR cycle step 1, 1 cycle of denaturation (95 °C, 1 min); step 2, 40 cycles of sequential denaturation (95 °C, 45 s), annealing (56 °C, 40 s), and DNA extension (72 °C, 1 min); step 3, 1 cycle of final extension (70 °C, 10 min) before infinite holding at 4 °C, in a GeneAmp PCR System 9700 Thermal Cycler (Applied Biosystems, Thermo Scientific, South San Francisco, CA, USA). Purified PCR amplicons (Wizard SV Gel and PCR Clean-up System, Promega) were analyzed on a DNA illuminating dye (GelStar TM Nucleic Acid Gel Stain, ratio 5 µL stain solution: 50 mL gel solution, Lonza Walkersville Inc., Walkersville, MD, USA) supplemented agarose gel (1.6% agarose, 1× Tris-borate-EDTA buffer) using a UV transilluminator (Ultra-Lum UV Transilluminator Muvb-20, ULTRA-LUM INC., Carson, CA, USA). Subsequently, the amplicons were subject to 1-directional Sanger sequencing using 515F primer (GTGCCAGCMGCCGCGGTAA) [63], chromatogram editing (MegaX, older version), and bacterial nucleotide identification (NCBI Blastn, default choose search set and program selection). The bacterial identity of each edited nucleotide sequence was called at 100% nucleotide query coverage, 0 E value, and 100% percent identity.

2.5. Soil Nutrients and pH Assessment

Individually, soil nitrogen (N), phosphorus (P), potassium (K), and pH were determined using a soil test kit (LaMotte^®, Chestertown, MD, USA) [64].

2.5.1. NPK Test

Accordingly, extraction mixtures (prepared in the kit-provided graduated tubes) containing an adjusted amount of sample (1–2 g), and extraction reagent were homogenized (1 min) and settled, and a clear solution containing nutrients was quantitatively assessed for N, P, and K, separately, in accordance with their color interpretative charts after being subject to their respective chemical indicator and test reagent reactions and the formation of different color intensities. Each nutrient was quantified according to three classes as instructed by the kit’s manufacturer, including low, medium, and high. N:L, 0–33.6 kg/ha; M, 33.6–67.3 kg/ha; H, +67.3 kg/ha. P:L, 0–56 kg/ha; M, 56–112.1 kg/ha; H, +112.1 kg/ha. K:L, 0–134.5 kg/ha; M, 134.5–224.2 kg/ha; H, +224.2 kg/ha.

2.5.2. pH Test

Briefly, reaction mixtures containing 1.5 g of sample and pH indicator solution were prepared in graduated tubes (provided in the manufacturer’s kit), homogenized, settled (10 min), and the colors formed were translated into pH values in accordance with the manufacturer’s pH color chart.

2.6. Literature Acquisition

Reported information on plant growth-promoting bacteria within the past four decades was acquired using online search engines, including Google.com and https://pubmed.ncbi.nlm.nih.gov/ (accessed 31 October 2025), and cross-referencing of peer-reviewed articles, including recent review articles [17,21]. Bacterial genus, species, and roles contributing to plant growth benefits, including soil stability, soil healthiness (i.e., water- and mineral-rich), and plant resistance to infections and malnutrition, were extracted. Each species or group of species (i.e., sp., spp., designated species name) was called by its specific function(s) reported in the literature (Supplementary Table S1).

2.7. Statistical Significance

Using the t-test and ANOVA for independent samples, two and four, respectively, the means of bacterial species were assessed for their significant difference (p < 0.05). The tests are available online: http://vassarstats.net/anova1u.html (accessed 30 December 2025).

3. Results

3.1. Benchwork: Diverse Bacteria of Domesticated and Pristine Soils Tested

3.1.1. Soil-Dependent Bacterial Diversity

Different soil samples were analyzed for bacterial isolation and identification using a holistic isolation method (i.e., non-enrichment alone, enrichment alone, and their heated combinations, including non-enrichment and heat process and enrichment and heat process). Of 21 abundantly represented bacterial isolates examined (Table 1), a total of 15 different bacterial genera (seven) and various species of Aeromonas (1), Bacillus (7), Lactobacillus (1), Lelliottia (1), Pseudomonas (1), Staphylococcus (3), and Stenotrophomonas (1) were identified (Table 1). Comparatively, the bacterial genus Bacillus was immensely prevalent among other isolated bacterial genera (i.e., Staphylococcus, Lactobacillus, Lelliottia, Aeromonas, Stenotrophomonas, and Pseudomonas) (Table 2, Figure 4) in all soil samples examined whether domesticated or pristine), but two species, Bacillus cereus and B. huizhouensis, were exclusively found in all pristine soil samples, including black, light black, and white (Table 1 and Table 2). Further, Aeromonas sp., Lelliottia sp., Staphylococcus spp./Lactobacillus sp., and Pseudomonas were exclusive to pristine soil samples, whereas Stenotrophomonas was acquired only from domesticated (red) soil (Table 2, Figure 4). In addition, each white (SMGM15) and black (SMGM33) soil bacterial isolate exhibited homologous 16S rRNA amplicons (Table 1) among Staphylococcus and Lactobacillus species and among two species of Bacillus, respectively.

Table 1. Total bacteria recovered in each soil sample using the holistic isolation method.

Isolate ID	16S Bacterial ID	Black 1	Light Black 1	Red 1	White 1	NE 2	E 2	H 2	E + H 2
SMGM1	Bacillus thuringiensis	√	×	×	×	√	×	×	×
SMGM5, SMGM34, SMGM41, SMGM43, SMGM45	Bacillus cereus	√	√	×	√	√	×	√	√
SMGM8, SMGM37, SMGM44	Bacillus megaterium	×	×	√	×	√	×	√	√
SMGM31, SMGM32, SMGM35, SMGM40, SMGM42	Bacillus huizhouensis	√	√	×	√	×	×	√	√
SMGM33 a	Bacillus niacin	√	×	×	×	×	×	√	×
SMGM33 b	Bacillus drentensis	√	×	×	×	×	×	√	×
SMGM36	Bacillus amyloliquefaciens	×	×	√	×	×	×	√	×
SMGM15 a	Stahylococcus xylosus	×	×	×	√	√	×	×	×
SMGM15 b	Staphylococcus succinus	×	×	×	√	√	×	×	×
SMGM15 c	Staphylococcus saprophyticus	×	×	×	√	√	×	×	×
SMGM15 d	Lactobacillus plantarum	×	×	×	√	√	×	×	×
SMGM17	Lelliottia sp.	√	×	×	×	×	√	×	×
SMGM21	Aeromonas caviae	×	√	×	×	×	√	×	×
SMGM25	Stenotrophomonas sp.	×	×	√	×	×	√	×	×
SMGM29	Pseudomonas sp.	×	×	×	√	×	√	×	×

¹ These colors represent the color of the soil used in this study. Pristine, black, light black, white; domesticated, red. ² These letters represent diverse bacterial isolation methods used in this study. NE, non-enrichment; E, enrichment; H, heated (i.e., non-enrichment); E + H, enrichment and heated. SMGM33 ^a and SMGM33 ^b are from one isolate that shares highly homologous 16S rRNA sequences. SMGM15 ^a, SMGM15 ^b, SMGM15 ^c, and SMGM15 ^d are from one isolate that shares highly homologous (100% nucleotide query coverage, 0 E value, and 100% identity) 16S rRNA sequences. Keys: √, detected; ×, not detected.

Table 2. Bacterial availability in different soil types ¹ (in this study).

Bacterial ID	Domesticated	Pristine
Bacillus thuringiensis	-	√
Bacillus cereus	-	√
Bacillus megaterium	√	-
Bacillus huizhouensis	-	√
Bacillus niacin	-	√
Bacillus drentensis	-	√
Bacillus amyloliquefaciens	√	-
Stahylococcus xylosus	-	√
Staphylococcus succinus	-	√
Staphylococcus saprophyticus	-	√
Lactobacillus plantarum	-	√
Lelliottia sp.	-	√
Aeromonas caviae	-	√
Stenotrophomonas sp.	√	-
Pseudomonas sp.	-	√

¹ Include domesticated and pristine soils. Keys: √, detected; -, not detected.

Figure 4. Diverse bacteria acquired from pristine vs. domesticated soil. Each group of soil samples (i.e., pristine vs. domesticated) was processed using four isolation methods prior to inoculating bacterial dilutions on agar media, incubating at 35 °C until visible bacterial colonies formed, and then streaking the bacterial colonies for purification, followed by 16S rRNA bacterial identification. Data bars represent the total diverse bacteria acquired from each pristine and domesticated soil sample using four isolation methods. Each bar represents a mean ± SD of four technical replications of a biological sample. Means with similar capital/non-capital letters are not significantly different; means with different capital/non-capital letters are significantly different (p < 0.05). ¹ Two Bacillus species from heated (H) soil were identified from one colony. ² Staphylococcus and Lactobacillus spp. were identified from one colony.

3.1.2. Soil Color-Dependent Bacterial Diversity

Of all seven Bacillus spp., three (B. thuringiensis, B. niacin, and B. drentensis) were exclusively isolated from black soils and two (amyloliquefaciens and megaterium) from red (Table 3). Other bacterial isolates were differentially distributed in all soil samples, such as black (Lelliottia sp.), light black (Aeromonas caviae), red (Stenotrophomonas sp.), and white (Pseudomonas sp. and Staphylococcus spp. or Lactobacillus plantarum) (Table 3, Figure 5).

Table 3. Bacterial availability in different soil colors ¹ (in this study).

Bacterial ID
Bacillus thuringiensis	√	-	-	-
Bacillus cereus	√	√	-	√
Bacillus megaterium	-	-	√	-
Bacillus huizhouensis	√	√	-	√
Bacillus niacin	√	-	-	-
Bacillus drentensis	√	-	-	-
Bacillus amyloliquefaciens	-	-	√	-
Stahylococcus xylosus	-	-	-	√
Staphylococcus succinus	-	-	-	√
Staphylococcus saprophyticus	-	-	-	√
Lactobacillus plantarum	-	-	-	√
Lelliottia sp.	√	-	-	-
Aeromonas caviae	-	√	-	-
Stenotrophomonas sp.	-	-	√	-
Pseudomonas sp.	-	-	-	√

¹ Include black, light black, red, and white soils. Keys: √, detected; -, not detected.

Figure 5. Diverse bacterial genera acquired from different soil samples (i.e., determined by soil colors). Each soil sample was processed using four different isolation methods (i.e., non-enrichment alone, enrichment alone, heated alone, and enrichment followed by heat treatment) prior to inoculating bacterial dilutions on agar media, incubating (35 °C) until visible bacterial colonies formed, and bacterial colonies were streaked for purification followed by 16S rRNA bacterial identification. Data bars represent the total diverse bacteria acquired in each soil sample using four different isolation methods. Each bar represents a mean ± SD of four technical replications of a biological sample. Means with similar capital/non-capital letters are not significantly different; means with different capital/non-capital letters are significantly different (p < 0.05). ¹ Two Bacillus species from heated (H) soil were identified from one colony. ² Staphylococcus and Lactobacillus spp. were identified from one colony.

3.1.3. Isolation-Method-Dependent Bacterial Diversity

Comparatively, the number of bacterial genera isolated in each type of soil was remarkably reduced upon heat processes (i.e., one genus identified), including heat process alone (i.e., non-enrichment and heated) and sequential enrichment and heat process (Table 4, Figure 6), as opposed to non-heat processes (i.e., non-enrichment alone and enrichment alone) (i.e., six genera identified). Among Bacillus spp., diverse isolation conditions, including non-enrichment alone (i.e., one species identified), non-enrichment and other heated conditions (i.e., non-enrichment + heat treatment and enrichment + heat treatment) (i.e., two species identified), non-enrichment followed by heated conditions (i.e., three species identified), and either one of the two heated conditions (i.e., one species identified), were exclusive for the isolation of its various species (Table 4). Other bacteria were isolated exclusively by non-enrichment alone (i.e., Staphylococcus spp. or Lactobacillus plantarum) and enrichment alone (Lelliottia sp., Aeromonas caviae, Stenotrophomonas sp., and Pseudomonas sp.) conditions (Table 4, Figure 6).

Table 4. Bacterial availability in different isolation methods ¹ (in this study).

Bacterial ID	NE	E	H	E + H
Bacillus thuringiensis	√	-	-	-
Bacillus cereus	√	-	√	√
Bacillus megaterium	√	-	√	√
Bacillus huizhouensis	-	-	√	√
Bacillus niacin	-	-	√	-
Bacillus drentensis	-	-	√	-
Bacillus amyloliquefaciens	-	-	√	-
Stahylococcus xylosus	√	-	-	-
Staphylococcus succinus	√	-	-	-
Staphylococcus saprophyticus	√	-	-	-
Lactobacillus plantarum	√	-	-	-
Lelliottia sp.	-	√	-	-
Aeromonas caviae	-	√	-	-
Stenotrophomonas sp.	-	√	-	-
Pseudomonas sp.	-	√	-	-

¹ Include non-enrichment alone (NE), enrichment alone (E), heated alone (H), and enrichment and heated (E + H). Keys: √, detected; -, not detected.

Figure 6. Diverse bacteria acquired using different isolation methods. Non-enrichment (NE), enrichment (E), heat treatment (H), and enrichment followed by heat treatment (E + H) isolation methods were used to process each soil sample prior to inoculating bacterial solution on agar media, incubating (35 °C) until visible bacterial colonies formed, and morphologically distinct bacterial colonies were streaked for purification followed by 16S rRNA bacterial identification. Data bars represent the total species of each bacterial genus acquired using each of the four isolation methods. Each bar represents a mean ± SD of four technical replications of a biological sample. Means with similar capital/non-capital letters are not significantly different; means with different capital/non-capital letters are significantly different (p < 0.05). ¹ Two Bacillus species from heated (H) soil were identified from one colony. ² Staphylococcus and Lactobacillus spp. were identified from one colony.

3.1.4. Nutrient Contents of Diverse Soil Samples (i.e., Different Colors, Pristine vs. Domesticated)

Both groups of soil were analyzed for pH and growth essential nutrients. Pristine soil samples, including black, light black, and white soil, exhibited comparably non-distinctive pH and a range of trace to medium amounts of phosphorus, nitrogen, and potassium (Table 5). However, domesticated soil (i.e., red soil) possessed a medium-to-high range in the nutrients examined (Table 5).

Table 5. Nutrient composition (kg/ha) of pristine vs. domesticated soil (in this study).

Test	Light Black a,b	Black a,b	Red a,b	White a,b
Nitrogen (N)	Low	Low	High	Low
Phosphorus (P)	Medium	Low	Medium	Low
Potassium (K)	Medium	Low	Medium	Medium
pH	8	8	7	8

^a Pristine samples include light black, black, and white color soil. Domesticated sample includes red color soil. ^b Definition of different nutrient levels: N, low, 0–33.6 kg/ha; medium, 33.6–67.3 kg/ha; high, +67.3 kg/ha. P, low, 0–56 kg/ha; medium, 56–112.1 kg/ha; high, +112.1 kg/ha. K, low, 0–134.5 kg/ha; medium, 134.5–224.2 kg/ha; high, +224.2 kg/ha.

3.2. Metadata: Diverse Plant Growth-Promoting Bacteria

3.2.1. Plant Growth-Promoting Bacteria Metadata

An in silico review of recent articles revealed a group of plant growth-promoting bacteria. Of 33 bacterial genera of three functional groups, including soil stability (1, 3%), soil fertility (10, 30%), and plant health (26, 79%), 11 (33%), 22 (67%), and 3 (9%) were Gram-positive, Gram-negative, and endospore-forming bacteria, respectively (Table 6). Three bacterial genera (Paenibacillus, Pseudomonas, and Rhizobium) contribute to soil fertility and plant health and one (i.e., Actinomyces) to soil stability and plant health (Table 6).

Table 6. List of reported (in the literature) plant growth-promoting bacteria and their role(s).

Bacterial Genus	a Species #	Gram	Endospore	Soil Stability	Soil Fertility	Plant Health	Reference
Acinetobacter	4	-	×	×	×	√	[65,66,67,68]
Actinobacteria	1	+	×	×	×	√	[68]
Actinomyces	1	+	√	√	×	√	[69]
Aeromonas	3	-	×	×	×	√	[70,71,72,73,74]
Arthrobacter	3	+	×	×	×	√	[24,75,76]
Achromobacter	1	-	×	×	×	√	[67,77]
Azospirillum	1	-	×	×	√	×	[78]
Azotobacter	3	-	×	×	×	√	[19,79,80]
Bacillus	29	+	√	×	×	√	[3,17,69,76,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102]
Bacteroides	1	-	×	×	×	√	[67]
Bradyrhizobium	1	-	×	×	×	√	[103,104,105,106]
Brevibacterium	1	+	×	×	×	√	[107]
Burkholderia	1	-	×	×	√	×	[108,109]
Chloroflexi	1	-	×	×	×	√	[68]
Enterobacter	3	-	×	×	√	×	[35,110,111]
Erysipelatoclostridium	1	+	×	×	×	√	[67]
Exiguobacterium	1	+	×	×	×	√	[112]
Herbaspirillum	3	-	×	×	√	×	[35,108]
Inquilinus	1	-	×	×	×	√	[113]
Lactobacillus	10	+	×	×	×	√	[114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140]
Lelliottia	2	-	NA	×	×	√	[141,142]
Paenibacillus	2	+	√	×	√	√	[143,144]
Pantoea	2	-	×	×	√	×	[145,146]
Providencia	1	-	×	×	√	×	[147]
Pseudomonas	30	-	×	×	√	√	[3,30,65,73,76,80,90,96,145,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166]
Rhizobium	4	-	×	×	√	√	[36,167,168]
Serratia	3	-	×	×	×	√	[76,161,169]
Sinorhizobium	1	-	×	×	√	×	[35]
Sphingobacterium	1	-	×	×	×	√	[4]
Staphylococcus	3	+	×	×	×	√	[24,170]
Stenotrophomonas	3	-	×	×	×	√	[113,171,172]
Stratosphericus	1	+	×	×	×	√	[90]
Zobellella	1	-	×	×	×	√	[24]

^a Each taxon was called by its function(s) reported in the literature. NA, not available. Keys: -, Gram-negative bacterium defined by its thin cell wall; +, Gram-positive bacterium defined by its thick cell wall; ×, bacterium that does not form endospore or does not have a beneficial role in soil (as listed in the table); √, bacterium that forms endospore or has a beneficial role (as listed in the table).

3.2.2. Plant Growth-Promoting Bacteria Metadata vs. Benchwork

Further comparison of total bacterial genera (i.e., reported and in this study) exhibited four commonly available plant growth-promoting bacteria, Bacillus, Staphylococcus, Stenotrophomonas, and Pseudomonas (Table 1 and Table 6). Additionally, three bacterial taxa, Aeromonas sp., Lactobacillus sp., and Lelliottia sp., were exclusively identified in pristine soil in this experimental study (Figure 7).

Figure 7. Total plant growth-promoting bacterial genera discovered and reported in this study (i.e., both pristine and domesticated) and in the literature, respectively. The specific species of bacteria can be found in Supplementary Table S1.

4. Discussion

4.1. Benchwork: Diversity of Soil Bacteria

4.1.1. Soil Profile-Dictated Bacterial Distribution and Study Legitimacy

In comparison to reported plant growth-promoting bacterial community [2], the holistic assessment in this study reveals differential profiles of total bacterial genera and species in soils distinguished by their color and domestically disturbed vs. pristine status, thereby suggesting the availability of bacterial community of a kind that could be conducive to plant growth. Surprisingly, no bacterium occurs commonly in both pristine and disturbed soils, suggesting that the community structure of soil bacteria that promotes plant growth could be diverse, that the structure could be environmentally dictated, and that a novel all-season functional bacterial structure/profile could be constructed and applied for enhancing plant growth in the field as opposed to the currently available structures, whose functionality is closely environmentally modulated [173,174]. Multiple bacterial identities of SMGM15 (i.e., S. xylosus, S. succinus, S. saprophyticus, and L. plantarum) and SMGM33 (B. niacin and B. drentensis) (Table 1) could be attributed to their non-distinguishable 16S gene amplicons by 515F primer 1-directional sequencing (in this study) [63], thereby suggesting the need to locate genus- and species-specific 16S rRNA fragments to distinguish genetically closely related bacteria. Per prior reports, while Staphylococcus species (i.e., S. succinus and S. saprophyticus) [170], B. drentensis [101], and L. plantarum [114] possess plant growth-promoting implications, neither soil-originating B. niacin [175] nor meat-fermenting S. xylosus [176] has been directly associated with plant growth promotion [170,177]. Further, pristine and domesticated soil-associated bacterial exclusivity (in this study) could be attributed to natural and artificial manipulations by climate [14] and synthetic chemicals (i.e., fertilizer and pesticide) [15,173,174] as previously reported. Remarkably, the pristine samples selected in this study differentially exhibit plant growth-promoting bacterial genera reported in the literature, including Bacillus sp. and the pristine soil-exclusive bacterial genera Lactobacillus [140], Lelliottia [141,142], Pseudomonas [2], Staphylococcus [24,170], and Aeromonas [70,73] spp., of which Bacillus and Pseudomonas spp. are known to be primarily applied in biofertilizer formulation [2], thereby strengthening the legitimacy of soil samples selected for this study in the Black Belt of Alabama and bacterial contrast profiles of two groups of samples (i.e., domesticated vs. pristine). In addition, prior reports reported the environmental originality of these pristine soil exclusive bacteria, including Lactobacillus [178], Lelliottia [179], and Aeromonas spp. [72], thereby further validating this study’s findings. On the other hand, the domesticated sample selected in this study exhibited a group of soil bacterial genera of very restricted diversity, including Bacillus [177,180] and Stenotrophomonas spp. [2,113,181], which, according to prior studies, possess plant growth-promoting functions. Hence, the presence of an artificial formulation (i.e., bacterial inoculant applications) of the bacterial community in the domesticated land selected in this study and bio-antagonistic activity could lead to bacterial dominance-biased detection and reduced diversity, respectively. Among two Bacillus spp. that were exclusively detected (in this study), B. amyloliquefaciens was reported to be a commercially available bacterial inoculant and to have antimicrobial activity [17], thereby concurring with our bacterial profile findings of domesticated soil (in this study).

4.1.2. Soil Color-Dictated Bacterial Distribution

This study selectively employed the direct comparison of soil colors against the Munsell color chips and the assigning system to confirm the color of various soil samples, attributed to its official adoption [182], portability, accessibility, and comparable result interpretability [182], as opposed to instrument deployment (i.e., colorimeter) [183]. Researchers related the color to organic matter content, metal availability (i.e., iron), drainage, and aeration [183,184]. Accordingly, researchers [184] correlated red and white (referred to as “gray”) soil with the availability of iron in ferric vs. ferrous species, respectively, attributed to their environmental aeration and drainage conditions. Bacterial capability in iron metabolism has been reported in species of Stenotrophomonas [181], Pseudomonas [185], Lactobacillus [186], Bacillus [187], Aeromonas, and Staphylococcus. Of these, two species of Bacillus (megaterium and amyloliquefaciens) as well as Stenotrophomonas sp. (Table 3) were exclusively detected in ferric-rich, well-drained red soil in this study. The differential distribution/abundance of a subset of these bacteria (genus or species exclusive) in iron-rich soil in this study, as indicated by soil color [184], could be driven by bacterial inherited preference for metabolism pathway [188] and water activity [189]. On the other hand, researchers [184] have interpreted black soil, including different intensities of black soil in this study, as organic-rich.

4.1.3. Isolation Process-Dictated Bacterial Distribution

Various enrichment and heat processes applied in this study exhibit diverse bacterial cultivation profiles (i.e., enrichment and/or heat processes), suggesting the availability of diverse soil bacteria at generic and specific ranks (Table 1 and Table 4), including presumptive plant growth-promoting bacteria, in physiology and metabolism, and that its absence (i.e., specific cultivation conditions) could compromise findings’ accuracy. This concurs with the previously reported modified two-step enrichment method, which used enrichment and heat processing alternately to successfully recover diverse bacteria groups, both within and outside a genus/species and among endospore-forming [190] and non-endospore-forming bacteria [31,32], which would otherwise be nonculturable. Remarkably, of the seven endospore-forming Bacillus species (29) detected in this study, four cultures were exclusively heat-dependent in the isolation step (Table 4). Thus, for the first time, our culture-based study suggests the need for a holistic approach to unravel the complex structure of plant-growth-benefiting biofertilizers.

4.1.4. Diverse Soil Nutrient and pH Contents

Nutrient and pH contents were distinct in pristine soil alone (i.e., nutrient contents) and between pristine and domesticated soil (i.e., both pH and nutrient contents) examined in this study. Other researchers indicated black color soil as organic-rich [183,184]. Further, Zhang et al. [191] reported a positive correlation between fertilized soil with inorganic compounds (i.e., NPK) and organic carbon content in the soil and humic acid, thereby suggesting the low organic carbon content of the black, organic-rich indicative soil (in this study) and that all pristine soil varieties, including light black, black, and white, are friendly to cotton given its low nitrogen need [192] relative to that of other important crops, including corn (which needs 4–5× more N) [193]. In this study, the nutrient variations, including the organic matter indicative color, in pristine soil even within one sampling site could be attributed to soil water holding capacity [2,184], aeration [184], and land topology (i.e., slope) [194]/vegetation type (i.e., grassland vs. scrubland) [195,196,197], which could facilitate water movement-driven nutrient loss [49], erosion-driven nutrient loss (i.e., favorable for increasing slope) [49,194], and nutrient containment capability (i.e., favorable for grassland) [195], respectively. It is significant that the white soil, indicative of loss of organic matter, was sampled at multiple erosion-conducive high-level sites [194] in a pristine sampling area in our study. Additionally, other groups determined a positive relationship between land slope, erosion, and vegetation type [195,196,197,198,199,200] and land slope (i.e., gentle slope, 0–25°) and vegetation type (i.e., grassland) [201,202]. Relatively pristine soil’s (i.e., black, light black, and white) pH and nitrogen content are consistently lower than domesticated soil (in this study). This phenomenon could be attributed to the land being disturbed artificially with NPK fertilizers to favor cultivation. The inverse correlation between soil pH and N availability (i.e., increased N availability = reduced pH) (Table 5) concurs with a previous report [184], thereby validating this study. The different levels of N relative to P and K in domesticated soil in this study could be attributed to the standardized NPK application ratio set for crop-specific cultivation purposes [203,204]. In particular, N, P, K, and pH of 60–105, 75+–100+, 171–400, and 6, respectively [205], are essential for cotton plantation states, including Alabama, the seventh largest cotton-producing state by production yield in bales [42], thereby recommending the adjustment of one or more elements in the pristine soil to revitalize production [206]. It is worth noting that the fertility of cultivated land, including pristine land, does not necessarily correlate positively with organic-rich, dark, arable soil [182,183], as demonstrated here (Table 5), pointing out the need for testing N, P, and K prior to cultivation. The fact that pristine soil is not necessarily compatible with cultivation could be attributed to fertility distortions by extreme natural events, including inclement weather and biological invasion, that are indirectly catalyzed by routine human activity.

4.2. Metadata: Diversity of Plant Growth-Promoting Bacteria

4.2.1. Reported Plant Growth-Promoting Bacteria

Notably, thus far, reports of plant growth-promoting bacteria have been inconsistent as to the availability of bacterial genera and species associated with healthy plants. Consequently, the present study recollects the most recent review report [2] alongside reports spanning 1981–2025 that are associated with plant growth-promoting bacteria that are not revealed in one or more reports to compile a list of bacteria for potential holistic formulation for an efficient biofertilizer community structure that could functionally prevail in all environmental conditions.

4.2.2. Metadata vs. Benchwork Data

Additionally, this bacterial list consists of plant growth-promoting bacteria identified in this holistic study, including Lactobacillus and Lelliottia spp., which are not reported in Wang et al.’s review report [21], thereby justifying the need for this report to update the status of biofertilizer research and development, and its need-improvement area (i.e., identifying study methodology as a prime area for improvement).

5. Conclusions

Degradation of cultivated soil is inevitable, but farmland can be biologically regenerated using microorganisms [2]. The present study comparatively analyzes a group of recently (1981–2025) reported soil-benefiting bacteria and a group of total bacteria recovered using a holistic approach (i.e., by non-enrichment alone, enrichment alone, and heat-process supplementation). Holistic analysis suggests that the availability of soil bacteria is dependent upon recovery conditions and soil type, that using only a single method could render inaccurate results in bacterial community profile discovery, and that these bacteria (i.e., those exclusively recovered in pristine soil in this study) could functionally contribute to synergistic bioregeneration of farmland.