Glyphosate bioremediation using a newly isolated Bacillus albus strain F9D: mechanisms and kinetic studies

Abstract

Glyphosate is widely used as an herbicide around the world. The extensive application of glyphosate, however, has serious adverse effects on living systems. Therefore, the elimination of residual glyphosate pollution has become an urgent issue worldwide. In the present study, a novel bacterial strain named F9D was identified as Bacillus albus, based on its physio-biochemical characteristics and 16S rDNA analysis. This strain can completely degrade glyphosate (400 mg/L) within 5 days. An effective, rapid, and stable detection method for glyphosate and aminomethylphosphonic acid (AMPA) was developed using ultra-performance liquid chromatography–tandem mass spectrometry technology (UPLC-MS/MS). The degradability of glyphosate by the degrading strain F9D was optimized, considering various conditions, as follows: initial pH (5–9), incubation temperature (20–40℃), glyphosate concentration (50–800 mg/L), and inoculation amount (1–5%). The strain also demonstrated strong degradation ability in soil and water–sediment systems: 78.1% glyphosate (400 mg/kg) and 83.2% glyphosate (200 mg/kg), respectively, degraded in soil and water–sediment systems within 5 days of incubation. Furthermore, the F9D strain is capable of degrading 50–800 mg/L of glyphosate and AMPA under various treatments. Hence, the notable ability of B. albus strain F9D to degrade glyphosate makes it a highly promising candidate for the removal of this emerging contaminant from the environment on a large scale.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12934-025-02758-1.

Introduction

Glyphosate (N-phosphonomethylglycine, C₃H₈NO₅P) has been shown to be a highly efficient, non-selective, and broad-spectrum systemic herbicide since its introduction in the 1970s [1, 2]. Due to its stable physical and chemical properties and high weed control efficiency, glyphosate has been used in agriculture, forestry, livestock, and urban greening. At present, it is the most widely used herbicide in the world, with the largest production and annual sales [3]. The mode of action of glyphosate is mainly to inhibit the activity of 3-enolpyruvylshikimate-5-phosphate synthase (EPSPS), interfering with its synthesis and, subsequently, inhibiting the synthesis of aromatic amino acids and secondary metabolites, ultimately leading to the chlorosis, yellowing, wilting, and death of plants [4, 5]. The shikimic acid pathway is a metabolic pathway unique to bacteria, fungi, algae, and plants. It is mainly associated with the synthesis of aromatic amino acids in bacteria and fungi, and is also related to the synthesis of important secondary metabolites in plants [6, 7]. Due to its unique mechanism and high herbicidal activity, the scope and use of glyphosate has increased every year, playing an important role in the herbicide industry.

Glyphosate was once considered an environmentally friendly herbicide with low toxicity and residues. However, due to the large-scale and irregular use of glyphosate, a large amount of glyphosate remains in the natural environment, including soil and water, which can harm both human health and ecosystems [8–10]. The half-life of glyphosate in water/soil environments is reported to be 9-500 days [11, 12]. The majority of glyphosate can be adsorbed by the soil, due to its strong hydrophilicity. It can then accumulate in the upper layer of the soil and precipitate downward, polluting surface and groundwater [13, 14]. Moreover, in some studies, glyphosate has been shown to inhibit the growth of indigenous micro-organisms in the soil and promote the growth of plant pathogenic fungi, resulting in an alteration of the diversity and structure of soil microbial communities [15]. Toxicological testing of glyphosate in mice found that 50 mg/kg glyphosate decreased sperm count in male mice, increased the rate of premature delivery and miscarriage in female mice during pregnancy, and impaired fetal development in female mice [16, 17]. The issue of glyphosate safety has generated great controversy worldwide, especially the carcinogenic case against Monsanto in the United States in March 2019, which brought attention on the safety of glyphosate to a climax. Therefore, the elimination of residual glyphosate pollution has become an urgent issue worldwide.

As the C–P bond of glyphosate is relatively stable under electromagnetic radiation, acidic, or other environmental conditions, microbial degradation may provide a suitable solution for the remediation of glyphosate contamination [18]. Detailed research on glyphosate biodegradation has documented two major pathways for glyphosate degradation: the first involves the cleavage of the C–N bond by glyphosate oxidoreductase, leading to the formation of aminomethylphosphonic acid (AMPA) and glyoxylate; meanwhile, the second pathway involves cleavage of the C–P bond, which is converted to inorganic phosphorus and sarcosine, under the action of C–P lyase [19, 20]. Sarcosine can be further decomposed into formaldehyde and glycine under the action of sarcosine oxidase. Formaldehyde can enter the tetrahydrofolate (THFA) cycle and be metabolized, and glycine can be directly used by the strain for amino acid synthesis (Fig. 1) [21]. Therefore, the isolation and characterization of highly efficient glyphosate-degrading strains holds great potential for application in bioremediation and wastewater treatment.

Fig. 1.

Biodegradation pathways of glyphosate

The present study focuses on the isolation and characterization of a newly isolated bacterial strain named F9D, which is capable of degrading high concentrations of glyphosate. The degradation kinetics of glyphosate and its degradation product AMPA were elucidated, and the degradation products and pathways mediated by the F9D strain were inferred. Additionally, the effects of F9D strain on soil microbial communities during glyphosate degradation were investigated in glyphosate-contaminated soils. This study aims to elucidate the mechanisms of glyphosate degradation by the F9D strain, thereby highlighting its great potential for environmental remediation and providing a reference for the future application in the remediation of glyphosate-contaminated environments.

Materials and methods

Chemical reagents and media

Glyphosate 100% and aminomethylphosphonic acid (AMPA) 98% were purchased from Sigma-Aldrich. Liquid chromatography-tandem mass spectrometry (LC-MS)-grade acetonitrile, ammonium acetate, and ammonium hydroxide were purchased from Fisher Scientific, United States. Glyphosate and AMPA were dissolved in deionized water as stock solutions (10,000 mg/L), stored in shaded bottles at 4 ^oC, sterilized by membrane filtration, and added to the medium to obtain the desired concentrations. All other chemicals and solvents were of analytical grade and were purchased from Guangzhou Dongju Experimental Instrument Co., Ltd, Guangdong Province, China.

Mineral salt medium (MSM) containing: (NH⁴)₂SO₄ (2 g/L), MgSO₄·7H₂O (0.2 g/L), CaCl₂·2H₂O (0.01 g/L), FeSO₄·7H₂O (0.001 g/L), Na₂HPO₄·12H₂O (1.5 g/L), and KH₂PO₄ (1.5 g/L). Luria-Bertani (LB) medium containing: tryptone (10 g/L), yeast extract (5 g/L), and NaCl (10 g/L). Both culture media were adjusted to pH 7.0 and sterilized at 121 ^oC for 20 min.

Enrichment and isolation of glyphosate-degrading bacteria

The soil samples used to isolate the glyphosate degraders were collected from an insecticide factory in Guangzhou, Guangdong Province, China. A total of 5 g of the soil sample was placed in a 250 mL Erlenmeyer flask containing 50 mL of sterilized MSM enrichment medium amended with 100 mg/L of glyphosate solution. The enrichment culture was incubated at 30℃ in a rotary shaker at 180 rpm for 7 days. Afterwards, 5 mL of the enrichment culture was transferred into 50 mL of fresh MSM medium containing 200 mg/L of glyphosate for another 7 days of incubation. The enrichment culture was successively transferred into fresh enrichment media containing higher glyphosate concentrations (i.e., 400, 800, and 1000 mg/L) and further incubated for 7 days, as in the previous procedure. After enrichment, 0.2 mL of bacterial culture sample was aliquoted for gradient dilution, and then spread evenly on glyphosate (500 mg/L)-containing MSM solid plates. After incubation at 30℃ for 2 days, individual visible colonies with different morphology were picked and streaked repeatedly on MSM solid plates having the same glyphosate concentration. Finally, three purified individual colonies were selected and cultured overnight in LB liquid medium, then stored in 15% glycerol by volume at -80℃ and used for further experimental studies [22, 23].

Bacterial identification

The bacterial isolate was characterized and identified on the basis of morphological and physio-biochemical characteristics, as well as 16S rDNA gene analysis. The two-day colonies of F9D were observed under a light microscope (Olympus, Japan) and examined for size, color, surface, edge, and texture. Furthermore, the whole genome of the degrading strains was extracted and used as a template for DNA for gene sequencing analysis. The 16S rDNA gene was PCR-amplified with universal primer pairs: 27F (5’-AGAGTTTGA TCCTGGCTCAG-3’) and 1429R (5’-GGTTACCTTGTTACGACTT-3’) [24, 25]. The PCR amplification products were detected by 1% agarose gel electrophoresis. The single-band PCR products were purified by ExoSAP-IT™ (purchased from ThermoFisher Scientific), while the PCR products with non-specific bands were cut and purified into a gel. For the purified products, bidirectional Sanger sequencing was performed, and the sequencing service was provided by Suzhou Jinweizhi Biological Technology Co., Ltd. After sequencing was completed, the results were compared with the genes available in the Genbank Nucleotide Library by a nBLAST search in the National Center for Biotechnology Information (NCBI). Multiple alignments of the 16S rDNA were performed using the CLUSTALX 1.8.3 software and the phylogeny tree was analyzed using the MEGA 6.0 software [26, 27].

UPLC-MS/MS analysis

In the present study, a method was developed to measure glyphosate residues using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) (Waters, USA). The chromatographic conditions were as follows: ACQUITY UPLC HSS T3 chromatographic column (1.7 μm, 2.1 mm × 100 mm); flow rate, 0.3 mL/min; sample size, 5 µL; column temperature, 35℃; mobile phase A: 2 mM ammonium acetate (including 0.1% ammonia); mobile phase B: methyl alcohol, gradient elute (Table 1). For sample processing before UPLC-MS/MS analysis, the sample was centrifuged at high speed (12,000 rpm) for 5 min, following which the supernatant was diluted 1000 times with deionized water and filtered through a 0.22 μm micro-porous membrane.

Table 1.

Gradient elute procedure

Time (min)	Flow rate (mL/min)	Mobile phase A (%)	Mobile phase B (%)
0	0.3	99	1
1.5	0.3	99	1
3.5	0.3	10	90
4	0.3	10	90
6	0.3	99	1

Growth and degradation assays

The strain F9D was cultured in LB broth and the metabolically activated strain was used for further experiments. Moreover, the culture was incubated at 30℃ for 12 h in a rotary shaker at 180 rpm. The bacterial cells in the late exponential growth phase were harvested by centrifugation (5 min, 4000 rpm) at 4℃ and washed twice in 0.9% normal saline (N-saline) before inoculation [22]. A series of experiments were conducted to investigate the bacterial growth and biodegradation efficiency of the F9D strain under different treatments. The effects of pH of the medium (5.0, 6.0, 7.0, 8.0, and 9.0), incubation temperature (20, 25, 30, 35, and 40℃), initial concentration of glyphosate (50, 100, 200, 400, and 800 mg/L), AMPA concentration (50, 100, 200, 400, 800, and 1600 mg/L), and percentage of degrading strain inoculated (1, 2, 3, 4, and 5%) were assessed. The optimal culture conditions for F9D have been carefully explored and will be utilized in our subsequent experiments.

Identification of glyphosate metabolites

The F9D strain was grown in a 250 mL Erlenmeyer flask with 50 mL of sterilized MSM containing various concentrations of glyphosate (50, 100, and 200 mg·L^− 1) as the sole carbon and energy source at 30℃ and 200 rpm for 120 h. Samples were collected at 24 h intervals [28]. The glyphosate residue was removed from the flask and analyzed by UPLC-MS/MS. The ultra-high-performance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap/MS) (ThermoFisher Scientific, USA) was equipped with a heated electrospray ionization interface. In addition, the ACQUITY UPLC HSS T3 column (1.7 μm, 2.1 × 100 mm) was offered by Waters Technologies (ThermoFisher Scientific, USA). The system was regulated by Xcalibur software, and the collected data were processed by Metworks software (ThermoFisher Scientific, USA). The specific detection and analysis conditions were referred to previous studies [15].

Kinetic analysis

Kinetic degradation experiments with different initial concentrations of glyphosate and aminomethylphosphonic acid (25, 50, 100, 200, 400, 600, and 800 mg/L) by F9D were performed at 30℃ and 200 rpm on rotary shakers. The substrate inhibition model adapted from Luong [29] [Eq. 1] was used to determine the specific degradation rate (q) at different initial concentrations of glyphosate:

1

where q_max is the maximum specific degradation rate, K_i is the substrate inhibition constant, K_s is the half-saturation constant, and S is the inhibitor concentration. The q value was calculated from the gradient of a semi-logarithmic plot of glyphosate concentration.

The degradation process of glyphosate in liquid media or soil was fitted to the first-order kinetic model [30, 31] [Eq. 2]:

2

where C₀ is the initial concentration of substrate, C_t is the amount of substrate at time t, k is the degradation rate constant, and t is the degradation time.

The theoretical half-life (t_1/2) value of glyphosate was calculated using Eq. 3:

3

where ln (2) is the natural logarithm of 2 and k is the degradation rate constant.

Biodegradation of glyphosate in soil by strain F9D and its impact on indigenous soil microbial communities

The soil samples were collected from the top layer (0–20 cm) in a farm where glyphosate and fertilizers had not been used for more than 5 years. The farm is located at South China Agricultural University, Guangdong Province, China (E113°37’45’’, N23°17’05’’). To investigate the degradation kinetics of glyphosate in soils, the F9D strain was inoculated into sterile or non-sterile soils and cultured in a dark thermostatic chamber at 30℃ for 5 days, according to a previous method with slight modifications [12, 15]. In each plastic container, 0.4% glyphosate solution (10 g/L) was sprayed onto the surface of 200 g of either sterile or non-sterile soil, achieving a final glyphosate concentration of 400 mg/kg in the soil. After thorough mixing, the bacterial suspension (4 mL/case) was added to the soil samples. The controls contained samples of sterile or non-sterile soil but with sterile water instead of the bacterial suspension. The humidity of all soil samples was maintained at approximately 40% by daily addition of sterile deionized water. A total of 2 g of each soil sample was collected every day for glyphosate residue measurement. Each soil sample was placed in 40 mL KOH (0.6 mol/L) for 30 min for ultrasonic extraction, then adjusted to pH 7 and centrifuged at 4000 rpm for 15 min. Then, 1 mL of the supernatant was removed and centrifuged again at high speed (12,000 rpm) for 10 min, and finally filtered through a 0.22 μm micro-porous filter membrane. The detection method was the same as for the previous method, using UPLC-MS/MS.

To delve deeper into the relationship between the F9D strain and the native soil microbes, soil samples weighing 5 g each were systematically gathered at intervals of 0, 1, 3, and 5 days. These samples were then processed for 16S rDNA sequencing by LC Biotechnology Co., Ltd, a service provider based in China. Each treatment was conducted with three biological replicates. Following the protocols of Walters et al. and Logue et al. [32]– [33], DNA was extracted from diverse samples using cetyltrimethylammonium bromide (CTAB). The 16S rDNA gene was targeted for amplification with primers 27 F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). Purification of the PCR products was achieved with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and their quantities were determined using the Qubit system (Invitrogen, USA). The libraries thus obtained were sequenced on the NovaSeq PE250 platform. Normalization of sample relative abundances was conducted using the SILVA (release 138) classifier. Sequence alignment was performed with BLAST, and annotations for key sequences were refined using the SILVA database. For visual representation, diagrams were generated with the R package (version 3.5.2) [34].

Biodegradation of glyphosate in water-sediment system by strain F9D

The soil samples were collected from the same location where the soil matrix degradation was studied. The treatment of F9D was consistent with the previous degradation tests. A total of 200 mg/L glyphosate (10 g/L) was added to each 250 mL Erlenmeyer flask containing 48 mL MSM medium and 5 g soil (soil/water ratio of 1:10). After thorough mixing, 1 mL of bacterial suspension was added to each flask. To the controls, instead of bacterial suspension, 1 mL of sterile deionized water was added. Sediment–water samples (1 mL) were collected from the 250 mL Erlenmeyer flasks every 24 h until day four for biodegradation analyses.

Statistical analysis of data

Statistical analysis of the data was conducted using Origin 2021 and Microsoft Excel software. All results are presented as the mean ± standard error of the mean (SEM). Tukey’s Honestly Significant Difference (HSD) test was employed to determine significant differences, with a significance threshold set at p < 0.05.

Results and discussion

Isolation and screening of strain F9D

According to the results of UPLC-MS/MS analysis, one of the isolates, designated as F9D, showed the highest glyphosate degradation efficiency (more than 98% of glyphosate was degraded in just 5 days). Hence, isolate F9D was chosen for further biochemical, molecular, and glyphosate biodegradation studies.

Identification of strain F9D

Isolate F9D was characterized as Gram-positive by Gram staining. Colonies on LB were unpigmented (white), round, opaque, waxy, and smooth with irregular margins (Fig. 2A) and scanning electron microscope images of F9D are shown in Fig. 2B. The physiological and biochemical tests conducted on the F9D strain revealed a series of positive outcomes in Table 2, including anaerobic growth capabilities, efficient utilization of glucose for fermentation, and robust enzymatic activities in catalase, oxidase, hydrogen peroxide production, amylase activity, V-P determination, gelatin liquefaction, nitrate reduction, as well as the utilization of D-glucose, arabinose, maltose, and sucrose. These findings indicate that the F9D strain is well-adapted to anaerobic environments and possesses strong metabolic capacities for breaking down various carbon sources and metabolic intermediates. However, limitations were observed in the utilization of citrate and sorbitol, suggesting that the F9D strain has inherent challenges in converting acetic acid derived from citrate and effectively metabolizing sorbitol.

Fig. 2.

Characteristics of strain F9D. A: Colony morphology of the F9D strain after 48 h of culture; B: Scanning electron micrograph image at 24,987 magnification; C: Phylogenetic tree based on 16S rDNA sequences of strain F9D. The phylogenetic neighbor joining tree was constructed using MEGA (version 11) and was bootstrapped 1000 times. Numbers in parentheses represent GenBank accession numbers. Numbers at nodes indicate bootstrap values. The bar represents sequence divergence

Table 2.

Physiological and biochemical identification results for strain F9D

Identification Index	F9D	Identification Index	F9D
Anaerobic growth	+	Nitrate reduction	+
Glucose fermentation	+	Citrate utilization	-
Catalase	+	D-glucose utilization	+
Oxidase	+	Arabinose utilization	+
Hydrogen peroxide activity	+	Maltose utilization	+
Amylase activity	+	Sorbitol utilization	-
V-P determination	+	Sucrose utilization	+
Gelatin liquefaction	+	Gram staining	+

The 16S rDNA gene sequences of strain F9D were obtained and submitted to GenBank (http://www.ncbi.nlm.nih.gov). They showed the highest similarity (99%) to those of B. albus (GenBank accession: MN093403). A phylogenetic tree was constructed based on the 16S rRNA coding gene sequences of the closest relatives (Fig. 2C). Based on these two methods, the F9D strain was identified as B. albus.

The potential application of B. albus for environmental pollutant bioremediation has been underexplored. This study presents initial evidence demonstrating the effective degradation of glyphosate by B. albus under different conditions. B. albus has a wide range of applications in other areas of environmental remediation [35–37]. In practical applications, Kim et al. employed B. albus SMG-1 as probiotics in the cultivation of Litopenaeus vannamei (Pacific white shrimp). This improvement in water quality subsequently enhanced the growth and overall health of L. vannamei [36]. Similarly, Kishor et al. demonstrated the remarkable capabilities of B. albus MW407057, which exhibited efficient decolorization of methylene blue dye and significant reduction in chemical oxygen demand (COD). Further toxicity assessments confirmed that the dye solution treated with B. albus exhibited significantly reduced toxicity, as evidenced by a seed germination rate of 90% [38]. These findings collectively suggest that B. albus poses minimal ecological and environmental safety risks, making it a promising candidate for widespread practical application and adoption. Further in-depth research on various applications of B. albus will be carried out in the future. In the future, B. albus F9D will undergo further comprehensive research for diverse applications, such as aquaculture probiotics, mycotoxin-degrading bacteria, and microorganisms that break down organic pollutants [39, 40].

Growth and degradation assays with strain F9D

The growth and degradation ability of the F9D strain were investigated in MSM using 50 mg/L glyphosate as the sole carbon source. The results indicated that the F9D strain exhibited growth without a lag phase, suggesting its capability to utilize glyphosate as a growth substrate (Fig. 3). Glyphosate degradation was dependent on bacterial cell density, but the rate of glyphosate degradation did not increase rapidly in the logarithmic phase. An obvious phenomenon was that most of the glyphosate was degraded by day 4 or 5. In contrast, there was no significant change in glyphosate concentration in the non-inoculated controls.

Fig. 3.

Biodegradation of glyphosate (50 mg/L) in MSM by strain F9D. Legend: ■, glyphosate control; ●, glyphosate biodegradation; ▲, cell growth. Data represent mean values of three replicates with standard deviation

The F9D strain was inoculated into MSM with 200 mg/L glyphosate at different pH values (5.0, 6.0, 7.0, 8.0, 9.0) and incubated for 5 days at 30 °C. The glyphosate degradation activities of F9D at different pH values are shown in Fig. 4A. The results indicated that alkaline conditions favored glyphosate degradation. At a pH of 9.0, F9D could degrade all glyphosate in only 3 days. In contrast, the degradation activity was weakened under weakly acidic conditions (pH 5.0–6.0).

Fig. 4.

Effects of initial pH (A), incubation temperature (B), initial glyphosate concentrations (C), and inoculation amounts (D) on glyphosate degradation by strain F9D. The error bars indicate the standard error of the mean

The F9D strain was incubated with glyphosate (200 mg/L) at a pH of 6.38 for 5 days at different temperatures (20, 25, 30, 35, and 40℃). As shown in Fig. 4B, the optimal temperature for growth and glyphosate degradation was 30℃. Nevertheless, F9D also showed good performance under other temperature conditions, and the degradation rates were all above 70%. These conditions are common in most areas where glyphosate abuse occurs, indicating the promising potential of F9D for glyphosate bioremediation [41–43].

The glyphosate degradation activities of F9D at different initial glyphosate concentrations (50, 100, 200, 400, and 800 mg/L) are shown in Fig. 4C. At different initial concentrations of glyphosate, the tests consistently demonstrated a common trend: as the glyphosate concentration rises, the degradation efficiency decreases. At different initial concentrations of glyphosate, the tests showed the same tendency: as the glyphosate concentration increases, the degradation efficiency decreases. According to recent studies, Zhang et al. reported a highly efficient glyphosate-degrading bacterial strain, Pseudomonas alcaligenes Z1-1, which completely degraded 200 mg/L of glyphosate within 7 days and exhibited a maximum tolerance concentration of 800 mg/L [44]. In addition, Zhang et al. discovered the YS622 microbial consortium, currently recognized as the fastest glyphosate degrader, capable of completely degrading 50 mg/L glyphosate within 36 h [45]. However, under high-concentration conditions (800 mg/L glyphosate), the degradation efficiency of YS622 remains inferior to that of F9D. In contrast, F9D demonstrates remarkable performance by degrading over 70% of glyphosate at 800 mg/L within 5 days. These findings highlight F9D as a standout performer in the field of glyphosate bioremediation.

The glyphosate degradation activity of F9D at different inoculation amounts is shown in Fig. 4D. The culture was added to MSM containing glyphosate concentrations of 200 mg/L at inoculation amounts of 1, 2, 3, 4, and 5% (v/v), and incubated at 35℃ for 5 days. The degradation activity was positively correlated with the amount of inoculation. Experiments were conducted to investigate the optimal conditions for F9D, specifically pH=9, 30℃, glyphosate concentration of 50 mg/L, and inoculum of 5%.

AMPA, being the most commonly identified glyphosate intermediate in the natural environment, holds significant importance in research endeavors and cannot be overlooked [46–48]. Therefore, investigating the relationship between the F9D strain and AMPA is of critical importance. The AMPA degradation capability of F9D at different initial concentrations (50, 100, 200, 400, 800, and 1600 mg/L) is shown in Fig. 5. According to the results, F9D is capable of degrading AMPA. Under a lower initial concentration of AMPA, more than 60% of AMPA can be degraded by F9D, which reflects its promising potential in glyphosate-contaminated environments. To the best of our knowledge, the isolate F9D represents the first reported strain within the genus Bacillus capable of completely degrading glyphosate and its primary metabolite, AMPA, at a concentration of 100 mg/L.

Fig. 5.

Effect of initial aminomethylphosphonic acid (AMPA) concentration on degradation of AMPA by strain F9D. The error bars indicate the standard error of the mean

Degradation kinetics of glyphosate and aminomethylphosphonic acid by strain F9D

The F9D strain rapidly degraded and utilized glyphosate up to a concentration of 800 mg/L, while a lag phase was observed at higher glyphosate concentrations (Fig. 6A). Based on the kinetic analysis, the degradation of glyphosate by this strain exhibits a concentration-dependent behavior. Among the models evaluated, the Luong model demonstrated superior predictive accuracy, as evidenced by its lower Root Mean Square Error (RMSE), higher adjusted R² (adR²), and Backward/Forward (BF/AF) values closer to 1. Additionally, the Luong model incorporates a unique parameter (Sm), which defines the critical substrate concentration for inhibition. This parameter provides a more comprehensive description of substrate inhibition kinetics, especially at high glyphosate concentrations [49–51]. Therefore, the substrate inhibition model [Eq. (1)] adapted from the Luong model was employed to fit the specific degradation rate (q) at various initial concentrations of glyphosate. From the value of q and the initial glyphosate concentration, the kinetic parameters, including K_i, K_s, and q_max, for the substrate inhibition model were established as 388.1477 mg/L, 7.6897 mg/L, and 0.6577 d^− 1, respectively.

Fig. 6.

Degradation kinetics of glyphosate and AMPA with different initial concentrations by strain F9D: (A) Relationship between initial glyphosate concentration and specific degradation rate by strain F9D; and (B) relationship between initial AMPA concentration and specific degradation rate by strain F9D

AMPA is considered a significant degradation intermediate of glyphosate, known for its toxicity and resistance to biodegradation [52]. Thus, a strain capable of degrading both glyphosate and AMPA is required for bioremediation. In this study, F9D tolerated and degraded AMPA up to a concentration of 800 mg·L^− 1. With an increase in the initial AMPA concentration, the degradation rate of F9D was decreased, suggesting that this chemical may act as a partial inhibitor of F9D. The kinetic curve of AMPA degradation at different initial concentrations is shown in Fig. 6B. The values of K_i, K_s, and q_max for the AMPA inhibition model were determined to be 546.4162 mg/L, 4.1874 mg/L, and 0.5048 d^− 1, respectively.

Glyphosate metabolism of strain F9D

In the existing research on glyphosate-degrading bacteria, two main pathways have been identified for bacteria to degrade glyphosate. In the first pathway, the main degradation products are AMPA and glyoxylic acid. In the second pathway, glyphosate is degraded into formaldehyde and glycine [5]. Although most of the glyphosate-degrading bacteria degrade glyphosate through a single metabolic pathway, there exist a few strains which utilize both metabolic pathway, such as Ochrobactrum anthropi GPK3 and B. cereus CB4 [53, 54]. In the present study, the degradation metabolites resulting from glyphosate breakdown by the F9D strain were meticulously analyzed utilizing UPLC-MS/MS, and the presence of AMPA was unequivocally confirmed. As shown in Fig. 7, glyphosate was initially degraded to AMPA and glyoxylic acid through cleavage of the C–N bond by glyphosate oxidoreductase. Glyoxylic acid will be used in the tricarboxylic acid (TCA) cycle, and AMPA will be further degraded into several metabolites which can be used by the F9D strain. The toxicity of glyphosate degradation products was evaluated using Toxicity Estimation Software Tool (T.E.S.T.), which determined the Fathead minnow (Pimephales promelas) LC50, Daphnia magna LC50, and bioconcentration factors [55, 56]. Notably, the intermediates consistently demonstrated higher median lethal concentration (LC50) values and lower bioconcentration factor values (Figure S1), suggesting that the biodegradation of glyphosate is a detoxification process. These results highlight the considerable potential of utilizing F9D for the remediation of glyphosate-contaminated environments.

Fig. 7.

Proposed pathway for degradation of glyphosate by strain F9D

Biodegradation of glyphosate in soils and water–sediment system by strain F9D

To explore the ability of B. albus F9D in the bioremediation of glyphosate-contaminated soils, biodegradation with F9D was carried out in both sterile and non-sterile soils. The results shown in Fig. 8A indicate that the F9D strain possesses glyphosate degradation ability in soils. After incubation in both sterile and non-sterile soils for five days, the F9D strain degraded more than 75% of glyphosate. Moreover, there was little difference between the two experimental groups, indicating that the native bacterial community has little influence on the activity of F9D. As described previously studies [57], native micro-organisms may have a synergistic or competitive effect on non-indigenous species. Generally, the degrading micro-organisms isolated under experimental conditions usually fail to degrade pollutants due to the abiotic and biotic stresses [58, 59]. However, in our study, the F9D strain efficiently degraded glyphosate in soils without any further treatment, indicating that this strain holds promising potential and advantages as a bioremediation organism for eliminating glyphosate residues from various environments.

Fig. 8.

Degradation of glyphosate in soil and water–sediment system by strain F9D: (A) Degradation of glyphosate in sterile and non-sterile soils inoculated with strain F9D; and (B) degradation of glyphosate in sterile and non-sterile water–sediment (mixture) system inoculated with strain F9D

To further elucidate the degradation capacity of the F9D strain, an experiment was conducted under artificially simulated harsh aquatic conditions. After incubation in the water–sediment system for four days, F9D still demonstrated degradation activity, where more than 70% of glyphosate was degraded in both sterile and non-sterile water–sediment systems (Fig. 8B). Nevertheless, the results showed that the controls in the non-sterile water–sediment system also degraded nearly 70% of glyphosate. Therefore, it is plausible that the indigenous microbial community has absorbed a portion of glyphosate to some extent. The experimental group in the non-sterile water–sediment system had better degradation activity in first three days, demonstrating the degradation ability of the F9D strain. Moreover, the experimental group in the sterile water–sediment system had already degraded more than 65.8% of the glyphosate within one day. The evidence presented above is good proof of the degradation ability of strain F9D. As shown in Table 3, the kinetic parameters for glyphosate degradation in both sterile and non-sterile soils, when inoculated with strain F9D, demonstrating its effectiveness. Thus, this strain can be considered a potential and efficient candidate for the bioremediation of glyphosate-contaminated aquatic environments.

Table 3.

Kinetic parameters of glyphosate degradation in sterile and non-sterile soils inoculated with strain F9D

Treatment	Regression equation	K (d− 1)	t1/2 (d)	R 2
Sterile soils + glyphosate	Ct = 405.11692e− 0.08077t	0.08077	8.6	0.95156
Sterile soils + glyphosate + F9D	Ct = 387.08765 e− 0.2751t	0.2751	2.5	0.98174
Non-sterile soils + glyphosate	Ct = 400.72964e− 0.15817t	0.15817	4.4	0.98038
Non-sterile soils + glyphosate + F9D	Ct = 366.18258e− 0.30393t	0.30393	2.3	0.91053

Impact of strain F9D on indigenous soil microbial communities

The makeup of the microbial community is indicative of the distribution and relative prevalence of diverse microbial species within an ecosystem. Thus, scrutinizing the shifts within the soil microbial community subsequent to the introduction of the glyphosate-degrading bacterium F9D provides a precise depiction of the intrinsic changes occurring within the population of degrading microorganisms [60]. To elucidate the ecological dynamics of microbial community restructuring, comparative bioinformatics analyses were performed on rhizosphere samples collected prior to and following inoculation with the F9D microbial consortium. Soil samples were analyzed at four distinct time points: 0, 1, 3, and 5 days post-inoculation. The profile and heat map depicting the progression of the microbial community structure are presented, with the default display of the abundance rankings for the top 30 most significant species (Fig. 9).

Fig. 9.

Impact of B. albus F9D on indigenous microbial community structure in glyphosate-contaminated soil. (A) Heat map of soil microbial communities at the phylum level; (B) Heat map of soil microbial communities at the class level; (C) Stacked bar chart of soil microbial communities at the phylum level; (D) Stacked bar chart of soil microbial communities at the class level

The results demonstrate that in soil affected by glyphosate contamination, the abundance of Firmicutes at the phylum level rose from 1.06% at day 0 to 1.70% at day 1 post-inoculation with F9D. Correspondingly, at the class level, the abundance of Bacilli saw an increase from 0.8% at day 0 to 1.54% at day 1. This outcome is consistent with the findings from a sterile water-sediment system, where more than 65.8% of the glyphosate was degraded within the first day.This could be attributed to two main reasons: Firstly, the strain F9D, a member of the phylum Firmicutes and class Bacilli, has effectively outcompeted native microbes in the glyphosate-contaminated soil, successfully colonized the area, and exhibited remarkable remedial properties. In other words, the inoculation of F9D into the contaminated soil led to its rapid proliferation, which was in consistent with the rapid degradation of glyphosate. Wu et al. observed analogous outcomes in their study where Burkholderia sp. A11 was utilized for the remediation of soil contaminated with the organophosphorus insecticide acephate [61]. Secondly, at the phylum level, a notable and significant surge in the bacterial abundance of Proteobacteria and Verrucomicrobia was observed within a single day. Within the Proteobacteria phylum, Pseudomonas spp [62]. and Agrobacterium [63] have been consistently reported to play crucial roles in the degradation of glyphosate and its metabolite AMPA. Given these observations, it is hypothesized that the bacteria from these two phyla may act in concert to drive the degradation of glyphosate. This synergistic effect is likely mediated by the recruitment of these bacteria through the action of F9D, thereby enhancing the overall degradative process.

Alpha diversity is a measure of the variety within a specific environment or ecosystem, focusing on species richness, evenness, and sequencing depth. It is typically assessed using indices such as Chao1 and Shannon to reflect the diversity’s richness and evenness [27]. This study performed α-diversity analysis using the Chao1 and Shannon index for the samples (Figure S2), and the results demonstrated that both the Chao1 and Shannon indices revealed a significant decline in the richness and evenness of microbial species in soil samples within 1 day of glyphosate addition. In contrast, the introduction of F9D to the soil effectively preserved a high level of microbial richness and evenness. The experimental results were corroborated by the principal component analysis (Figure S3). In essence, F9D’s exceptional glyphosate degradation capacity, combined with its ability to maintain soil microbial diversity, positions it as a highly promising candidate for remediating glyphosate-contaminated soils.

Conclusion

The degradation of glyphosate, a widely used herbicide, by the novel bacterium B. albus F9D was systematically investigated under various growth conditions. Through optimized experimental designs, the most favorable conditions for the degradation of glyphosate by F9D were identified as follows: a pH of 9, an incubation temperature of 30℃, an initial glyphosate concentration of 50 mg/L, and an inoculum density of 5%. To the best of our knowledge, this study represents the first documented instance of simultaneous degradation of glyphosate and its major metabolite, AMPA, by the bacterium B. albus. A preliminary exploration of the strain’s degradation ability in soils and water-sediment systems was subsequently conducted based on these findings. This strain has remarkable potential for application in bioremediation and wastewater treatment. In addition, the reaction conditions studied in this work provide valuable reference for the application of glyphosate bioremediation. However, further research must be carried out to confirm the growth of F9D in natural environments and to determine its potential for bioremediation of glyphosate-contaminated terrestrial and aquatic environments. Furthermore, future research should focus on exploring the glyphosate-degrading genes and enzymes mediated by strain F9D. Exploring in-depth molecular work in the future could aid in designing more precise microbial technology for glyphosate bioremediation in contaminated soil and water.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

All authors contributed to the manuscript revision and approved the submitted version. Conceptualization, W.Z. and S.C.; methodology, W-J.C.,M.L.,S-F.C, and Y.Z.; formal analysis, W-J.C,M.L., S-F.C., and Y.Z.; investigation, W-J.C., M.L., S-F.C., and Y.Z.; resources, W-J.C,M.L., S-F.C., and Y.Z.; data curation, W-J.C,M.L., S-F.C., and Y.Z.; writing-original draft preparation, W-J.C., M.L., S-F.C., and Y.Z.; writing-review and editing H.S., M.H.A.,K.B., S.M., M.A.G.,W.Z., and S.C; supervision,W.Z.

Funding

This study was financially supported by grants from the Yunnan Fundamental Research Projects, China (202301AT070797, 202301BE070001-060).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.