Introduction

Phenazine-1-carboxylic acid (PCA) originally derives from the secondary metabolites of Pseudomonas species in the soil1. In recent years, PCA has been widely used in agriculture as the main active ingredient of a new bio-fungicide, shenqinmycin, due to its high yield, broad spectrum, and highly effective antibacterial activity2. However, PCA causes some degree of damage to non-target organisms by generating reactive oxygen species (ROS) and interfering with the respiratory electron transport chain. Additionally, PCA is a precursor substance for various phenazine compounds with similar antibacterial mechanisms2,3, and it is also an intermediate metabolite of these compounds4,5. Therefore, the environmental risk of PCA has attracted increasing attention from researchers, and the transformation and degradation of PCA urgently need to be studied6.

Laboratory and field studies demonstrate that biodegradation plays a major role in the dissipation of organic pollutants in soils. Recently, several different genera of microorganisms, such as Sphingomonas5,7, Rhodococcus8,9, Mycobaterium and Nocardia4 capable of degrading PCA have been reported and the activities of their free cells have been validated in the laboratory. However, degrading microorganisms are usually exposed to several environmental stresses, including temperature increments and high osmotic pressures, etc., during in-situ remediation, resulting in a decreased in the number of viable cells and enzymatic reaction efficiency10,11. To date, there have been few studies on the environmental stress tolerance of PCA-degrading strains. Therefore, there is a high demand for efficient PCA-degrading strains that can withstand various stresses.

For successful in-situ remediation of contaminated environments, in addition to the environmental tolerance of the microorganisms themselves, another method to enhance the survival rate of exogenous degrading bacteria and increase their activity is to immobilize them12,13. Currently, there is an increasing amount of research on the adsorption carrier materials for immobilization, such as sawdust, wheat straw, and biochar14,15. Among them, biochar prepared by pyrolysis of plant materials has a high specific surface area and internal porosity, and it possesses an excellent adsorption capacity for ions, compounds, and microorganisms. It is widely used to improve soil quality, increase agricultural yields, and alleviate environmental issues16,17,18.

Encapsulation with sodium alginate (SA) is a simple and cost-effective method for immobilizing microorganisms19. Moreover, SA is a natural substance that is both beneficial to microorganisms and environmentally friendly. Immobilization using SA can protect microorganisms from environmental stress, enhance their survival rate, and allow them to be reused in multiple reactions, thus reduce operating costs20. One obvious drawback is that the diffusion of substrates and products may be restricted in larger-sized gel beads, affecting the reaction rate21. This drawback can be complemented by biochar possessing excellent exchange capacity for substrates and microorganisms22. It is proposed that the co-work of SA and biochar may greatly improve the performance of microbial immobilization. To date, there have been only a few reports on the application of co-immobilization with SA and biochar. The synergy between SA and biochar in agricultural pollution bio-remediation is worth exploring.

Pesticide-degrading bacteria play a crucial role in alleviating phytotoxicity by degrading pesticides, enhancing plant resistance, and improving soil quality23. Numerous studies have shown that free cells of pesticide-degrading bacteria can mitigate the harmful effects of pesticides on non-target plants. For example, Zhang et al. demonstrated the enhanced degradation of chlorimuron-ethyl in the rhizosphere by Hansschlegelia zhihuaiae S113, establishing a mutually beneficial relationship between S113 and cucumber24. However, few studies have examined the mitigating effect of immobilized cells on plant toxicity, particularly regarding plant germination.

Based on the theory of detoxifying pollution by carrying degradable microorganisms with biochar, we hypothesize that immobilized SA-biochar-bacterial beads can overcome traditional limitations through structural stability, microenvironment optimization and dual adsorption-degradation mechanisms, offering a scalable and sustainable solution for environmental remediation. Thus, we first isolated a strain with heat and osmotic tolerance that can effectively degrade PCA. This strain was then co-immobilized on SA-biochar carriers, and the process was optimized to achieve better performance and more repetitions. Finally, a seed germination test was designed to evaluate the ability of immobilized SA-biochar-bacterial beads to alleviate phytotoxicity. The method of co-immobilization with SA and biochar represents a paradigm shift by integrating material science and microbial ecology. This study provides an effective reference for in-site pollution remediation.

Results

Isolation and screening of the bacteria for PCA degradation

Six PCA-degrading strains, named WH101, WH102, WH103, WH104, WH105, and WH106, were isolated from rhizosphere soil samples in Nanjing. The degradation of PCA as the sole carbon source was investigated (Fig. 1).

Fig. 1
figure 1

The degradation of 0.10 mM PCA by different strains. The symbols used in the graph represent different strains as follows: (ring), PCA control; (triangle), PCA with strain WH101; (Right-Pointing Triangle), PCA with strain WH102; (black filled ring), PCA with strain WH103; (down-pointing triangle), PCA with strain WH104; (diamond), PCA with strain WH105; (Left-Pointing Triangle), PCA with strain WH106. Error bars represent the standard errors from three replicates. A (black asterisk) indicates a statistically significant difference between the PCA treatment-line and PCA control-line(P < 0.05).

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Strains WH101, WH104, WH105, and WH106 exhibited lag phases in PCA degradation within the first 18–24 h, indicating that the enzymes involved in the initial steps of PCA degradation may be inducible, which hinders rapid PCA degradation. On the other hand, strains WH102 and WH103 demonstrated a rapid degradation to PCA, with their degradation process occurring without any lag phase. This indicates that they are suitable for the fast remediation of PCA-contaminated sites. Analysis of the degradation rates of the six PCA-degrading strains revealed that strain WH103 exhibited the best degradation effect on PCA, with a degradation rate exceeding 99% for 0.10 mM PCA after 27 h of cultivation. Although the degradation efficiency of strain WH102 was slightly lower than that of strain WH103, reaching 97.13% for 0.10 mM PCA after 36 h, it still showed significant effectiveness in degrading PCA. Strains WH102 and WH103 were selected for further studies on environmental tolerance.

Stress tolerance of PCA-degrading strains WH102 and WH103

Further research on the degradation characteristics of strains WH102 and WH103 revealed that the optimal pH and temperature for their growth were 7.0 and 30 °C, respectively (data not shown). When strain WH102 was incubated at 42 °C for 3 days and then subjected to spot tests on MMP [the mineral salts medium (MSM) with 0.10 mM PCA] agar plates, its viability decreased significantly (Fig. 2a). The addition of 0.7 M NaCl to MMP agar plates further reduced the number of bacteria of WH102.

Fig. 2
figure 2

Identification of thermotolerant and osmotic pressure-tolerant strain. The (a) and (b) were 5 µL aliquots of tenfold serial dilutions of strain WH102 and strain WH103 were spotted on MMP [the mineral salts medium (MSM) with 0.10 mM PCA] agar plates with 0 M and 0.7 M NaCl, respectively. The spotted MMP plates were incubated for 3 days at 30 °C and 42 °C, respectively.

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Strain WH103 was the only one that grew well on MMP agar plates at 42 °C and in the presence of higher concentrations of NaCl, showing only a minimal decrease in the total number of bacterial colonies (Fig. 2b). From this, it can be inferred that strain WH103 is thermotolerant and tolerant to osmotic pressure.

Identification of PCA degrading bacteria WH103

The colonies of strain WH103, which has the best degradation activity and stress tolerance, grown on the MMP agar plate were smooth, convex and milk-white (Fig. 2b). It is a round-shaped bacterium about 0.8–1.1 μm in diameter (Fig. 3). It was negative for oxidase, starch hydrolysis, methyl red and nitrate reductase tests, and positive for phenylalanine aryl aminase, tyrosine aryl aminase and L-lactic acid salinization tests (data not shown). These characteristics were consistent with the general properties of the Rhodococcus species.

Fig. 3
figure 3

The strain WH103 captured by a scanning electron microscope.

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The 16 S rRNA gene sequence of strain WH103 was amplified and sequenced by General Biol. Co. Ltd (Chuzhou, Anhui). The phylogenetic tree, as shown in Fig. 4, was constructed based on the 16 S rRNA gene sequence of strain WH103 (Table S1, GenBank accession number is PQ390419). Strain WH103 shows the closest genetic relationship to Rhodococcus, with similarities of 99.08%, 98.62% and 98.61% to Rhodococcus sp. WH99, Rhodococcus sp. D32 and Rhodococcus equi HF-1, respectively. Based on these preliminary results, strain WH103 was identified as Rhodococcus species.

Fig. 4
figure 4

Phylogenetic tree constructed on the basis of 16 S rRNA gene sequences of strain WH103. The Neighbour-joining phylogenetic tree based on 16 S rRNA gene sequences shows the relationships of strain WH103 with related taxa. Bootstrap values (%) are indicated at the nodes, and the scale bar represents 0.01 substitutions per site.

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Optimization and characterization of the immobilized SA-biochar-bacterial beads

The morphological appearance of SA-biochar-bacterial beads is shown in Fig. S1. The structure parameters of biochar and biochar-immobilized beads were measured. The specific surface area of the biochar derived from the trunk of masson pine is 603.9 m2 g−1, the pore volume is 0.051 cm3 g−1, and the mass fractions of the main elements C, H, O, and N that constitute the biochar are 92.6%, 1.2%, 6.2%, and 0.025%, respectively. The specific surface areas of the SA-biochar-bacterial beads and SA-bacterial beads are 25.92 and 3.29 m2 g−1, respectively.

Optimization of parameters such as temperature, pH and biochar amount is crucial for process development. An orthogonal test was conducted and the results of the immobilization of strain WH103 were analyzed using the extreme difference method, as shown in Table 1. The experimental results from Table 1 were further processed by the range analysis to determine the most significant parameters for the degradation rate of PCA in MMP liquid medium by SA-biochar-bacterial beads. Based on the results, we can conclude that the primary and secondary factors affecting the process were amounts of biochar > temperature > pH. Combining with the results of the single factor experiment (data not shown), the maximum rate of PCA degradation was achieved when the cells immobilized in biochar were cultured at 30 °C and an initial pH of 7.0. The degradation rate decreases significantly at temperatures above or below 30 °C. When SA-biochar-bacterial beads were incubated at a pH below 6.5, the rate of PCA degradation was lower. However, there was no significant difference between pH 6.5 and pH 7.5.

Table 1 Orthogonal array design for PCA degradation by SA-biochar-bacterial beads.
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The amount of biochar was the most significant factor affecting the degradation rate of PCA. 99.26% of 0.10 mM PCA was depleted after 21 h of incubation when 75 mg of biochar was added. However, in the absence of biochar, PCA degradation by SA-bacteria beads with a lower degradation rate was observed, with only 77.24% of 0.10 mM PCA was depleted after 21 h of incubation (Fig. 5). Thus, we could conclude that the PCA degradation rates were highest in the SA-biochar-bacterial beads, followed by free cells and then SA-bacteria beads. Based on the results above, the run A2B2C3 was selected as the best group for preparing and using SA-biochar-bacterial beads.

Fig. 5
figure 5

The degradation of 0.10 mM PCA by different subject. The symbols used in the graph represent different subject as follows: (ring), PCA control; (black filled ring), PCA with free cells of strain WH103; (Hexagon), PCA with SA-bacteria beads; (black filled Hexagon), PCA with SA-biochar-bacterial beads. Error bars represent the standard errors from three replicates. A (black asterisk) indicates a statistically significant difference between the PCA treatment-line and PCA control-line (P < 0.05). A (red asterisk) indicates a statistically significant difference between the PCA with SA-biochar-bacterial beads-line and PCA with free cells of strain WH103-line (P < 0.05). A (blue asterisk) indicates a statistically significant difference between the PCA with SA-biochar-bacterial beads-line and PCA with SA-bacteria beads-line (P < 0.05).

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Reusability of immobilized strain WH103 cells

The immobilized cells can be reused for degradation of 0.10 mM PCA. The immobilized SA-biochar-bacterial beads can be reused in 12 consecutive reaction cycles (21 h per cycle) without any loss of biocatalytic activity under optimal conditions (Fig. 6). PCA levels were lower than 1% of the initial concentration at the end of each cycle. The metabolites were collected from the culture media above mentioned by filtering to remove particles.

Fig. 6
figure 6

Repetitive batch degradation of PCA by immobilized bacteria. The columns in the bar chart represent different subject as follows: (Rectangle), PCA with SA-bacterial beads; (black filled Rectangle), PCA with SA-biochar-bacterial beads. Error bars represent the standard errors from three replicates. A (black asterisk) indicates a statistically significant difference between the PCA with SA-biochar-bacterial beads-column and PCA with SA-bacteria beads-column (P < 0.05).

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Phytotoxicity assessment

The sample upon degradation was tested for its phytotoxicity by a seed germination assay. The assessment of seed germination and root development showed the influence of degraded products on plant growth and development (Table 2). The seeds treated with respective treatments were measured for the percentage germination after 7 days, and the length of the root (radical) and shoot (plumule) were recorded.

The phytotoxicity analysis revealed that the seed germination rate of degradation products was significantly higher than that of PCA treated seeds. The metabolites may be structurally simple and less toxic or the metabolites are further degraded till mineralized completely. In addition, the metabolites may exhibit reduced fat solubility, limiting their penetration into plant cells and subsequent destruction of membrane integrity. Some metabolites act as signaling molecules to prime plant defenses without causing direct harm. All plant varieties used in the study still had more than 15% of their root and shoot germinated when treated with MMP media. This might be because the possibility of PCA acting as a micronutrient in MMP media. These toxic compounds accumulate in the plants and enter the food chain. In summary, the biodegradation of PCA can effectively alleviate plant toxicity and it is both efficient and environmentally friendly.

Table 2 Average root length in Cm, percentage relative seed germination (RSG), relative root growth (RRG) and germination index (GI) of four types of seeds.
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Discussion

PCA is produced by some strains of Pseudomonas and a few other bacterial genera. The producers and degraders of PCA in the rhizosphere are more abundant and diverse compared to other areas. Therefore, as reported, multiple PCA-degrading bacteria have been isolated from the rhizosphere, including aerobic degrading bacteria such as Rhodococcus8,9, Sphingomonas5,7 and Mycobacterium and Nocardia4. Among them, Rhodococcus sp. stands out due to its strong environmental adaptability and multiple intracellular oxidases25,26.

Bacteria must overcome a wide range of other environmental stresses to survive. To withstand such adverse environments, bacteria have evolved complex stress-sensing defense mechanisms that involve the temporary production of specific proteins and physiological changes27,28. Prasad et al. concluded that bacteria undergo significant changes including the induction of stress proteins and the accumulation of compatible solutes such as betaine, carnitine, and trehalose during osmotic stress. However, there are only a few reports describing the physiological stress responses in degrading bacteria. In this study, only strain WH103 grew well on MMP agar plates at 42 °C with higher concentrations of NaCl, with just a minimal decrease in the total number of bacterial colonies. All the results indicated that strain WH103 exhibited the excellent degradation activity and stress tolerance, suggesting its potential for large-scale bioremediation of PCA-contaminated soil. The superior stress tolerance of Rhodococcus sp. WH103 compared to WH102 likely stems from a combination of genetic, physiological, and metabolic adaptations. To confirm these hypotheses, genomic sequencing (e.g., identifying stress-related operons) and transcriptomic profiling under stress conditions are recommended. Comparative studies with WH102 could pinpoint key genetic differences, as demonstrated in strain Pediococcus pentosaceus analyses29.

The use of free cells for bioremediation has many disadvantages, while microbial immobilization offers numerous important advantages, such as higher cell density, long-term reuse, and increased efficiency30. For these reasons, immobilized cells have been used in the degradation of a variety of important chemicals. It should be noted that the optimal degradation conditions for immobilized cells may be different from those for free cells, but the reasons why external conditions affect the degradation of immobilized cells may be similar to those for free cells31. In our study, the optimal degradation conditions for immobilized cells were similar to those for free cells. When the temperature exceeds or falls below the optimum temperature for microbial growth, the growth metabolism of the strain will be affected and the activity of the enzyme will decrease, resulting in a decrease in the ability to degrade the substrate32. In a similar way, Lambert concluded that the growth and metabolism of the strain would be affected and the activity of the enzyme would decrease when the optimum pH deviates. This was shown by the experimental effect of pH on microbial growth, which in turn affects the degradation performance33,34. Our study, which integrated single-factor experiments with orthogonal optimization, elucidated the effects of temperature and pH on the immobilized pellets of strain WH103. It was found that temperature is the second important factor for the degrading efficiency of immobilized pellets, with the optimal range being 25–35 ℃. The pH within a certain range (6.5–7.5) had very little influence on the degradation effect, possibly because the immobilized material had a certain buffer effect against acids and bases.

Biochar is a carbon-rich product obtained by pyrolyzing of biomass under anoxic or anaerobic conditions at a relatively low temperature (less than 700 ℃)35. It has a high specific surface area and a good pore structure, making it widely used in soil fertility improvement and pollutant remediation due to its low cost and environmental friendliness36. In this study, the specific surface area of the immobilized SA-biochar-bacterial beads can be increased by adding the prepared Masson pine trunk biochar to them. This increase is conducive to the colonization of bacteria on the immobilized SA-biochar-bacterial beads and the increase of the microbial biomass of the biochar-immobilized beads. As shown in Fig. 7, bacteria primarily attach and grow on the surface of the immobilized SA-biochar-bacterial beads. Additionally, biochar facilitates the comprehensive adsorption of pollutants, enhances contact opportunities between pollutants and degrading bacteria, and protects microorganisms, ultimately improving the degradation rate37. Compared with the traditional SA immobilization method without the addition of biochar, the immobilized SA-biochar-bacterial beads mainly rely on the adsorption of biochar to remove the substrate in the initial stage of degradation. Activating bacteria enhances the microbial degradation effect, leading to a more significant impact. This results in the continuous adsorption-degradation-adsorption process of the biochar-immobilized bacteria, ultimately greatly improving the degradation rate.

Fig. 7
figure 7

The technological process of PCA degradation by SA-biochar-bacterial beads.

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Moreover, the exchange of substrate and dissolved oxygen poses a challenge to the immobilized bacteria. By adding biochar to the SA-bacteria treatment, we can not only the rich pore structure on its surface can be utilized to improve permeability, but also it accelerates the exchange rate among bacteria, substrate and oxygen, ultimately enhancing the mass transfer performance of the biochar-immobilized bacteria and the activity of the bacteria38,39,40. In this study, the primary and secondary PCA degradation rates were highest in SA-biochar-bacterial beads, followed by those of free cells, and then SA-bacteria beads. This phenomenon is similar to the one observed by Qiao et al., who immobilized pyridine-degrading bacteria with bamboo charcoal. Throughout the entire process, the degradation rate of free bacteria is consistently lower than that of the SA-biochar-bacterial beads, but higher than that of the SA-bacteria treatment without the addition of biochar37. Yin et al. reported that a novel polydopamine (PDA)-modiffed iron/polylactic acid (PLA)/biochar composite (PDA@OBC-nZVI) coupling with Shewanella oneidensis MR-1 (MR-1) was successfully synthesized to remove 1,1,1-trichloroethane (1,1,1-TCA) from simulated groundwater. 83.14% of 100 mg⋅L− 1 1,1,1-TCA was removed by PDA@OBC-nZVI + MR-1 within 360 h. The degradation rate is about 25% higher than that degraded by PDA-nZVI + MR-141. Our results align with and extend previous research on microbial immobilization for pollutant degradation, with notable improvements in stability and efficiency. SA-biochar-bacterial beads achieved complete degradation of PCA (below detection limits) within optimized conditions. Our co-immobilized system combined biochar’s adsorption capacity with SA biocompatibility, accelerating degradation by 28.51% compared to single SA systems.

To date, only a few reports have been published on the application of co-immobilized cells in alleviating phytotoxicity42,43,44,45. The phytotoxicity analysis by Swarnkumar et al. showed that the degraded product was less toxic compared to that of crude dye treated seeds46. This indicates the presence of lower-toxicity or non-toxic byproducts after treating Reactive Red 120 with strain VITSAJ5, in contrast to the untreated Reactive Red 120 solution. Our findings are consistent with this result. However, there is a significant gap in our understanding of how the synergistic effects between bacteria and biochar can enhance the remediation of phytotoxicity.

Despite promising results, this study has several limitations: (1) Scalability: Transitioning from lab to industrial scale poses challenges such as even distribution of beads and maintaining consistent conditions. (2) Economic feasibility: Costs of biochar, sodium alginate, and long-term bacterial culturing may hinder large-scale use. (3) Field application: Complex soil environments, competing microorganisms, and weather conditions could impact performance.

Future research could focus on investigating the pot experiment as well as field application. The high degradation rate and reusability of the SA-biochar-bacterial beads make large-scale bioremediation more feasible and cost-effective, thus reducing the long-term environmental pollution from agricultural chemicals. The process shows potential for wide-scale application without causing additional environmental stress, compared to traditional chemical-based pollution control methods. By alleviating PCA-induced phytotoxicity, as demonstrated in the seed germination assays, it helps in fostering healthier crop growth. This directly contributes to sustainable agriculture by reducing the harmful effects of pesticides on non-target organisms, ensuring better yields and crop quality. If PCA can be effectively degraded, it may lead to more sustainable use of bio-fungicides like shenqinmycin.

Conclusions

Screening experiments were carried to select the most active PCA-degrading bacteria, Rhodococcus WH103, which showed tolerance to high temperature and high osmotic pressure. Under the conditions of 30 °C and pH 7, 75 mg of biochar was added for co-immobilization. SA-biochar-bacterial beads degraded over 99% of 0.10 mM PCA within 21 h and exhibited reusability for up to 12 cycles. Notably, the SA-biochar-bacterial beads significantly alleviated phytotoxicity of PCA by seed germination assay. The results of this study provide a theoretical basis for the practical application of immobilized microbial technology to repair PCA contamination. Through the pot experiments and field applications, the SA-biochar-bacterial beads will have significant practical applications and high commercialization. Future research could focus on exploring other pollutant-degrading bacteria or testing in diverse environmental conditions.

Materials and methods

Chemicals and media

The PCA (98%) was purchased from Shanghai Qinba Chemical Co., Ltd. (Shanghai, China). All the chemicals used in this study were of the highest analytical grade. Luria-Bertani (LB) medium contained the following ingredients per liter: tryptone 10.0 g, yeast extract 5.0 g, NaCl 10.0 g. MSM contained the following ingredients per liter: NaCl 1.0 g, K2HPO4 1.5 g, KH2PO4 0.5 g, NH4Cl 1.0 g, MgSO4 0.2 g. MMP was generated by supplementing MSM with 0.10 mM PCA as the sole carbon source unless otherwise stated. Solid media were prepared by adding 15 g of agar per liter to the respective liquid media. The initial pH of the media was adjusted to 7.0 unless otherwise stated and autoclaved at 121 °C for 30 min.

Biochar derived from the trunk of Masson pine tree was purchased from Henan Xingnuo Environmental Protective Materials Co., Ltd. (Henan, China) and sieved through a 1 mm sieve. It was then washed several times with deionized water and dried at 75 °C for 24 h. The elemental contents of biochar, including carbon (C), hydrogen (H), and nitrogen (N), was determined by an elemental analyzer (Flash EA 1112, Thermo Finnigan, Italy), and the oxygen (O) content was obtained by subtraction. The specific surface area of the biochar was determined using the nitrogen adsorption-desorption method on an Autosorb-1-C analyzer (Quantachrome Instruments, USA).

Isolation of efficient PCA-degrading strains

Strain isolation was carried out using the enrichment culture method as previously described, with some modifications47,48. Soil samples collected from a rice field in Nanjing, China (31.65°N, 119.07°E), were used as initial inoculants to enrich PCA-degrading bacteria.

Five grams of soil samples was added into a 250 mL flask containing 100 mL MMP and incubated at 30 °C, 150 rpm for three days. From this, 5 mL of the enriched culture was transferred to fresh 100 mL MMP liquid medium and incubated under the same conditions for another three days. After three successive rounds of such enrichment, the content of PCA was determined by high-performance liquid chromatography (HPLC). Compared with the blank group, there was a significant decrease in PCA content, indicating the presence of enriched culture with degradation activity. The enriched liquid with degradation activity was then diluted in a gradient of 10− 4 to 10− 8, and 0.10 mL of each dilution was spread on MMP solid media. The plates were incubated upside down in a constant temperature incubator at 30 °C until colonies grew. Single colonies exhibiting distinct morphological characteristics were selected for isolation and purification. The purified colonies were then cultured and inoculated into 100 mL of MMP liquid medium, respectively. The bacterial colonies were then incubated at 30 °C and 150 rpm for 3 days to evaluate their PCA degradation activity. Six colonies exhibiting significant degradation activity were preserved at -80 °C for further experiments. They were named WH101, WH102, WH103, WH104, WH105 and WH106, respectively.

After pre-culturing single colonies in LB liquid medium at 30 °C and 150 rpm until reaching the late-exponential phase, the bacterial cell pellets were collected by centrifugation at 6000×g for 5 min at room temperature. They were subsequently washed and resuspended in MMP liquid medium to achieve a final OD600 of 1.00, which served as the initial inoculants for the subsequent experiments.

Seeking PCA-degrading strains with stress tolerance

To further screen for the thermotolerant and osmotic pressure-tolerant strains, the strains were grown in MMP liquid medium to an OD600 of 1.0 and serially diluted tenfold with sterile water. Then, 5 µL aliquots of each dilution were spotted onto MMP agar containing 0.0 M NaCl or 0.7 M NaCl and incubated at 42 °C for 3 days. A blank control group incubated at 30 °C for 3 days without adding NaCl was also set up. Strains that exhibited robust growth on the MMP agar with 0.7 M NaCl at 42 °C compared to the control group were identified as be thermotolerant and osmotic pressure-tolerant.

Identification of the strain WH103

The strain with the best degradation activity and stress tolerance was confirmed to be WH103. Strain WH103 was identified according to Bergey’s Manual of Determinative Bacteriology49 and through 16 S rRNA sequence analysis. Genomic DNA was extracted using the high-salt-concentration precipitation method50, Subsequently, the 16SrRNA gene was amplified following standard PCR procedures51. The 16SrRNA gene sequence was compared with the National Center for Biotechnology Information (NCBI) database using the BLAST search tool on the website (https://submit.ncbi.nlm.nih.gov/BLAST) and then submitted to GenBank. After multiple alignments of the data by Clustal X (version 2.1)52,53, phylogenetic analysis was carried out using MEGA software (version 6.0). The phylogenetic tree was constructed by the neighbor-joining method54.

Immobilization of strain WH103 cells

As previously described with some modification, strain WH103 mixed with biochar was immobilized in calcium alginate gel beads55,56. First, strain WH103 was grown in MMP liquid medium at 30 °C and 150 rpm until it reached an OD600 of 1.0. The culture was centrifuged at 6000 rpm for 5 min and the cell pellets were washed two times with sterile 0.85% (w/v) saline solution (SSS) to collect wet cells. Wet cells (5 g) were thoroughly resuspended in SSS, and the total volume was completed to 50 mL to prepare a cell suspension. Then, combine the biochar and bacteria mixture with an equal amount of SA (2% w/v, prepared by dissolving SA in SSS at 70 °C) and stir for 5 min. Next, the mixture was dropped into a well-stirred sterile CaCl2 solution (3.5%, w/v) using a syringe. Upon contact with CaCl2, each SA drop solidified, forming beads that encapsulated the strain WH103 cells and biochar. Finally, the immobilized SA-biochar-bacterial beads were left to harden for 30 min at 4 °C after they were washed with SSS to remove excess calcium ions and un-encapsulated cells. The average bead diameter was approximately 2–3 mm. All beads displaying obvious degradation activity were stored at -20 °C and utilized for subsequent experiments. The first blank control group (CK1) consisted of free cells of the same biomass, while the other control group (CK2) consisted of immobilized SA-bacterial beads with 50 mL of cell suspension added without biochar before being mixed with an equal amount of SA. The remaining steps were the same as described above.

Optimization of immobilizing parameters and reaction conditions

The degradation rate of PCA in MMP liquid medium by 2.0 g SA-biochar-bacterial beads was measured. The best immobilization conditions were studied using single-factor and orthogonal array design methods. Three operating parameters, namely temperature (15, 20, 25, 30, 35, 40 °C), pH (6.5, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0) and amounts of biochar (0, 25, 50, 75, 100, 125, 150 mg) were determined to screen the best immobilization conditions for PCA degradation. Optimization was carried out using the L9 (34) orthogonal array design.

Repeated batch degradation test of SA-biochar-bacterial beads

The batch degradation of 0.1 mM PCA by SA-biochar-bacterial beads and SA-bacterial beads in MMP liquid medium under the optimized conditions was estimated, respectively. The degradation time for each cycle was 21 h, and the temperature maintained at 30 °C and the solution shaken at 150 rpm. At the end of the degradation process, the immobilized beads were removed from the solution, then washed with physiological saline and transferred to fresh MMP liquid medium. The above steps were repeated and MMP cultured media degraded by SA-biochar-bacterial beads were collected for phytotoxicity assessment.

Phytotoxicity assessment

The toxicity of the MMP liquid medium and MMP cultured media degraded by SA-biochar-bacterial beads was tested on plants through a seed germination assay. Four-commonly used plant varieties, Abelmoschus esculentus, Oryza sativa, Vigna radiata and Vigna mungo were selected for the study based on both agricultural significance and ecological relevance to the study. Selected seeds were surface sterilized and placed on Petri plates with wet filter paper soaked in distilled water, MMP liquid medium, and the MMP cultured media degraded by SA-biochar-bacterial beads, respectively. The seeds were then observed for germination for a period of 7 days. Toxicity was measured in terms of the percentage of germination and lengths of plumule and radical after 7 days. Relative Root Growth (RRG), Relative Seed Germination (RSG) and Germination Index (GI) were calculated by the following formula57,58.

$$\:\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{S}\text{e}\text{e}\text{d}\:\text{G}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n}\:\left(\text{\%}\right)=\frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{e}\text{e}\text{d}\text{s}\:\text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{e}\text{d}\:\text{i}\text{n}\:\text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}}{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{e}\text{e}\text{d}\text{s}\:\text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{e}\text{d}\:\text{i}\text{n}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\times\:100$$
(1)
$$\:\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{R}\text{o}\text{o}\text{t}\:\text{G}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n}\:\left(\text{\%}\right)=\frac{\text{m}\text{e}\text{a}\text{n}\:\text{r}\text{o}\text{o}\text{t}\:\text{l}\text{e}\text{n}\text{g}\text{h}\text{t}\:\text{i}\text{n}\:\text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}}{\text{m}\text{e}\text{a}\text{n}\:\text{r}\text{o}\text{o}\text{t}\:\text{l}\text{e}\text{n}\text{g}\text{t}\text{h}\:\text{i}\text{n}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\times\:100$$
(2)
$$\:\text{G}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n}\:\text{i}\text{n}\text{d}\text{e}\text{x}\:\text{\%}=\frac{\text{R}\text{S}\text{G}\times\:\text{R}\text{R}\text{G}}{100}$$
(3)

Analytical methods

To analyze PCA, the cultures were centrifuged at 12,000 × g for 5 min. Supernatants were filtered through 0.2-µm-pore-size filters and subjected to HPLC analysis using a system (Dionex UltiMate 3000, USA) equipped with a C18 reverse phase column (4.6 × 250 mm, 5 μm). The mobile phase was a mixture of methanol (A), acetonitrile (B), and water (C) in a ratio of 40:30:30 with a flow rate of 0.8 mL min− 1. The column temperature was set at 30 °C, and the injection volume was 20 µL. Column elution was monitored by measuring absorbance at 367 nm for 15 min.

The statistical analyses of the data were performed using one-way analysis of variance (ANOVA). The level of significance was p\0.05. Statistical analyses were conducted using the SPSS 20.0 software program.

Land use permit statement

The permission has been obtained from the landowner to collect the samples.