Identification, degradation characteristics, and application of a newly isolated pyridine-degrading Paracidovorax sp. BN6-4 Open Access

Heterocyclic nitrogen compounds are organic compounds that contain a heterocyclic structure in their molecules. Among them, pyridine is a common six-membered heterocyclic compound widely used in the manufacture of pesticides, pharmaceuticals, and petroleum products (Qiao & Wang 2010). It is a volatile, odorous, and difficult to degrade organic pollutants (Meng et al. 2017). Due to their high solubility in water and high chemical stability, pyridine compounds are susceptible to entering groundwater systems by diffusion, leading to an increased risk of contamination of the aquatic environment (Watson & Cain 1975). Moreover, pyridine is moderately acutely toxic to living organisms and may cause biotoxicity, teratogenicity, and carcinogenicity. As a result, the U.S. Environmental Protection Agency has listed pyridine as one of the priority pollutants (Watson & Cain 1975; Shi et al. 2019). In addition to its ecosystem hazards, pyridine is strongly inhibitory to microorganisms and is difficult to degrade by natural oxidative decomposition, which increases the difficulty of self-purification of surface water and environmentally sound treatment of wastewater. Therefore, there is an urgent need to develop cost-effective pyridine treatment technologies to effectively remove or reduce the concentration of pyridine and lessen its negative impact on the environment and ecosystems to protect water resources and ecosystem health.

In addition, the mechanisms of microbial degradation of pyridine have only been extensively studied to a limited extent. Wang et al. (2018) isolated Paracoccus sp. NJUST30 during the degradation of pyridine, which produced intermediates such as 2,4-dihydroxy-2H-pyridine-3-one and 2-carboxylic acid, revealing a biodegradation pathway involving hydroxylation, pyridine ring cleavage, carbonylation, and carboxylation for pyridine. Shukla & Kaul (1974) found that Corynebacterium sp. generates formic acid, amines, and succinic semialdehyde during the degradation process. Deng's research found that Achromobacter sp. DN-06 degrades pyridine through a direct ring-opening mechanism, concurrently involving N-C2 and C2–C3 ring-opening pathways (Deng 2011). These studies indicate that the current understanding of the biodegradation mechanism of pyridine is limited, and different microbial species exhibit variations in pyridine degradation mechanisms. Some Paracidovorax sp. exhibit notable tolerance to organic solvents and have been predominantly employed for degrading organic compounds like polycyclic aromatic hydrocarbon (Singleton et al. 2009) and 4-nitrotoluene (Ju & Parales 2011). Nevertheless, research on the degradation of pyridine by acidophilic bacteria remains limited. Therefore, there is a compelling need for an in-depth investigation into the degradation traits of pyridine by acidophilic bacteria and the associated metabolic mechanisms.

In this study, a new efficient pyridine-degrading bacterium was isolated from the sludge at the coking plant's effluent, and it was molecularly identified as Paracidovorax sp. Although pyridine biodegradation investigations have been described, since Paracidovorax sp. is being used for pyridine degradation for the first time, a thorough investigation of their degradation properties and pathways is required. The purpose of this study was to investigate the effects of different initial pyridine concentrations, temperature, pH, and other environmental factors on the degradation of pyridine by the strains. Based on the intermediate products produced during pyridine biodegradation, a new degradation pathway for pyridine biodegradation by Paracidovorax sp. BN6-4 was proposed for the first time. In addition, pyridine-efficient degrading bacteria combined with carriers were applied to treat real coking wastewater. Strengthen the proliferation of indigenous microorganisms, increase microbial diversity, and change the structure of the microbial community. It can effectively degrade organic matter in wastewater and remove COD in wastewater. This study provides valuable supplementary information for the microbial pyridine degradation pathway and offers a new resource and theoretical basis for exploring efficient microbial pyridine remediation technology.

The bacterial enrichment medium (EM) used for cultivating pyridine-degrading bacteria is a minimal salt media (MSM) containing pyridine and composed of the following components: MgSO4·7H₂O 0.2 g, Na₂HPO₄ 4.26 g, KH₂PO₄ 2.65 g, CaCl₂ 0.02 g, 1 mL trace elements, distilled water 1,000 mL, pH = 7, pyridine concentration adjusted as per experimental requirements. Sterilized at 121 °C for 20 min. Trace elements stock solution: KI 0.005 g, MnSO₄·4H₂O 0.2 g, CuSO₄·5H₂O 0.02 g, ZnSO₄·7H₂O 0.2 g, Na₂MoO₄·2H₂O 0.25 g, H₃BO₃ 0.008 g, FeCl₃ 0.1 g, diluted to 100 mL with water.

Luria Bertani (LB) medium is employed to amplify bacterial cultures for the investigation of their degradation characteristics and metabolic pathways. It comprises peptone 10 g, yeast extract 5 g, NaCl 10 g, diluted to 1L with deionized water, pH = 7.4–7.6, sterilized at 121 °C for 20 min. The solid medium, used for strain preservation, was prepared by incorporating agar into the LB medium.

Pyridine-degrading strains were isolated from the sludge at the Chongqing Coking Plant. To enrich pyridine-degrading bacteria, 90 mL of MSM and 2 g of sludge were introduced into 150 mL conical flasks, with pyridine as the sole carbon and nitrogen source. The initial pyridine concentration was 30 mg/L, and every 7 days, 10 mL of the enrichment solution was transferred to a new enrichment medium. The concentration of pyridine in the medium was gradually increased to 300 mg/L (30, 90, 180, 240, 300 mg/L). To isolate pyridine-degrading bacteria, 1 mL of the enrichment medium suspensions was aseptically transferred to a sterile test tube and diluted with sterile water (10⁻¹ ∼ 10⁻⁸). Subsequently, the diluted bacterial suspension was evenly spread onto solid LB plates containing inactivated bacteria. These plates were placed in a constant-temperature incubator at 30 °C for 48 h. Well-developed colonies with distinctive morphologies were selected for further purification through 3–4 rounds of streaking. These purified isolates were then transferred and maintained on a solid LB slant medium. The isolated strains were subsequently inoculated into MSM containing pyridine and incubated at 30 °C with a pH of 7 and a rotational speed of 150 rpm. The concentration of pyridine in the medium was determined after 48 h of incubation. Among these strains, the one exhibiting the highest pyridine degradation rate was labeled as BN6-4.

The strain BN6-4 was subjected to Gram staining to observe its cellular morphology under an optical microscope. For the identification of the obtained pyridine-degrading strain, a 16S rRNA gene sequence analysis was performed. A culture of the strain in an LB liquid medium for 1 day was used for DNA extraction. A 300 μL aliquot of bacterial culture was processed using the Ezup column soil DNA extraction kit to extract the strain's DNA. The bacterial DNA was then subjected to PCR amplification using the universal bacterial primers 27F (5′-AGAGTTTGATCATGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACTACTT-3′). Following gel electrophoresis confirmation of PCR products, they were sent to Sangon Biotech for sequencing. Sequencing results were uploaded to the NCBI database for gene alignment, providing a preliminary determination of the strain's species. Furthermore, a phylogenetic tree was constructed using MEGA 7.0. Metabolic characteristics of the microorganism were assessed through biochemical experiments, including sugar metabolism, enzyme activity, and redox reactions.

The pyridine degradation experiment using strain BN6-4 was carried out in 500 mL conical flasks, each containing MSM with pyridine. Initially, the pyridine-degrading bacterium BN6-4 was cultured in an LB liquid medium at 30 °C with a rotational speed of 150 rpm for 48 h. After incubation, the culture solution was centrifuged at 10,000 rpm for 5 min, and the supernatant was discarded. The bacterial cells were then washed two to three times with a sterilized inorganic salt medium. Following these washes, the bacterial cells were resuspended in MSM to reach an optical density (OD600) of 1 at a wavelength of 600 nm, resulting in the preparation of the seed solution. Subsequently, this seed solution was used for the pyridine degradation experiments in the conical flasks containing MSM. The degradation characteristics of BN6-4 on pyridine were investigated through single-factor experiments. In the experiment to assess the impact of initial concentrations, a 5% BN6-4 seed solution was inoculated into MSM containing varying initial pyridine concentrations (500, 1,000, 1,500, 2,000, 2,500 mg/L). The incubation was conducted at 30 °C, with a pH of 7 and agitation at 150 rpm for 48 h. Samples were collected to measure the pyridine concentration, and the degradation rate was subsequently calculated. In the temperature experiments, the incubation temperature was set at 20, 25, 30, 35, and 40 °C. The pH was maintained at 7, and the rotational speed was set at 150 rpm. For the experiment investigating the effect of inoculum amount on microbial pyridine degradation, the inoculum amounts were set at 0.1, 0.5, 1, 3, and 5%, respectively. The BN6-4 seed culture was inoculated into MSM containing 1,000 mg/L pyridine at a 5% inoculation rate, with shaking at 150 rpm and incubation at 30 °C. In the pH experiment, the initial pH was adjusted to 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 using 1 mol/L HCl and 1 mol/L NaOH. Samples were taken after 48 h of incubation to measure pyridine concentration and calculate the degradation rate.

Samples during pyridine degradation by the strain were filtered through a 0.45 μm filter membrane, and 30 mL of filtrate was extracted three times with an equal volume of ethyl acetate under neutral and acidic conditions, respectively. The extraction solution was filtered through anhydrous sodium sulfate and then concentrated to 1 mL using a rotary evaporator. Specific intermediates in the metabolism were determined by GCMS-QP-2010. Samples were first taken at different times of pyridine degradation, filtered through a 0.45 μm membrane, and then extracted three times with equal volumes of ethyl acetate in 30 mL of filtrate under neutral and acidic conditions, respectively. The extracts were filtered through anhydrous sodium sulfate baked at 400 °C for 7 h and then concentrated to 1 mL using a rotary evaporator. The extracts were analyzed by GC-MS under the following operating conditions: inlet and detector temperatures of 250 °C, flow rate of 1 mL/L, heating program of 0 °C/min to 250 °C, holding time of 3 min, and a carrier gas of He. The conditions for mass spectrometry (MS) analysis included an ionization voltage of 70 eV, a full-ion scan at 50–625 m/z, an ion source of 250 °C, and an electronic capability of 70 eV. Peak detection and mass spectral peak integration were performed on the detected sample mass spectral data. The molecular formulas and structures of compounds are determined by comparing the mass spectra with those of known compounds in the NIST20 spectral library.

The ability of strains and carriers combined to treat coking wastewater was evaluated in shake flask experiments. PBS granules and activated carbon powder were heated at 185 °C and combined with stirring to make the carriers. The biodegradation of coking wastewater was organized into four groups: CK, which served as the control and received 100 mL of coking wastewater; J, which involved the addition of 5% (w/v) strain BN6-4; Z, which involved the addition of 5% (w/v) carriers; and JZ, which involved the addition of 5% (w/v) of carriers and strain BN6-4. The running time was 6 days, and the conditions of the operation were 30 °C and 150 rpm. Samples were taken every 2 days to analyze the COD removal rate. The samples were centrifuged and the supernatant was discarded for NextSeq Illumina.

The pyridine concentration was determined using UV spectrophotometry. This involved scanning the UV spectrum of pure pyridine to identify its maximum absorption wavelength, which was found to be 256 nm. Five pyridine concentration gradients (0, 10, 50, 100, and 150 mg/L) were prepared, and their absorbance was measured to construct the pyridine standard curve. The bacterial suspension was introduced into the pyridine inorganic salt medium, and samples were collected to measure the absorbance after a specified incubation period. The concentration was then determined by fitting the absorbance value to the standard curve (Li & Zhao 2001). The pyridine and COD degradation rates were calculated by the formula ((C0 − C1)/C0) × 100%, where C0 is the concentration of pyridine after degradation in the control group (mg/L), and C1 is the concentration of pyridine after degradation in the experimental group (mg/L). In this study, three separate typical organic matter degradation tests were conducted. The results for COD, biomass (OD600), and pyridine concentration are expressed as mean ± SD (standard deviation).

After five rounds of consecutive enrichment, a highly efficient pyridine-degrading bacterium, BN6-4, was isolated from sludge samples collected from a coking plant. Following 24 h of cultivation on LB solid medium, BN6-4 exhibited morphological characteristics on LB agar plates, as depicted in S1. In the image, BN6-4 formed large, circular colonies with well-defined edges, displaying an orange-pink color and a smooth surface. The morphological characteristics of BN6-4 under an optical microscope are shown in S1B, where it appeared rod-shaped and tested Gram-positive.

Partial physiological and biochemical test results for strain BN6-4 are presented in S3. Among the three carbon sources tested, strain BN6-4 demonstrated the ability to utilize glucose and sucrose but not lactose. Additionally, strain BN6-4 exhibited positive reactions in oxidation enzyme tests, starch hydrolysis tests, lipase tests, urease tests, methyl red tests, V-P tests, and Gram staining reactions. The 16S rDNA of strain BN6-4 was amplified through PCR, and sequencing resulted in an 855bp-long sequence. This obtained sequence was then compared to sequences already registered in the GenBank database using BLAST. A phylogenetic tree was constructed using MEGA-X software (as shown in S4), which provided initial identification of BN6-4 as belonging to the Paracidovorax sp.

The amount of microbial inoculum impacts the density and activity of the microbial population, which influences the pace and efficiency of pyridine breakdown. Figure 2(b) shows the effect of inoculum size on pyridine degradation by BN6-4. With an increase in the quantity of microbial inoculum, the efficiency of pyridine breakdown improved. The highest degradation efficiency of 97.94 ± 0.23% was observed for pyridine at 5% inoculum concentration. This is because higher inoculum concentration helps to initiate the degradation process faster at the initial stage. In addition, high inoculum populations of microorganisms may be more resistant to the toxicity of organic matter, whereas high inoculum levels may minimize the detrimental effects of toxicity on microorganisms. Consequently, the higher the microbial population, the more likely it is that the organic matter will be completely degraded and the more likely it is that the concentration of residues will be reduced. Higher the microbial population, the more likely it is that the organic matter will be completely degraded and the concentration of residues reduced.

The pyridine molecule undergoes ionization at different pH conditions, thus changing its solubility and reactivity. Under acidic conditions, pyridine may exist in a molecular form, while under alkaline conditions it may exist in an ionic form. This ionized state may impact pyridine's bioaccessibility when degraded by microbes. In addition, microorganisms exhibit the highest activity in a specific pH range. Different microbial strains may have different optimal pH ranges, and the pH of the medium plays a crucial role in microbial growth, metabolism, enzyme activity, and ecological interactions, so choosing the right pH range optimizes the efficiency of microbial degradation of pyridine and ensures that the microorganisms can maximize their degradation potential in the most suitable environment. Figure 2(c) shows the effect of pH on the degradation of pyridine by BN6-4. The bacterium was able to degrade pyridine with more than 80% efficiency in the pH range of 6–10. It is noteworthy that BN6-4 showed superior degradation performance under alkaline (7–10) conditions as compared to acidic conditions (5–7). The optimum pH value for pyridine degradation by BN6-4 was pH 7 with a maximum degradation efficiency of 95.27 ± 0.42%. This indicates that the enzyme activity of BN6-4 is the most suitable for the growth of the bacterium under neutral conditions. The degradation rate of pyridine was 83.79% at pH 10, but the degradation rate decreased sharply to 18.08 ± 6.5% when the pH was increased to 11. This indicates that pH is very critical for microbial degradation of pyridine.

Temperature is a key environmental factor affecting microbial growth and substrate degradation because important components of microbial cells (e.g., proteins and nucleic acids) are temperature sensitive. Different microorganisms and enzymes have different optimum temperatures. Figure 2(d) shows the effect of temperature on pyridine degradation by BN6-4. At 20 °C, the degradation rate of pyridine was only 8.79% ± 1.51%, which indicated that pyridine degradation was significantly inhibited at low temperatures, suppressing the growth and metabolic rate of the microorganisms. The highest removal rate of pyridine was achieved at 30 °C with 99.05 ± 0.22%. This indicates that 30 °C is the optimum temperature for microbial growth and the microbial degradation activity is the highest. It is noteworthy that a decrease in degradation rate was observed at 35 °C, but there was an increase in degradation rate at 40 °C. This may be because pyridine is less toxic and more easily degraded at a high temperature of 40 °C.

GC-MS analysis was employed to identify the major metabolites formed during pyridine degradation by strain BN6-4 (Figure 4). Table 1 summarizes the main intermediate metabolites detected during the biodegradation of pyridine, along with their molecular formulas, structures, retention times, and molecular weights. Among these, B1, with an m/z of 79 and a chemical formula of C₅H₅N, was identified as pyridine, with a retention time of 7.15 min. B2, identified as N-Ethylurea with an m/z of 88 and a molecular formula of C₃H₈N₂O, may be the result of an amino group substituting the oxygen in pyridine during oxidative ring opening. B3, urea, with an m/z of 60, is generated by the carbon–nitrogen bond cleavage of N-Ethylurea. Metabolite B4, acetamidoacetaldehyde, with an m/z of 101 and a molecular formula of C₄H₇O₂, is formed when pyridine undergoes N-C2 and C2–C3 ring opening simultaneously. B5, N-Hydroxymethylacetamide, with an m/z of 89 and a molecular formula of C₃H₇NO₂, is reduced from acetaldehyde acetamidoacetaldehyde.

Coking wastewater is produced during the refining operations of many industrial products, including coal coking, coal gas purification, as well as the processing of copper and steel. Its chemical composition is notably influenced by the coal's constitution and the characteristics of the manufacturing procedures. Typically, coking wastewater contains high concentrations of both inorganic and organic pollutants, such as ammonia, sulfides, phenolic resins, polycyclic aromatic hydrocarbons (PAHs), and nitrogen-containing heterocyclic compounds. Pyridine, indole, and quinoline are common examples of nitrogen-containing organic pollutants in this context (Ma et al. 2015; Zhao & Liu 2016). Microbial immobilization techniques find extensive application in the treatment of complex pollutants. For example, Niu et al. (2023) work involved immobilizing the J2 strain with biochar, achieving a degradation rate of 98.66 ± 0.47% for wastewater containing 2,000 mg/L of pyridine. Additionally, research by Cao suggests that employing immobilized mixed microbial communities for degrading PAH in wastewater can effectively harness the role of microorganisms in treating recalcitrant wastewater and improve water quality (Cao 2022).

In this study, a novel pyridine-degrading strain BN6-4 was successfully isolated from coking sludge. The BN6-4 was identified by 16S rDNA as Paracidovorax sp. This strain was capable of growing solely on pyridine. Furthermore, Paracidovorax sp. BN6-4 demonstrated significant pyridine resistance and rapid breakdown rates. Within 48 h, BN6-4 achieved a degradation rate of 97.49 ± 1.59% for pyridine at an initial concentration of 1,500 mg/L. Furthermore, BN6-4 demonstrated growth capabilities across a temperature range of 20–40 °C, with optimal degradation efficiencies consistently above 90%. Between 30 and 40 °C, BN6-4 was found to thrive within a pH range of 5-10, with a maximal degradation rate of 95.27 ± 0.42% at pH 7. Additionally, increasing the inoculum size led to faster deterioration, with a rate of 97.94 ± 0.23% at 5% inoculum size. Through GC-MS analysis, a novel pyridine degradation pathway for strain BN6-4 was proposed, involving pyridine hydroxylation, N-C2, and C2–C3 ring-opening reactions. After the BN6-4 was applied to real coking wastewater, the results showed that the combination of Paracidovorax sp. BN6-4 and the carrier increased the removal rate of COD in real coking wastewater, changed the structure of the microbial community, stimulated the growth of indigenous microorganisms, and improved the degradation ability of the microbial community on pollutants.

This work was supported by the Strategic Biological Resources Capacity Building Project of the Chinese Academy of Sciences [grant number KFJ-BRP-009-004] and the Key Research and Development Program of Sichuan Province [grant number 2020YFS0021].

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.