1 Introduction

Antibiotics have been extensively used in both animals and humans for treating microbial infections (Priya et al. 2022) and have even been used as feed additives for promoting growth in livestock animals (Nosanchuk et al. 2014). In recent years, antibiotics have been found as residues in water and soil environments (Leichtweis et al. 2022), food (Xue et al. 2022), and even humane blood (Picó et al. 2019), causing a long-term risk for water and soil ecosystem environments because of the continuous use of antibiotics. There are a wide variety of biological or physicochemical treatment techniques for antibiotics, e.g., adsorption by various adsorbents, photodegradation using different light sources, and biodegradation by bacteria or other microorganisms (Hz et al. 2022; Zhang et al. 2022; Zhao et al. 2022b). Generally, low-cost biochar adsorbents can remove antibiotics at concentrations ranging from a few to several  hundred µg mL−1 (Biswal and Balasubramanian 2022; Xiang et al. 2019; Zhang et al. 2020), which is considered to be one of the most efficient methods in water and soil environments. However, to our knowledge, using  biochar alone cannot completely remove pollutants in aqueous solution in the environment (Biswal and Balasubramanian 2022). Researchers have also proved that biological and chemical processes alone are not effective for the removal of antibiotics from aquatic environments (Ge et al. 2021). Therefore, to enhance the pollutant removal efficiency, researchers have begun to develop a technology that uses biochar combined with microbiological and/or photocatalytic methods to improve the removal of pollutants in the aqueous  solutions.

The porous structure and large surface area of biochar not only adsorb hazardous substances (He et al. 2021) but also provide a good refuge and shelter for microorganisms to reproduce and grow (Choppala et al. 2012; Cooper et al. 2020). Meanwhile, the elements in biochar can supply organic matter or inorganic nutrients for microorganisms to reproduce and grow (Cooper et al. 2020; Zhao et al. 2020). Additionally, the interface electronic effects of biochar have been proven, which can also improve the photodegradation of organic pollution (Wei et al. 2020). Owing to this outstanding performance related to microorganisms and photocatalytic activity, biochar combined with microbial and photodegradation technology has been increasingly applied in aqueous solutions to remove organic pollution. However, biochar releases dissolved organic matter into the water or soil environment when biochar is applied, which results in environmental behavior changes in pollutants (Bao et al. 2019; Yang et al. 2021b). An increasing number of studies have proved that BDOM components mainly include phenols, polycyclic aromatic hydrocarbons, and nitrogen-, oxygen- and sulfur-containing heterocyclic compounds (Chen et al. 2017; Qu et al. 2016). Although these substances also cause a threat to the environment, they not only act as nutrients for the reproduction and growth of microbes (Choppala et al. 2012; Cooper et al. 2020) but also improve the photocatalytic activities for the degradation of organic pollutants (Wei et al. 2020). Thus, biochar combined with microbial and photocatalytic technology could solve the secondary pollution caused by BDOM released from biochar. Due to these special characteristics of BDOM affecting the particular mechanisms of interaction between biochar and pollutants in the environment, BDOM has sparked a strong research interest during the last several years.

Many studies have illustrated that biochar was used to treat water or amend soil, which emphasized that BDOM clearly increased the pH (Chen et al. 2016; Khodadad et al. 2011; Zhang et al. 2018), nutrition and chemical elements (Kosin 2009; Šipailienė and Petraitytė 2018) and released some organic matter or inorganic  nutrients (Smebye et al. 2016). In addition, BDOMs from biochar as exogenous nutrients  are decomposed by microbiota, and they also modify the metabolism of microorganisms by inducing their growth (Zhang et al. 2018). Our previous studies have proven that the physicochemical characteristics of BDOM are directly dependent on the pyrolysis temperature of biochar (Yang et al. 2020), which  is the same as that of Li et al. (2022) and Lee et al. (2018). In particular, BDOMs are mainly composed of protein, lignin, and some incomplete combustion of PAHs. While increasing the pyrolysis temperature of biochar, the number of molecules in BDOM decreases significantly, and BDOMs are the main component of hyperpolymer carbohydrates. Moreover, BDOMs could act as exogenous substances to promote and induce functional genes, affecting the biodegradation efficiency of SMX and CAP (Yang et al. 2021a). These results indicate that the components of BDOMs are important influences not only on the biodegradation of antibiotics, but also on the genes of bacteria.

Based on a previous study on the influence of BDOM on the degradation of pollutants, especially phototransformation conditions and bacterial growth (Chen et al. 2022; Gui et al. 2017; Wu et al. 2021; Xue et al. 2019; Yang et al. 2021c, e; Zhang et al. 2017; Zhao et al. 2022a, c; Zhou et al. 2018), it was found that there were no studies about how the molecular types and/or components of BDOM controlled or affected the degradation of pollutants and transcriptional metabolism for bacteria under visible light. Therefore, BDOMs derived from biochar at different temperatures were strengthened to investigate the change rules of bacterial growth and the components of fatty acid for the membrane, the copy number of genes, and the transcriptome of genes during the degradation of SMX and CAP.

In the present study, the whole scheme of mechanisms and experimental design for this study are shown in Additional file 1: Fig. S1, and the three main aims included the following: (1)  to study the effects of BDOM on the degradation of antibiotics by P. stutzeri and S. putrefaciens during BC and MBC solutions under visible irradiation; (2)  to investigate the response of bacterial growth, components of the cell membrane, copy number of genes, and transcriptome during antibiotics biodegradation processes; (3)  to reveal the main molecular types and components of BDOM affecting bacterial functional genes during antibiotic biodegradation processes using various analyses, including PCA, RDA, Venn and interaction network analysis. This research provided the application potential of   using biochar technology in combination with microbial and photodegradation for remediation and photodegradation and provided theoretical data for risk assessment in the biochar-remediation process under natural conditions.

2 Materials and methods

2.1 Chemicals and microorganisms

Reagent grade SMX and CAP (assay 98%, the structures are shown in Additional file 1: Tables S1–S3), HPLC-reversed-phase C18 column (4.6 × 150 mm, 5 µm) and other experimental consumables were obtained from Anpel Laboratory Technologies Inc. (Shanghai, China). Sterile normal saline and phosphate buffered saline (PBS, 0.2 M) were purchased from Solomen Lab Shopping Biotechnology Technology Limited Company (Tianjin, China).   HPLC grade acetonitrile was obtained from Concord Technology Limited Company (Tianjin, China). All solutions were prepared using deionized (DI) water (Tianjin, China) in this study.

The two strains Pseudomonas stutzeri (P. stutzeri, stain no. ATCC17588) and Shewanella putrefaciens (S. putrefaciens, stain no. ATCCBAA-1097) were gram-negative and purchased from Guangdong Culture Collection Center, China. The microbial culture conditions of liquid nutrient agar medium (NA), liquid nutrient medium (NM), and mineral liquid medium (MM) were used in this study. The detailed culture conditions are shown in Additional file 1: Text S1.

2.2 Preparation of biochar and modified biochar and biochar-DOM extraction

The preparation methods of BC, MBC and BDOM were all the same as those in our previous study (Yang et al. 2021a). In brief, rice straw from the rice field was used to make biochar at pyrolysis temperatures of 350, 500, and 700 °C, and the samples were denoted as BC350, BC500 and BC700, respectively (Additional file 1: Text S2). Then, 1:1 HCl and HF were used to modify BC350, BC500 and BC700 twice, and the corresponding modified   biochars named MBC350, MBC500, MBC700 were collected (Additional file 1: Text S3). BDOMs are aqueous solutions that remove biochar, and the detailed method is shown in the supporting information (Additional file 1: Text S4). BDOMs from biochar at different pyrolysis temperatures (350, 500, and 700 °C) were  denoted by BDOM350, BDOM500, and BDOM700, respectively.

2.3 Experimental designs

2.3.1 Bacterial suspension preparation

The third generation of P. stutzeri and S. putrefaciens (the detailed cultures are shown in Additional file 1: Text S5) and sterile normal saline were used to prepare bacterial suspensions. Ultraviolet spectrophotometry (model no. TU-1810; Beijing Purkinje General Instrument Co., Ltd., China) was used to measure the concentrations of the bacterial suspension at an optical density (OD) of 660 nm, and the bacterial suspensions were constantly diluted to an absorbance of approximately 0.65, which indicated that the bacterial concentration was approximately 5.2 × 108 CFU mL−1 in the suspensions. Then, the bacterial suspensions were stored at 4 °C for the subsequent experiments.

2.3.2 Batch experiments

The degradation efficiency for a range of initial SMX and CAP concentrations, including 0.2, 0.5, 1, 2, 4, 6, 10 and 30 mg L−1, was determined in our previous study (Yang et al. 2021a), and the results indicated that the degradation efficiency was optimal when the concentration was 10 mg L−1. Based on the previous study, the initial concentration of SMX and CAP in all experiments in this study was also chosen to be 10 mg L−1. In general, SMX and CAP  exist extensively in natural environmental samples. In the water environment, SMX concentrations ranged from 0.1 to 124.5 ng L−1, while CAP was reported to reach 266 ng L−1 (Danner et al. 2019). SMX and CAP have also been detected in soils at concentrations ranging from 0.1 to 2683 ng g−1 (Ji et al. 2012; Yi et al. 2019). Although the initial concentrations of SMX and CAP were much higher than the residual concentrations in the environment, they were used in the laboratory to highlight the effects and differences. Additionally, the effects and mechanisms of BDOM on the removal of SMX and CAP by using biochar combined with microbes and photodegradation were the main aims of this study. Hence, an initial concentration of 10 mg L−1 for SMX and CAP in all experiments was chosen.

The entire experimental setup is shown in Additional file 1: Fig. S2. The wavelength of xenon lamp irradiation was ranged from 380 to 780 nm, which was mainly visible light, and the variation in the irradiation spectrum with wavelength is shown in Additional file 1: Fig. S3, which was the same as that in our previous study (Yang et al. 2021d). Additionally, the intensity of xenon lamp irradiation was 100 mW cm−2 as detected by a GEL-NP2000 High Light Optical Power Meter (China Education Au-light Co., Ltd). To  prepare and sterilize the MM solutions, BC and MBC were added into 50 mL sterile centrifuge tubes at concentrations of 100, 250, 500, 1000, and 2000 mg L−1. Alternatively, BDOM solutions were used to prepare and sterilize the MM solutions, which contained the BDOM at 2, 4, 6, and 10 mg L−1. Then, the P. stutzeri and S. putrefaciens suspensions of 2% (v:v) were added to the above sterilized solution. The fermentation samples were cultured under xenon lamp irradiation for 12 h and alternating light for 12 h with shaking at 180 rpm min−1 at 30 ± 1 °C, and the total culture was maintained for 72 h. Then, the SMX and CAP stock solution (100 mg L−1)  was added to make the samples contain SMX and CAP at 10 mg L−1, which was proved to be the optimal biodegradation for P. stutzeri and S. putrefaciens in our previous study (Yang et al. 2021a). The samples were shaken at 180 rpm min−1 at 30 ± 1 °C for 12 h to biodegrade SMX and CAP. Then, the samples were crushed using a cell breaker (model no. JY92-IIDN; Scientz Co., Ltd., Ningbo, China), and the residual supernatant was passed through a 0.22 µm filtration membrane. Residual SMX and CAP were then measured using HPLC (Agilent Technologies 1260, USA). The control groups were all cultured under light-avoiding conditions. All experiments were performed in triplicate.

2.4 Experiments of bacterial growth cultured with BDOM

The samples of the experimental and control groups were obtained from the above, which were cultured with different concentrations of BDOM under xenon-lamp irradiation (12 h) and light avoidance (12 h) alternated for 72 h. Samples of 20 mL were centrifuged at 10,000 rpm min−1 and 4 °C for 20 min, and then the supernatant was removed. The residual samples were washed with 0.2 M PBS twice, and the precipitation fractions (bacteria) were dried for 24 h at 60 °C in a constant temperature oven (model no. GFL-70; Tianjin Labotery Instrument Co., Ltd., China). Finally, the bacterial dry weight was measured and calculated to elucidate the effects of the conditions on bacterial growth.

2.5 Experiments on the fatty acid of bacteria cultured with BDOM

The samples (5 mL) of experimental and control groups were obtained from the above, which were cultured with different concentrations of BDOM under xenon-lamp irradiation (12 h) and light avoiding (12 h) alternated for 72 h. The methods of extraction and analysis of fatty acid from P. stutzeri and S. putrefaciens were modified according to the methods of Law’s study (Law et al. 2018). The main steps contained saponification, methylation, extraction, and washing. The detailed description was the same as that in our previous study (Yang et al. 2021a) and is shown in Additional file 1: Text S6.

2.6 Experiments on the copy number of genes of bacteria cultured with BDOM

The samples (5 mL) of the experimental and control groups were obtained from the above, which were cultured with different concentrations of BDOM under xenon-lamp irradiation (12 h) and light avoidance (12 h) alternated for 72 h. Then, the bacterial DNA spin kit was used to obtain the DNA from the experimental samples and control groups. Additionally, the appropriate primers were designed, and qPCR experiments were carried out. The detailed reaction conditions are shown in Additional file 1: Text S7.

2.7 Experiments on the transcriptomic of bacteria cultured with BDOM

The samples (10 mL) of the experimental and control groups were obtained from the above. Samples were cultured with different concentrations of BDOM (2, 4, 6, and 10 mg L−1) under xenon lamp irradiation (12 h) and  light avoidance (12 h) alternated for 72 h. That was, a complete experimental period was 72 h. Then RNA was extracted from P. stutzeri and S. putrefaciens   using RNA spin kit to carry out the subsequent experiments. The detailed steps  are shown as below.

2.7.1 RNA extraction

Total RNA was extracted from the tissue using TRIzol® Reagent. The detailed steps were in accordance with the manufacturer’s instructions (Invitrogen) and genomic DNA was removed using DNase I (TaKara). Then RNA quality was determined by Bioanalyser (Agilent 2100) and quantified by the ND-2000 (NanoDrop Technologies). Only high quality RNA samples (OD260/280 = 1.8–2.0, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, ≥ 100 ng/μL, ≥ 2 μg)   were used to construct sequencing library.

2.7.2 Library construction and sequencing

RNA-seq transcriptome library was prepared following TruSeq™ RNA sample preparation Kit from Illumina (San Diego, CA, USA) using 2 μg of total RNA. In brief, ribosomal RNA (rRNA) depletion instead of poly(A) purification was performed by Ribo-Zero Magnetic kit (epicenter) and then all mRNAs were broken into short (200 nt) fragments by adding fragmentation buffer firstly. Secondly double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA, USA) with random hexamer primers (Illumina). When the second strand cDNA was synthesized, dUTP was incorporated in place of dTTP. Then, the synthesized cDNA was subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol. The second strand cDNA with dUTP is recognized and degraded by UNG enzyme. Libraries  were  size selected for cDNA target fragments of 200 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by TBS380, paired-end RNA-seq sequencing library  was sequenced with the Illumina HiSeq × TEN (2 × 150 bp read length). The processing of original images to sequences, base-calling, and quality value calculations were performed using the Illumina GA Pipeline (version 1.6), in which 150 bp paired-end reads were obtained. A Perl program was written to select clean reads by removing low quality sequences, reads with more than 5% of N bases (unknown bases) and reads containing adaptor sequences.

2.7.3 Bioinformatics analysis

The data generated from Illumina platform were used for bioinformatics analysis. All of the analyses were performed using the free online platform of Majorbio Cloud Platform (www.majorbio.com) from Shanghai Majorbio Bio-pharm Technology Limited Company. The major software, database, and parameters are shown in Additional file 1: Text S8.

2.8 Analysis of antibiotics and fatty acid

An analysis method of antibiotics by HLPC  was utilized to measure the residual concentrations of SMX and CAP in this study (Yang et al. 2021a). The detailed analysis method is shown in the Additional file 1: Text S9. The biodegradation efficiencies of SMX and CAP were calculated according to the residual concentrations of SMX and CAP in the final culture medium, and the formula is shown as followed: biodegradation efficiency (E%) = ((Initial concentration – Residual concentration)/Initial concentration) × 100%.

An analysis method of bacterial fatty acid by GC–MS (Agilent Technologies 5977B MSD) was utilized to measure the components and the content of fatty acid from P. stutzeri and S. putrefaciens cultured with different concentrations of BDOM under xenon-lamp irradiation (12 h) and  light avoidance (12 h) alternated for 72 h. The detailed steps have been reported in our previous work (Yang et al. 2021a), and the parameters were set as the same as those of in our previous study, as shown in Additional file 1: Text S9.

2.9 Quality control and data analysis

In this study, HPLC grade solvents and water were used to avoid contamination from other laboratory materials and solvents. Solvent blanks and procedural blanks were prepared and measured to avoid contamination between samples. A series of concentrations of SMX and CAP (calibration points: 0.5, 2.0, 4.0, 6.0, 10.0, 20.0 mg L−1 SMX, and 0.5, 1.0, 2.0, 4.0, 5.0, 8.0, 10.0 CAP using HPLC) was set to produce calibration curves and regression coefficients R2 > 0.999.

We used one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test to determine the statistical significance (p < 0.05) of each parameter among groups in this study. Three replicates were used to calculate the mean values and the corresponding standard error. All figures and Spearman rank correlation analysis were generated by OriginLab 8.0 (MA, USA) and SPSS, version 22.0 (IL, USA).

3 Results and discussion

3.1 Antibiotic degradation by bacteria cultured with BDOM under visible light

When P. stutzeri and S. putrefaciens were cultured in different concentrations of BC and MBC solutions under the alteration between xenon-lamp irradiation (12 h × 3) and light avoidance (12 h × 3), biodegradation efficiencies of SMX and CAP by the above two bacteria were investigated. The results showed that the alternation of light irradiation significantly improved the biodegradation efficiencies of SMX and CAP (Additional file 1: Fig. S4a). Furthermore, the biodegradation efficiencies of SMX and CAP increased significantly with increasing BC and MBC concentrations compared to the control groups, and the maximum biodegradation efficiencies reached more than 80% (Additional file 1: Fig. S2). However, it was found that the change rules of the biodegradation efficiencies of SMX and CAP showed an opposite trend as the baterial solutions with added BC or MBC pyrolysis temperatures increased. The biodegradation efficiencies of SMX and CAP in BC solutions were as follows: BC350 > BC500 > BC700 (Additional file 1: Fig. S4b, c), whereas those in MBC solutions showed the opposite order of MBC700 > MBC500 > MBC350 (Additional file 1: Fig. S4d, e).

Additionally, the effects of the concentrations of BC and MBC obtained at the different pyrolysis temperatures on antibiotic biodegradation under the alteration between xenon-lamp irradiation and light avoidance were estimated through the correlation between biodegradation efficiency and the different concentrations of BC and MBC solutions (Fig. 1). When the concentrations of BC increased from 100 mg L−1 to 2000 mg L−1, the biodegradation efficiencies of SMX and CAP showed a significant linear increase (R2 > 0.5806) (Fig. 1a). In contrast, the concentrations of MBC ranged from 500 to 1000 to 2000 mg L−1, and the biodegradation of SMX and CAP appeared to be linearly correlated (Fig. 1b). Thus, the biodegradation efficiencies of SMX and CAP by P. stutzeri or S. putrefaciens under the alteration of visible light and no light were different in the strains cultured between the BC and MBC. The different reasons for the biodegradation efficiency in the system between BC and MBC were explained in terms of the surface of BC and MBC and the BDOM.

Fig. 1
figure 1

Correlation analysis between the biodegradation efficiency of SMX and CAP by P. stutzeri and S. putrefaciens cultured with different concentrations of BC/MBC under the alteration between visible light irradiation and  avoiding-light conditions ((note: concentration range of BC/MBC is from 100, 250, 500, 1000 to 2000 mg L−1; visible light and an avoiding light time is 12 h × 3)

Full size image

The correlations between the biodegradation efficiency of SMX and CAP and the surface area of BC and MBC were analyzed (Fig. 2a, b). The biodegradation efficiencies were positively correlated with the surface of BC (r = 0.631, p < 0.01) (Fig. 2a) and MBC (r = 0.749) (Fig. 2b), indicating that the surface area of BC and MBC could be used as the carrier to increase the bacterial growth and improve the biodegradation efficiencies of SMX and CAP (Ashok et al. 2019). In particular, the content of BDOM was markedly linearly correlated with the biodegradation efficiency of SMX and CAP (r = 0.857, Fig. 2c), resulting in BDOM released from BC being a key factor influencing the biodegradation efficiency of SMX and CAP under the alternating conditions of xenon lamp irradiation and avoiding light. In addition, the BDOM contents from BC and MBC obtained at the different temperatures were different (Additional file 1: Table S4).

Fig. 2
figure 2

The analysis between the biodegradation efficiencies of SMX and CAP by P. stutzeri and S. putrefaciens and the surface areas of BC (a)/MBC (b) and the contents of BDOM (c) released from BC systems cultured under alternating conditions between xenon lamp irradiation and avoiding light. Figures b-c represent the biodegradation efficiencies of SMX and CAP by P. stutzeri and S. putrefaciens, respectively, which cultured with BDOM concentrations ranging from 2 to 10 mg L−1 under visible light conditions

Full size image

The change rule of biodegradation efficiencies of SMX and CAP by P. stutzeri and S. putrefaciens cultured with BDOM under visible light was significantly improved compared to the control groups without visible light (Additional file 1: Fig. S5). Some studies have reported that dissolved black carbon could enhance the aquatic phototransformation of chlortetracycline (Leal et al. 2013; Tian et al. 2019). Our previous study drew the same conclusion (Yang et al. 2021c). The results indicated that the rapid photodegradation phase occurred in the first 4 h, the photodegradation efficiency reached 80%, and the photodegradation efficiencies of SMX and CAP were over 90% after 12 h of xenon lamp irradiation. In contrast, in this study, the degradation efficiency of antibiotics ranged from approximately 50% to 90% in mineral liquid medium with different concentrations of BDOM solutions added with two strains under xenon lamp irradiation (12 h) and light avoidance (12 h) alternated at the total culture time for 72 h. Hence, these data confirmed that antibiotics were not completely mineralized after total culture for 72 h. Thus, the degradation of antibiotics in this study was mainly contributed to bacteria, and the residual concentrations of antibiotics after 4 h of light irradiation were not measured. This may be because the solution after adding the bacterial suspension in this study was more viscous and muddier than that without the bacterial suspension in our previous study, which would cause a decrease in the penetration of light, so the photodegradation of antibiotics was not the main reaction and was ignored in this study.

The biodegradation efficiencies of SMX and CAP significantly increased with increasing concentrations of BDOM (r = 0.836) (Fig. 2d, e). In addition, it was clearly found that in different pyrolysis temperature biochar-derived DOM solutions, the biodegradation efficiencies of SMX and CAP were as follows: BDOM350 > BDOM500 > BDOM700. It was presumed that BDOMs released from the low pyrolysis temperature biochar were more helpful to be used as nutrients for bacteria than at high temperatures under the alternation of visible light irradiation and light avoidance (Ji et al. 2020). Low-pyrolysis temperature took the biomass to transform biochar, which contained more organic carbon substances to be consumed by microorganisms (Li et al. 2022), resulting in BDOM from low-pyrolysis temperature biochar containing higher organic carbon substances (Yang et al. 2021a, 2020). These substances were easily transformed into carbon- containing small molecules under xenon lamp irradiation, even breaking down the original complex molecules into simpler ones (Maizel et al. 2017; Tadeu et al. 2021), which stimulated bacterial growth and metabolism (Fang et al. 2018; Li et al. 2018; Selvam et al. 2017), further enhancing the biodegradation efficiency of SMX and CAP.

3.2 Effects of BDOM on the growth, fatty acid and gene copy number of bacteria under visible light

The dry weight of P. stutzeri and S. putrefaciens cultured with different BDOMs when alternated exposed to xenon lamp irradiation and avoiding-light was significantly greater than that of the control group (avoiding-light conditions) (Additional file 1: Fig. S6a, b). In addition, the bacterial dry weight showed a significant increase with increasing BDOM concentrations (p < 0.05), indicating that the conditions of xenon lamp irradiation and avoiding light were conducive to the bacterial growth and amplification (Kuo et al. 2012; Meyer et al. 2018). Interestingly, the bacterial dry weight cultured with BDOM released from different pyrolysis temperatures was not significantly influenced (p > 0.05), showing that  BDOMs from different pyrolysis temperatures were used as nutrients for bacteria (Zhao et al. 2020).

The fatty acid components of the cell membrane of P. stutzeri and S. putrefaciens cultured with BDOM under xenon lamp irradiation and light avoidance conditions are shown in Additional file 1: Tables S4–S5. The fatty acid components of P. stutzeri did not change among different concentrations of BDOM solutions, and the main change trends of the fatty acid components of S. putrefaciens were generated C10:0, C11:0 and C14,cis-9, while C18,cis-9 disappeared compared to those of P. stutzeri. These results indicated that the cell membranes of P. stutzeri and S. putrefaciens showed different adaptive responses to the culture conditions of xenon lamp irradiation and light avoidance (Zuo et al. 2020). The total content of fatty acid from P. stutzeri cultured with BDOM350 clearly improved (Additional file 1: Fig. S6c), whereas the total content of fatty acid for S. putrefaciens cultured with BDOM350 and BDOM500 was enhanced (Additional file 1: Fig. S6d). In particular, the content of saturated fatty acid (SFA) in P. stutzeri and S. putrefaciens cultured in BDOM under xenon lamp irradiation and alternating light avoidance was significantly improved compared with that in the control group and linearly increased with increasing concentrations of BDOM (R2 > 0.8947) (Additional file 1: Fig. S6c, d). The culture condition of alternating visible light and avoiding light was conducive to the synthesis of SFA in the bacterial membrane (Dosoky et al. 2018), and BDOM promoted more active lipid metabolism of the bacterial cell membrane (Kong et al. 2020), thus increasing the fluidity and instability of the cell membrane (Min et al. 2020), making them better adapt to the stimulation of environmental changes.

The copy number of genes can also directly reflect the adaptability of bacteria to culture conditions (Tomanek et al. 2020). In the present study, the copy numbers of genes from P. stutzeri and S. putrefaciens cultured with BDOMs were inhibited (Additional file 1: Fig. S6e, f) compared to the control group in the BDOM, indicating that visible irradiation was not conducive for bacteria to generate the same genes to be adaptive to the environmental change (Tomanek et al. 2020). However, the gene copy number of P. stutzeri increased with increasing BDOM concentrations under xenon lamp irradiation and alternating light avoidance conditions, reaching a maximum value at BDOM concentration of 10 mg L−1 (Additional file 1: Fig. S6e). In contrast, the gene copy number of S. putrefaciens increased with increasing BDOM350 and BDOM500 concentrations ranging from 2 mg L−1 to 4 mg L−1 to 6 mg L−1 and then decreased at BDOM concentration of 10 mg L−1 (Additional file 1: Fig. S6f). Hence, BDOM concentrations influenced the gene copy number of P. stutzeri and S. putrefaciens to be adapted to the growing environment under the conditions of xenon lamp irradiation and light avoidance.

3.3 Effects of BDOM characteristics on the degradation of antibiotics

The characteristics of BDOM have been clarified in our previous study (Yang et al. 2021a, 2020, 2021d), and we used these parameters of BDOM, e.g., components, fluorescent regions and molecular types, to analyze the effect on the degradation of SMX and CAP using the PCA and RDA methods in Fig. 3 in this study. The molecular type of BDOM was the most important factor affecting the biodegradation of SMX and CAP, followed by the component groups and fluorescence regions (Fig. 3a). In addition, the RDA showed that the contribution degree of the SFA content to the biodegradation efficiency of SMX and CAP reached 90.9%. This is because the content of SFA significantly affected the fluid ability of the membrane, resulting in increasing exposure of microorganisms to contaminants (Zhang et al. 2018). The contribution degrees of the bacterial dry weight and the copy numbers of genes from P. stutzeri and S. putrefaciens for the biodegradation efficiency of SMX and CAP were 75.8% and 57.7%, respectively (Fig. 3b), indicating that microbial reproduction and growth were also the main factors affecting the biodegradation of pollutants (Kosin 2009; Šipailienė and Petraitytė, 2018). Hence, P. stutzeri and S. putrefaciens components had a significantly positive role in the biodegradation of SMX and CAP.

Fig. 3
figure 3

PCA (a) and RDA (b) between the biodegradation of SMX and CAP and the characteristics of BDOM or bacteria

Full size image

3.4 Transcriptomics of bacteria cultured with BDOM under visible light

To deeply understand the mechanisms by which BDOM affect the experimental groups and control groups, transcriptome responses of P. stutzeri and S. putrefaciens from experimental groups and control groups to visible light were systematically investigated. The quality control data of samples obtained from transcriptome sequencing and assembly are listed in Additional file 1: Table S6. The clean data were aligned to the reference genome using Trinity RNA-Seq (Conesa et al. 2016; Kim et al. 2015). Q20 (%) and Q30 (%) in the culture condition of xenon-lamp irradiation and   avoiding light alternated treatment, BDOM treatment and control matched up 98.2% and 94.5%, respectively, which met the requirements of further experiments of transcriptome.

Heatmap is a direct and basic visualization method to reveal hidden patterns (Gu et al. 2016), and the heatmap analysis of bacteria cultured in different BDOM under the alternation of visible light irradiation and light avoidance is shown in Additional file 1: Fig. S7, indicating that the heatmaps of P. stutzeri and S. putrefaciens were 2498 unignes (Additional file 1: Fig. S7a) compared to 1695 unignes in the control (Additional file 1: Fig. S7b). According to differentially expressed gene (DEG) changes, these unigenes were divided into three categories: cellular processes and signaling, information storage and processing, and metabolism (Additional file 1: Tables S7, S8). The unignes for P. stutzeri and S. putrefaciens under the condition of xenon lamp irradiation and alternating light avoidance were in far regions by PCA (Fig. 4a) compared to the control groups, indicating that xenon lamp irradiation induced P. stutzeri and S. putrefaciens to generate DEG.

Fig. 4
figure 4

PCA (a) and Venn (b) analysis between the DEG of P. stutzeri and S. putrefaciens exposed to xenon lamp irradiation and avoiding light conditions and the detailed number of DEG (c). Correlation analysis between the DEG of P. stutzeri and S. putrefaciens exposed to xenon lamp irradiation and avoiding light conditions cultured with different BDOM (d)

Full size image

The DEG among BDOM350-700 solutions under visible light and control groups without visible light was different in Fig. 4b, c. The DEG in the transcriptome of P. stutzeri and S. putrefaciens under xenon lamp and alternating light avoidance all increased significantly by Venn analysis (Fig. 4b). The upregulated genes were all more than the downregulated genes in Fig. 4c, and the DEG for P. stutzeri and S. putrefaciens cultured with BDOM350-700 under xenon lamp irradiation and/or light avoidance exhibited a reasonably high correlation (r > 0.797) (Fig. 4d). These results indicated that light not only triggers the synthesis of specialized metabolites but also can induce a regulatory effect on the expression of functional genes in the P. stutzeri and S. putrefaciens (Bamneshin et al. 2022). Markedly, the degree of deviation for upregulated and downregulated genes from the diagonal were different (Additional file 1: Fig. S8) (Arias et al. 2016). The points of upregulated and downregulated genes of P. stutzeri and S. putrefaciens cultured with BDOM350, BDOM500, and BDOM700 were few, and the degree of deviation from the diagonal was not significant. Moreover, the upregulated and downregulated genes and the degree of deviation from the diagonal cultured under xenon lamp and light avoidance alternating conditions significantly increased. These results revealed that the differences were visualized in gene expression patterns between BDOM-, culture condition-treated, and control groups. However, the upregulated and downregulated DEG genes of P. cultured with BDOM350, BDOM500, and BDOM700 under xenon lamp and light avoidance alternated were significantly changed, proving that the conditions of visible light irradiation and light avoidance alternated had a significant influence on the DEG of P. stutzeri and S. putrefaciens.

The index of TPM was used for quantitative expression level of functional genes of the bacterial transcriptome. Compared to the control groups which were cultured with BDOM under avoiding light conditions, the TMP values of drug resistant genes, drug transformation and metabolism genes, and biodegradation genes were all significantly improved for P. stutzeri and S. putrefaciens cultured with BDOM under visible light (Additional file 1: Tables S7, S8). These genes were considered to be involved in biological processes, constituent cellular components and molecular functions. Moreover, the key genes related to antibiotic, metabolic and catabolic processes were enriched under visible light (Wang et al. 2019) (Additional file 1: Fig. S9). Interestingly, genes for lipid oxidative and catabolism processes and cell membrane motility under visible light were all improved, and thus, biodegradation metabolism through functional genes of reoxidative and catabolism processes was promoted (An et al. 2020). Additionally, under BDOM solution and visible light, many bacterial enztmes are involved in basal metabolism, such as oxidoreductase activity and amide biosynthetic processes, which also induce antibiotic metabolism (Liang et al. 2020). Overall, the above results revealed that visible light significantly improved metabolic activity and growth rate by generating genes related to drug resistance, metabolism and biodegradation, which in turn facilitated SMX and CAP biodegradation.

3.5 Proposed biodegradation mechanisms of antibiotics

Figure 5 shows the interaction network analysis between the top 100 dominnat functional genes and the molecular types of BDOM, including CHO, CHON, and CHOS (p < 0.05). Markedly, the genes of molecular function from P. stutzeri and S. putrefaciens showed the high positive or negative correlations with the CHO type of BDOM. Additionally, the functional genes of cellular components and biological processes also showed a positive correlation with CHO type of BDOM. The correlation between the functional genes and the CHON and CHOS types of BDOM was not significant. In particular, the interaction network analysis between the components of BDOMs and the dominant genes (based on the description) is shown in Additional file 1: Fig. S10. The results indicated that the presence of aplihatics in BDOM had a significant effect on the enzymes of bacteria under visible light irradiation, followed by lignin. The number of fuctional genes affacted by tannic acid in BDOM was clearly lower. Taken together, these results revealed that under visible light, CHO-type BDOM mainly affected the functional genes of molecular function, cellular component, and biological process. Aplihatics in BDOM mainly impacted the enzymes and proteins for P. stutzeri and S. putrefaciens. These effects directly resulted in the biodegradation of SMX and CAP.

Fig. 5
figure 5

Correlation analysis between BDOM molecular types and the top 100 functional genes of P. stutzeri and S. putrefaciens

Full size image

Based on the above results, the proposed mechanisms of SMX and CAP biodegradation by P. stutzeri and S. putrefaciens cultured with BDOM under visible light are shown in Additional file 1: Fig. S11. The culture condition of visible light significantly photodegraded BDOM components to small molecules, which would be directly used for bacterial amplification and growth (Meyer et al. 2018). Meanwhile, visible light also stimulated the bacterial generation of DEG, which would improve the content of saturated fatty acid and the motility of antibiotic and then the biodegradation metabolism of antibiotic by reoxidative and catabolism processes (An et al. 2020), e.g., fatty acid oxidative and catabolism processes, lipid oxidative and catabolism processes, and cell membrane motility. Moreover, the upregulated genes and enzymes related to drug metabolism, such as sulfonamide and chloramphenicol drug metabolic and catabolic processes, were involved in the pathways of generating ATP for cell proliferation and metabolism, which were the precursors for the biosynthetic processes (Li et al. 2020; Wang et al. 2019), resulting in significant improvement of SMX and CAP biodegradation efficiencies.

4 Conclusion

Studying the biodegradation behaviors of SMX and CAP by P. stutzeri and S. putrefaciens and the effects and mechanisms of BDOM under visible light on the degradation of antibiotics are crucial for assessing biochar application in the environment. In this work, BDOM components could be directly used for bacterial amplification and growth under visible light conditions. Visible light conditions also stimulated microbes to generate DEG which improved the content of saturated fatty acid and the motility of antibiotics, resulting in the promotion of the biodegradation efficiency of antibiotics during the reoxidative and catabolism processes. However, compared with the control groups without visible light, the bacteria promoted the adaptation to visible light conditions by decreasing the copy number of genes, and thus, the gene copy number was significantly inhibited. Upregulated genes and enzymes related to drug metabolism, such as sulfonamide and chloramphenicol drug metabolic and catabolic processes, were generated, enriched and involved in the biodegradation pathways of antibiotics during the oxidative phosphorylation process. This study provided theoretical research data for the technology of biochar combined with photodegradation and/or biodegradation to remove pollutants and also provided a basis for risk assessment of microorganisms for biochar remediation of antibiotic-contaminated water environments.