The raw material for preparing biochar in this experiment was wheat straw (from Hebei Province, China). The preparation method of biochar adopts the method in references (Zuriaga-Agustí et al., 2013; Lyu et al., 2016). Briefly, the dried wheat straw powder was heated at 300, 500, and 600°C for 2 h in a stainless-steel reactor in a Muffle Furnace under the O₂-limited condition. After it was cooled to room temperature, and then passed through a 100-mesh sieve and washed with deionized water until the biochar was neutral. Keep it in a sealed bag and place it in a desiccator for later use. Biochar prepared at 300, 500, and 600°C were termed 300BC, 500BC and 600BC.

Shewanella oneidensis MR-1 was cultivated in LB medium at 30°C. After incubation for 16 h, 50 mL of the culture was centrifuged (6,000 g for 5 min), and the cell pellet was washed twice with anoxic bicarbonate buffer (pH 7) and suspended in the same buffer.

Microbial degradation experiments were conducted in 50 mL anaerobic bottles. The experiments were designed into 5 groups, divided into biochar combined with MR-1 group (called 300BC biotic, 500BC biotic, and 600BC biotic), MR-1 alone group (called only MR-1), and blank group. Each group has three parallel samples. The initial concentration of roxarsone was 1.0 mmol/L, the initial concentration of MR-1 (OD600) was 0.8, and 5 mmol/L of sodium lactate was added as an exogenous carbon source. The N₂/CO₂ (80:20) mixed gas was purged into the butyl-stopper glass bottles for 15 min to remove oxygen. The anaerobic flask was placed in a constant temperature shaker under dark conditions. The parameters of the shaker were set to 100 rpm and 30°C. When the reaction time was 12, 24, 36, 48, 84, 132, 168, and 192 h, the concentration of roxarsone in the sample was analyzed. When the reaction time was 0, 48, 72, and 100 h, the extracellular polymer was extracted, and the protein and polysaccharide content were determined.

In this experiment, the concentration of roxarsone was determined by a high-performance liquid chromatograph (Flexar, PerkinElmer, United States), and the wavelength was 264 nm. The chromatographic column used in the determination was a C18 column (4.6 mm × 150 mm). The mobile phase was composed of 0.05 mol/L potassium dihydrogen phosphate (KH₂PO₄), methanol, and 10% glacial acetic acid (V/V). The volume ratio was 95:5:0.1, and the mobile phase flow rate was set to 1.0 mL/min. The column temperature was 30°C.

Extracellular polymeric substances (EPS) was extracted by an improved cation exchange resin method (Liang Z. W. et al., 2010; Zuriaga-Agustí et al., 2013). 10 mL of bacterial suspension was placed in a centrifuge tube, centrifuged at 2,000 g for 15 min at 4°C, and the supernatant was discarded. Resuspend the pellet with 10 mL PBS buffer. Add 1 g of sodium type 732 type cation exchange resin (20–50 mesh) to the suspension, which was shaken at 40 rpm for 6 h at 4°C, and then centrifuged for 15 min with a high-speed centrifuge. The centrifuge parameters were set to 4°C and 10,000 g. After centrifugation, the supernatant was filtered with a 0.22-μm polyethersulfone filter, and stored at −20°C for testing. The sulfuric acid-anthrone method was used to determine the polysaccharides in EPS (Yuan et al., 2011). The BCA protein concentration determination kit was used to detect the protein content. To determine the relative concentration of MR-1 in different groups, 5 mL samples were taken from the treatment groups at 24, 48, and 84 h, the OD600 value was measured with a microplate reader.

The physical and chemical properties of biochar samples (300BC, 500BC, and 600BC) were analyzed by JOEL JSM-7800F field emission scanning electron microscope (FE-SEM), American Mike ASAP 2460 multi-station extended automatic rapid surface area analyzer, the Nicolet iS50 Fourier transform infrared spectrometer. The surface group in biochar samples are analyzed by solid-state ¹³C nuclear magnetic resonance (NMR) spectroscopy on a 400 MHz NMR spectrometer (Advance III WB 400, Bruker, Germany).

The degradation rates of roxarsone by MR-1 in the presence and absence of biochar were analyzed by one-way analysis of variance (ANOVA) followed by Duncan test using SPSS 27.0 software. The results were considered significant when the p value was less than 0.05.

The biotransformation of roxarsone in the presence and absence of biochar was shown in Figure 1. According to the results in Table 1, roxarsone was almost completely degraded in all groups within 68 h. It was found that the degradation rates of roxarsone at 68 h were: 600BC biotic > 500BC biotic > 300BC biotic > only MR-1, and the degradation rates were increased by 9.79, 6.84, and 3.23% with the addition of 600BC, 500BC and 300BC. Therefore, biochar had a promoting effect on removing roxarsone by MR-1. The ability of biochar to promote the degradation of roxarsone by MR-1 was followed as: 600BC > 500BC > 300BC. It was found that the change of ln(C₀/C_t) with time (t) showed a good linear relationship (C₀ is the initial concentration of roxarsone, C_t is the concentration of roxarsone remaining in the reaction when the reaction time is t), in line with the first-order kinetic reaction fitting equation. The kinetic curves of the removal of roxarsone by MR-1 in the presence and absence of biochar were shown in Figure 1B. The average rate constant fitted by the first-order kinetic equation was 0.017 h^–1 in the only MR-1 group, and the removal rates of roxarsone by MR-1 combined with biochar group were greater than that of only MR-1 group. According to the results in Table 2 the average rate constants in MR-1 combined with 300BC, 500BC, and 600BC groups were 0.020, 0.024, and 0.032 h^–1, which showed the order: 600BC biotic > 500BC biotic > 300BC biotic. It can be seen that when the pyrolysis temperature is in the range of 300–600°C, the higher the pyrolysis temperature of biochar, the greater the promotion effect of biochar on the removal of roxarsone by MR-1. Lehmann et al. (2011) summarized a large amount of literature and found that the addition of biochar in most studies can increase the biomass of microorganisms. On the other hand, Klüpfel et al. (2014b) found that in the process of biochar combined with MR-1 for oxidation-reduction, the biochar produced at the medium to high temperature pyrolysis temperature (400∼600°C) showed the highest ability to accept and supply electrons, while the low-temperature pyrolysis temperature (300°C) biochar has the worst effect. Because the pyrolysis temperature of biochar was related to its transfer rate of electrons, it affected the speed of MR-1’s reaction to remove roxarsone. Zhang et al. (2019) found that with the increase of pyrolysis temperature, the conversion ability of biochar was gradually changing from the supply of electrons to the dominance of electrons. The surface functional groups and condensed aromatic structure play an important role in the reaction.

In order to explore the effect of biochar on the secretion of the extracellular polymer by MR-1, the content of protein and polysaccharides at 48, 72, and 100 h in different treatments were measured, as shown in the Figure 2. It can be seen that the changing trend of the protein concentration and polysaccharides concentration was the same in the presence and absence of biochar. At the same time, the polysaccharides and protein content in the only MR-1 group was higher than that of biochar combined with MR-1 group. And in the presence of biochar, the polysaccharide and protein content in the 300BC biotic group was the highest, and that in the 600BC biotic group was the lowest. Therefore, the higher the pyrolysis temperature of the biochar, the lower the content of polysaccharides and proteins produced by MR-1 during the reaction. Because the poorer the living environment of MR-1 was, the more extracellular polymers were secreted, MR-1 can adapt to the surrounding environment to ensure survival (Vu et al., 2009). The results showed that MR-1 survives well in the presence of biochar, and the growth environment of 600BC was the best, followed by the growth environment of 500BC, and the growth environment of 300BC was the worst. This was because, on the one hand, biochar can provide nutrients for microorganisms; on the other hand, because of its large surface area, porous structure, and strong affinity for microorganisms, biochar can be used as a habitat for microorganisms (Lehmann et al., 2011; Wahla et al., 2020).

As shown in Figure 3, the OD values in the presence and absence of biochar decreased as the reaction time lasted, which inferred that the MR-1 is in the stationary and decline phase and the proliferation slowed down as the degradation of roxarsone. The OD values in the only MR-1 group decreased fastest. In the presence of biochar, the OD value of MR-1 in the whole reaction process was sorted: 600BC biotic group > 500BC biotic group > 300BC biotic group. Moreover, when the reaction reached 84 h, the OD values in the presence of biochar were much greater than that of only MR-1 group, especially the OD value of the 600BC biotic group was the largest. The living or dead states of MR-1 in the presence and absence of biochar was determined by Live/Dead assay using a laser scanning confocal microscope when the reaction reached 55 h (Supplementary Figure S1). In 600BC biotic group, most of the MR-1 exhibit green fluorescence, which indicated that the number of live MR-1 was much larger than the dead MR-1, and the growth activity is the best compared to other groups. The addition of biochar made the bacteria grow better, and the activity was better. The addition of biochar benefited to the MR-1 growth, which was consistent with previous research results. Liang B. Q. et al. (2010) studied the effect of biochar on microorganisms in soil, and found that the biomass of microorganisms in soils with high biochar content was 125% higher than that in adjacent soils with low biochar content.