2 Materials and methods

2.1 Carrier material

Almond shell biochar was produced through high-temperature pyrolysis of almond shells at 500 ℃, with a particle size of 4-8 mesh and an ash content of less than 10. The MBBR carrier was purchased from a local supplier and was a high-density polyethylene carrier with strong hydrophilicity, a large specific surface area, an excellent microbial growth environment, a high surface roughness, and a fast biofilm formation rate. The PPC carrier was a polyurethane porous gel carrier with an average diameter of 1cm, a bulk density of 12.6 kg/m3, a specific surface area (Sa) of 4000 m2/m3, good corrosion resistance, and high specific surface area, and was not easily deformed.
To test the physicochemical properties of carriers, firstly, pH measurement was conducted by preparing a carrier-water solution with a weight ratio of 1:50, where 2 g of carrier was added to 100 mL of water. The mixture was allowed to reach sample equilibrium by standing for 45 minutes, and the pH value of the solution was measured to determine the pH value of the carrier samples. Three replicates were prepared for each group and mixed appropriately. This method was based on previous studies (Bharti et al., 2019; Cao and Harris, 2010). Secondly, scanning electron microscopy (SEM) analysis was conducted by cutting biofilm-adhered carriers into 5 mm thick slices. The cut carriers were fixed in a 2.5% glutaraldehyde solution at 4 ℃ for 3 hours, washed repeatedly with a phosphate buffer solution to avoid residual fixative, and then dehydrated using ethanol solutions of different concentrations (30%, 50%, 70%, 100%) for 10 minutes each. Three rounds of dehydration with anhydrous ethanol were then conducted. The dehydrated carriers were dried, sprayed with gold, and mounted onto the scanning electron microscope sample stage for observation. This method was employed to investigate the surface morphology and roughness of the carriers.

2.2 Reactor equipment

The reactor system (Figure 1) consisted of an inlet tank, fixed microbial reactor, outlet tank, insulation tank, and lift pump.Shewanella bacteria were inoculated into all three reactors using almond shell biochar, MBBR carrier, and PPC carrier as fillers. The fixed microbial reactor was made of organic glass pipes with a diameter of 6 cm, a height of 50 cm, and an effective volume of 1.4 L. The inlet water was pumped into the fixed microbial reactor by a constant flow pump. The inlet water contained 100 mg/L RB5 synthetic wastewater, with sodium formate and MSM as the carbon and nutrient sources and operated in a continuous flow mode. The outer wall of the fixed microbial reactor was wrapped with rubber tubing, and water in the insulation tank circulated through the rubber tubing to the outer wall of the fixed microbial reactor to ensure stable operation at 30-35 ℃.

2.3 Reactor performance analysis

2.3.1 Measurement of decolorization rate

The hydraulic retention time in the reactor was maintained at 24 hours. The absorbance A0 of RB5 wastewater before decolorization, i.e., in the inlet bucket, was measured at 595 nm. At the outlet of the reactor, 10 mL of effluent was collected, centrifuged for 15 min at 4000 g, and the filtered solution was measured for absorbance A at the maximum absorbance value of RB5 (595 nm). The decolorization percentage was calculated using the initial (A0) and final (A) absorbances as follows:
\begin{equation} \begin{matrix}\text{Decolorization\ rate}\left(\%\right)=\frac{\left(A_{0}-A\right)}{A_{0}}\times 100\%\#\left(1\right)\\ \end{matrix}\nonumber \\ \end{equation}

2.3.2 Analysis of RB5 degradation pathways

In this study, we analyzed the differences in degradation pathways using three different techniques: Ultraviolet-Visible spectroscopic analysis (UV-Vis), Fourier transform infrared spectroscopy analysis (FTIR), and Liquid chromatography-mass spectrometry (LC-MS).
For the UV-Vis analysis, samples of influent and effluent (10 mL) from each reactor were collected and centrifuged at 4000 g for 15 minutes. After filtering through a 0.22 μm membrane filter, the samples were subjected to full wavelength scanning using a UV-Vis-NIR spectrophotometer to compare changes in the absorption peaks before and after dye decolorization.
For the FTIR analysis, samples of almond shell biochar, MBBR, and PPC reactor effluent (60 mL) were collected and centrifuged. The filtered liquid was evenly distributed in multiple plastic culture dishes and sealed with sealing film. The samples were then frozen overnight at -80 °C, removed from the freezer, and immediately dried in a drying oven until the sample moisture was completely vaporized. The dried samples were then analyzed using a Fourier transform infrared spectrometer.
For the LC-MS analysis, influent and effluent samples (10 mL) from the almond shell biochar, MBBR, and PPC reactors were taken and centrifuged at 4000 g for 15 minutes. The supernatant was concentrated by rotary evaporation and analyzed by LC-MS. Three replicates were performed for each sample. The LC-MS flow and parameter settings used were as follows: a liquid chromatography-mass spectrometry instrument was used for sample analysis, with the following conditions: chromatographic column: Acclaim™ 120–3 C18, 150 mm × 2.1 mm; mobile phase: pure water (A), methanol and 2.5 mM triethylamine acetate (TEAA, a mixture of acetic acid and triethylamine in equimolar amounts, B); flow rate: 0.25 mL/min; column temperature: 40 °C; mode: negative ion mode; scanning range: 50-1000 m/z. The mobile phase composition during elution was as follows: 0-30 min, 10→70% B; 30-40 min, 70→10% B; 40-45 min, 10% B. The column temperature was maintained at 40 °C. The injection volume was 50 μL.

2.4 Analysis of microbial community structure in reactors

For analysis of the microbial community structure, carrier materials from three stable biofilm reactors were sampled, with three replicates from each reactor, for a total of nine samples. High-throughput sequencing was conducted at BMK in Beijing using the Illumina HiSeq sequencing platform. The RB5-degrading bacterial communities were also isolated using the dilution spread method, with two replicates taken from each reactor for a total of six samples. These samples were also sequenced and compared with the sequencing data.

2.5 Statistical analysis

One-way analysis of variance (ANOVA) was used to analyze the effect of carriers on reactor performance. Statistical significance was determined at p <0.05.