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.