Study on treatment of acid red G with bio-carbon compound immobilized white rot fungi

As one of the main sources of industrial wastewater, printing and dyeing wastewater contains difficult to degrade substances and is toxic (Yang et al. 2011; Zhu et al. 2018). Azo dye is one of the main pollutants in printing and dyeing wastewater. Among them, monoazo dye acid red G (ARG) dye is an example of anionic dye with the characteristics of high chromaticity and biodegradation resistance. If discharged into the water environment, it will cause non-negligible harm to the ecosystem and human health. At present, the removal of azo dyes in wastewater includes physical method, chemical method and biological method. The physicochemical methods include adsorption method, precipitation, flocculation, membrane method, electrochemistry, advanced oxidation method, and so on (Huang 2016; Al-Dahri et al. 2020; Li et al. 2020; Paz et al. 2020). Microbiological methods have also been reported (Jinwook et al. 2016). Since microbiological method has the advantages of safety, low treatment threshold, less pollutant residue and economy, it has a broad application prospect in the treatment of dye wastewater.

Most of the intermediate products produced after decolorization of azo dyes are colorless aromatic amines, which cannot be further degraded by most bacteria. The lignin degrading enzyme synthesized by white rot fungi can degrade some refractory pollutants containing aromatic ring structure and produce CO₂ and H₂O (Wang et al. 2017). The reaction of various free radical chains is started to realize the oxidation of pollutants. Therefore, white rot fungi are widely used in the dye degradation of printing and dyeing wastewater (Senthilkumar et al. 2014; Lu et al. 2016; Zhang et al. 2018).

Although the treatment effect of microorganisms on pollutants is very ideal, the treatment effect will decline because free bacteria are easy to be affected by the environment. The method of immobilized microorganisms is used to solve the disadvantage that free bacteria are easy to be affected by the environment (Yu et al. 2015). Because biochar has the advantages of high porosity and large specific surface area, it has been studied at home and abroad to use biochar as the carrier of microbial immobilization (Abu Talha et al. 2017), so biochar is suitable to immobilize white rot fungi in order to improve the dye removal effect. Bharti et al. (2019) made biochar from casuarina seeds as an immobilization carrier, and studied the dye removal effect of biochar immobilized microorganisms in a packed bed bioreactor. Experiments showed that when the dye concentration was 50 mg/L, the batch reactor containing immobilized microorganisms had a removal rate of about 10% higher than that of the batch reactor containing free microorganisms.

At present, the research on biochar as a bacterial carrier to treat printing and dyeing wastewater and the immobilization of fungi is basically vacant. Therefore, the research on the use of biochar composite immobilization of white rot fungi has broad prospects. In this study, corn straw biochar was used as the carrier to immobilize white rot fungi through adsorption-embedding. The optimal preparation conditions of immobilized pellets were explored. Through batch experiments, under the optimal preparation conditions, the effects of different conditions such as the dosage of immobilized pellets, temperature, solution pH, shaking table speed, and initial dye concentration on the removal of ARG from wastewater were studied, the degradation products of monoazo dye ARG were studied and the degradation pathway was analyzed by characterization.

Phanerochaete chrysosporium was used in the experiment, which was from China Industrial Microbial Species Preservation Center (CICC), storage number 40719. The ARG used in the experiment was a commercially available dye. Corn straw biochar came from Nanjing Rongchuanglian Co. Ltd. Chemical reagents, such as potassium dihydrogen phosphate (KH₂PO₄), sodium alginate (SA), sodium chloride (NaCl), hydro-chloric acid (HCl), sodium hydroxide (NaOH), and ARG dye of analytical grade were provided by Sinopharm Chemical Reagent Co., Ltd., China.

In our previous research, the immobilization method was determined by comparative experiments (as shown in Figures 1–5 of Supplementary Material). Through the adsorption immobilization experiment and adsorption-embedding composite immobilization experiment, the results showed that if only corn straw biochar is used as the material for adsorption and immobilization of white rot fungi, although the removal efficiency of ARG can reach more than 93%, the mechanical strength of adsorption and immobilization is low, and corn straw biochar is easy to fall off from the material for adsorption and immobilization of white rot fungi, which is not conducive to the subsequent solid-liquid separation. The mechanical strength of corn straw adsorption embedding composite immobilization is higher than that of corn straw adsorption immobilization alone. The prepared materials are easy to separate solid and liquid, which is conducive to the repeated use of immobilized white rot fungi. The pellets prepared by composite immobilization have uniform size, excellent mechanical strength and mass transfer performance, and the removal rate of ARG can reach more than 90%, which is about 30% higher than that of free bacteria. The removal efficiency of ARG was the best after the immobilized pellets were cultured for 6 days, and the removal rate reached 97.62%. In summary, the method of adsorption-embedding composite immobilization was studied to immobilize white rot fungi.

A certain amount of SA was put into a 100 mL beaker, added deionized water and placed overnight. Corn straw biochar was ground into powder, washed with deionized water for three times and dried. It was called to take a certain amount and put it in a beaker. CaCl₂ solution with a certain concentration was put into a pressure cooker at 121 °C for sterilization for 30 min, dried and put into a sterile console. The bacterial suspension with OD₆₀₀ = 1 was added to the biochar and was placed in a constant temperature shaker for 2 h. The biochar and SA colloid adsorbed by white rot fungi were mixed and stirred, and then drop by drop into the solution containing CaCl₂ placed on the magnetic stirrer. After curing, they were washed with sterile 0.9% NaCl and stored in cold storage. There are many complex factors in the preparation of immobilized pellets, which may affect the properties of pellets. Therefore, SA concentration (1%, 2%, 3%, 4%, 5%), biochar content (0, 0.3%, 0.5%, 0.7%, 1.0%), addition amount of mycelium suspension (0.5%, 1.0%, 5.0%, 10.0%, 25.0%), CaCl₂ concentration (2%, 3%, 4%, 5%), and immobilization time (12 h, 24 h, 36 h, 48 h, 72 h) were used for single factor experimental design. Meanwhile, the mechanical strength, expansion coefficient, mass transfer performance and dye removal rate of immobilized pellets were used as evaluation indexes.

The randomly selected 100 immobilized pellets were put into a conical bottle containing 100 mL deionized water and put into a constant temperature oscillation incubator for oscillation at 250 r/min. After 24 h, the ratio of the number of complete pellets in the bottle to the total number of initial small pellets was observed. The mechanical strength was expressed by the crushing of pellets. Three groups of parallel tests were carried out.

The randomly selected 30 immobilized pellets were immersed in a beaker containing 100 mL deionized water for 8 h. The diameter of the immobilized pellets before and after immersion was measured with a vernier caliper, and the average diameter of the pellets was calculated. The ratio of the diameter after expansion to the diameter before expansion was the expansion rate. Three groups of parallel tests were carried out.

The randomly selected 50 immobilized pellets were added to 100 mL distilled water, 100 mg/L methylene blue solution was added dropwise in the water, the solution was stirred with a glass rod, the absorbance was adjusted to 0.5, and the blank deionized water was used as the blank reference. After 24 h, the absorbance of methylene blue solution at the maximum absorption wavelength of 665 nm was measured, and the adsorption rate of immobilized pellets to methylene blue was calculated, the mass transfer performance of immobilized pellets was characterized by the adsorption effect of immobilized pellets. Three groups of parallel experiments were carried out.

The spectrum of ARG was scanned by ultraviolet-visible spectrometer (UV-Vis) and the results showed that the maximum absorption wavelength of ARG was 532 nm. Before measuring absorbance, the sample needs to be centrifuged at 10,000 r/min for 10 min. After filtration, the sample is measured at the maximum absorption wavelength.

A₀ – The absorbance value at the characteristic wavelength of the dye at time 0; A_t – The absorbance value of the dye at the characteristic wavelength at time t.

Batch experiments were carried out to study the influence of immobilized pellet dosage, temperature, pH, and initial concentration of the dye on the dye elimination process. The dye solution with an initial concentration of 50 mg/L was formulated. The immobilized pellet dosage was in the range 20–500, temperature was 14 °C–46 °C, the pH of the solution was 3–9, the rotation speed was 0–180 r/min and the initial (ARG) concentration range was 20–500 mg/L.

An orthogonal experimental design of five factors, including immobilized dosage, temperature, pH, rotating speed, as well as wastewater concentration, and four levels were conducted to optimize the process parameters. Meanwhile, the removal rate of ARG was used as the evaluation index (Gao et al. 2008).

The form before and after the culture of the fixed pellet was observed by ordinary electron microscope (S-4800, Japan) and scanning electron microscopy (SEM) (ZEISS EVO 18, Germany). The ultraviolet spectrogenic spectrum was tested with UV-V is (Lambda750S, United Kingdom), the Fourier transformation infrared spectrum of the sample was measured using a Fourier transform infrared spectrometer (FTIR) (Nicolet6700, USA), and the liquid-mass conjunctive chromatograph of the sample was determined by a liquid chromatography-mass spectrometry (LC-MS) (1260-6420, USA).

The test materials were wheat seeds, which were soaked in distilled water for 8 h, and then 30 wheat seeds were selected and placed on the Petri dish covered with seedling paper. 1 mL degradation supernatant, 100 mg/L ARG standard solution, and distilled water were sprayed on the the bottom paper on germination and emergence every day, and cultured at room temperature. Three groups of parallel tests were carried out under each condition. After 5 days, the germination rate (%), the length of radicle and germ (cm) and the fresh weight of seeds (g) were detected (as shown in Table 1 of Supplementary Material).

The results showed that the original solution of ARG had great toxicity to the growth of wheat seeds and affected the germination rate of plants and the growth of embryo bacon, while the immobilized white rot fungus had a good detoxification effect on the dye ARG and could reduce the impact of the dye on the environment.

A certain amount of small pellet culture 5 d fixed by different SA concentrations (1%, 2%, 3%, 4%, 5%) was added to the ARG solution for decoloration. The biochar content was 0.5% (M: V), the bacterial suspension content (represented by OD₆₀₀ = 1) was 5% (V: V), the CaCl₂ solution concentration was 2% (M: V), and the immobilization time was 24 h.

As can be seen from Table 1, when SA concentration is 3% and 4%, the pellets are well formed and the size is relatively uniform. With the increase of concentration of SA, SA with Ca^{2 +} easier role, formed pellet hardness increases, but too high SA concentration can make the colloid viscosity too large, produce the tail phenomenon. Preparation is difficult and the pore size of the pellet will be smaller (Huang et al. 2019). As can be seen from Figure 1(a), the immobilized pellets with 3% and 4% SA concentration were quite complete. Three percent SA concentration was determined as the optimal concentration of immobilized pellets.

A certain amount of small pellet cultured by different biochar content (0, 0.3%, 0.5%, 0.7%, 1.0%) was connected to the ARG solution for discoloration. The concentration of SA was 3% (M: V), the concentration of bacterial suspension (OD₆₀₀ = 1) was 5% (V: V), the concentration of CaCl₂ solution was 2% (M: V), and the immobilization time was 24 h.

It can be seen from Table 2 that the more biochar content is added, the worse the mechanical strength of the pellets will be, but the mass transfer performance will be improved. According to Figure 1(b), 0.7% carbon content of the immobilized pellets of ARG removal rate reached 94.61%. Because of high carbon content, immobilized pellets pore increases, white-rot fungi can more in training process use the nutrients and oxygen in the environment. The more white-rot fungi biomass in same condition was, the higher removal efficiency of dye was (Bayramoglu et al. 2011). The optimum biochar content was 0.7%.

A certain number of pellets fixed by different mycelial suspension (0.5%, 1.0%, 5.0%, 10.0%, 25.0%) were cultured for 5 days, and then added into ARG solution for decolorization. The concentration of SA was 3% (M: V), the content of biochar was 0.7% (M: V), and the concentration of CaCl₂ solution was 2% (M: V). The immobilization time was 24 h.

It can be seen from Table 3 that the mass transfer performance of immobilized pellets decreases with the increase of the addition amount of mycelium suspension. As shown in Figure 1(c), the degradation rate of dye by fixed pellets firstly increased and then decreased with the increase of mycelial suspension addition. When the addition amount of mycelial suspension was 5.0%, the best degradation rate of dye by immobilized pellets was 94.61%. When the amount of mycelium suspension is too high, the biomass after immobilized culture is relatively small due to excessive microbial biomass and limited nutrients, and the removal rate of ARG is also reduced. Meanwhile, when the amount of mycelium suspension is too high, bacteria will leak from the immobilized pellets, which may cause secondary pollution to the water (Ruiz-Marin et al. 2009). The optimum addition amount of mycelium suspension was 5.0%.

A certain amount of small pellet culture 5 d fixed by different CaCl₂ solution concentrations (1%, 2%, 3%, 4%, 5%) was added to the ARG solution for decolorization. The concentration of SA is 3% (M: V), the content of biochar is 0.7% (M: V), the amount of bacterial suspension (represented by OD₆₀₀ = 1) was 5% (V: V), and the immobilization time was 24 h.

As can be seen from Table 4, with the increase of CaCl₂ concentration, the mechanical strength of the immobilized pellets also increased, and no damage of the pellets occurred after 24 h of shock. The expansion coefficient and the mass transfer performance decreases with the CaCl₂ concentration increasing. As shown in Figure 1(d), when the concentration of CaCl₂ was 4%, the immobilized beads had the best degradation effect on ARG, and the degradation rate reached 96.81%. When the concentration of cross-linking agent CaCl₂ is low, the cross-linking is insufficient, and the immobilized beads will cause the loss of white rot fungi. When the concentration of cross-linking agent CaCl₂ is too high, the structure of immobilized pellets is tight, which affects the growth of white rot fungi, and thus affects the degradation effect of immobilized pellets on ARG. The optimal concentration of CaCl₂ was 4%.

A certain number of immobilized pellets with different immobilization time (12 h, 24 h, 36 h, 48 h, 72 h) were cultured for 5 d and then connected to ARG solution for decolorization.

It can be seen from Table 5 that with the increase of time, the internal cross-linking of the immobilized pellets is sufficient, the mechanical strength of the immobilized pellets also increases, the structure of the immobilized pellets becomes tighter, and the mass transfer performance decreases relatively. According to Figure 2(e), when the immobilization time was 36 h, the immobilized pellets had the best degradation effect on ARG.

Under the abovementioned optimal conditions of preparation (3% SA concentration, 0.7% corn straw biochar, 5% white rot fungus mycelium suspension, 4% CaCl₂, and 36 h immobilization time), the immobilized pellets were prepared, and then it was characterized by SEM and bioelectron microscopy scan, and the results are presented in Figure 2.

The Brunauer, Emmett & Teller (BET) specific surface area method was used to measure the immobilized pellets with 0.05 g dry weight. The specific surface area and pore volume of the immobilized pellets were 17.96 m²/g and 0.095 m³/g respectively, which were favorable for the growth of white rot fungus and the degradation of dye by immobilized pellets.

According to the characterization diagram, it can be seen from Figure 2(a) that the immobilized pellets have rich pore structure and large specific surface area. It can be seen from Figure 2(b) that there are many folds on the surface of the immobilized pellet, which is conducive to pollutant adsorption.

The immobilized pellets after 5 d of culture were scanned by biological electron microscopy. According to Figure 2(c), the interior and surface of the immobilized pellets after 5 d of culture were covered with mycelia of white rot fungi, and the growth of white rot fungi broke through the outside of the immobilized pellets, which was conducive to the adsorption of dyes and biodegradation of immobilized pellets. At the same time, it can be seen from Figure 2(d) that the surface of immobilized pellets was covered with white rot fungus hypcelia and the hypcelia were wound and loose after 5 d of culture, which was conducive to the degradation of substances and oxygen into the immobilized pellets and improved the degradation effect of immobilized pellets on dyes.

The gradient of the immobilized pellet amount is set to 20, 60, 100, 200, 300, and 500. As shown in Figure 3(a), the decolorization efficiency of dye increases first and then decreases with the increase of the dosage of immobilized pellets. The more the dosage of immobilized pellet within 36 h was, the higher the degradation efficiency of ARG was. This is because the immobilized pellets contain more biomass of white rot fungi, more dyes are adsorbed at the same time, and the effect of degradation of dyes is better. When more than 36 h, the nutrient content in the solution was reduced due to the microbial degradation of the dye in the previous period, the nutrients required by the subsequent microorganisms were insufficient, the microorganisms died and produced certain toxic substances, which affected the subsequent immobilized white rot fungi to degrade ARG, and the degradation efficiency of ARG was reduced. From the economic point of view, the optimal dosage of immobilized pellets was 100 capsules/100 mL.

Each group of samples is processed in a thermostatic oscillating incubator with five temperature gradients (14 °C, 22 °C, 30 °C, 38 °C, and 46 °C). The oscillator speed during processing is 150 r/min. According to Figure 3(b), when the temperature was 30 °C, the immobilized pellets had the best degradation effect on ARG, with a degradation efficiency of 98.6%. At 96 h, the removal efficiency of ARG basically reached the maximum at each temperature, and then showed a stable trend with the extension of time. With the increase of temperature, the removal efficiency of the immobilized beads on ARG increased. When the temperature exceeded 30 °C, the degradation efficiency of the immobilized pellets on ARG decreased. This should be due to the poor high temperature resistance of some enzymes as the temperature increased. When the enzyme is inactivated, the amount of enzyme which can degrade dye effectively in unit time decreases and the degradation efficiency decreases. Therefore, the optimal removal temperature is 30 °C, which is consistent with the temperature of white rot fungus culture.

A certain number of fixed pellets were added and each group of samples were placed in a thermostatic culture oscillator with 5 pH gradients (3, 4, 5, 6, 7, 8, 9). The oscillator speed during processing is 150 r/min. According to Figure 3(c), the degradation efficiency of immobilized beads on ARG firstly increased and then decreased. When pH = 5, the immobilized beads had the highest degradation efficiency on ARG. When the environment is alkaline, the immobilized pellet degradation efficiency of ARG is less than 70%, the greater the pH, the lower the degradation efficiency, but the actual printing and dyeing wastewater is mostly alkaline wastewater, immobilized pellet also has a degradation efficiency of 70%. The optimal pH range of immobilized spheres for ARG degradation is 4–6, which is consistent with the optimal pH range for dye degradation and the growth of white rot fungi (Daassi et al. 2013; He et al. 2018). The effect of pH on the degradation of dyes by immobilized spheres may be related to the transport of dye molecules across cell membranes, which is considered to be the main cause of dye degradation (Ogugbue et al. 2011). The optimal pH for dye removal was 5.

A certain number of fixed pellets were added and each group of samples were placed in a thermostatic culture oscillator with four speed gradients (0, 90 r/min, 120 r/min, 150 r/min, and 180 r/min). According to Figure 3(d), the degradation efficiency of immobilized beads on ARG first increased and then decreased with the rotation speed. When the rotation speed was 150 r/min, the immobilized beads had the highest degradation efficiency on ARG, and the removal rate of immobilized beads on dye was 98.4% under this condition. Degradation of dyes by immobilized microorganisms requires the participation of O₂. Within a certain range, the greater the rotational speed was, the greater the mass transfer rate of O₂ at the water-gas interface was, resulting into the higher the oxygen content in water, and the higher the removal rate of dyes by immobilized microorganisms (Daassi et al. 2013). When the dye was treated at appropriate rotational speed, the increase of rotational speed was beneficial to the contact between immobilized white rot fungus and wastewater, promoted the substance diffusion and improved the efficiency. The optimal rotational speed was 150 r/min.

Each group of samples is treated in a thermostatic culture oscillator with five concentration gradients (20 mg/L, 50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, and 500 mg/L). It can be seen from Figure 3(e) that the degradation rate of ARG by immobilized spheres decreases with the increase of the initial dye concentration. When the initial concentration of ARG was 20 mg/L, the degradation efficiency of ARG by immobilized beads reached 99% for 120 h. When the initial concentration of ARG was 500 mg/L, the degradation efficiency of immobilized pellets for ARG was only 20.69% after 120 h, which may be because the higher the initial concentration of dye, the higher the organic load. Because the dye itself has certain toxicity, it inhibits the activity of white rot fungus cells and enzymes, and affects the degradation efficiency of the immobilized pellets (Ogugbue et al. 2011; Tan et al. 2014). The optimal initial concentration of dye is 20 mg/L.

As can be seen from Table 6, the influence of five factors on ARG is as follows: initial concentration of dye > temperature > dosage of dye > rotational speed > pH. The optimal combination is as follows: dosage of immobilized pellets is 200 capsules/100 mL, temperature is 30 °C, pH = 4.5, rotational speed is 150 r/min, and initial concentration of dye is 20 mg/L.

Figure 4 shows the change of the maximum absorption peak of the dye ARG during degradation. The absorption wavelength at the characteristic peak of ARG is 532 nm, which is the characteristic absorption peak of -N = N- azo bond and is the hair group of the dye (He et al. 2018). In the UV region, two characteristic peaks were generated, indicating the naphthalene ring structure in ARG at 364 nm and the benzene ring structure in ARG at 235 nm (Khalid et al. 2012; Hai et al. 2013). With the progress of ARG degradation process, the intensity of ARG's characteristic absorption peak at 532 nm decreased continuously, and the characteristic absorption peak disappeared at 48 h, indicating that the azo bond was broken and the chromogenic group was destroyed during the process. At the same time, the characteristic absorption peak intensity of benzene ring (235 nm) and naphthalene ring (362 nm) decreased continuously, indicating that the conjugate structure of benzene ring and naphthalene ring in ARG was destroyed, which also indicated that the dye structure was destroyed. At the same time, studies showed that if the removal of dye was by adsorption removal, the absorption peak intensity would be proportional to the decrease. In the case of microbial degradation, the characteristic peak disappears and new characteristic peaks may be formed at other wavelengths (Asad et al. 2007). These results indicate that the degradation of azo dye ARG by immobilized beads is mainly caused by the degradation of white rot fungi, and the molecular structure of ARG changes significantly before and after degradation.

As can be seen from Figure 5, the infrared spectra of ARG, 24 h degradation solution and 48 h degradation solution vary greatly between 400 and 4,000 cm⁻¹ (Lin & Juang 2009). For ARG, 3,473.00 cm⁻¹ represents the stretching vibration of -OH and -NH-, 1,619.43 cm⁻¹ represents the stretching vibration of azo bond -N = N-, the azo bond is the main color group of ARG, 1,558.30 cm⁻¹ is the stretching vibration of upper -C = C- of benzene ring framework. 1,496.36 cm⁻¹ represents the stretching vibration of -C = C- on the naphthalene ring skeleton; 1,403.74 cm⁻¹ represents the stretching deformation vibration of -S = O in the sulfonic acid group; 1,126.26 cm⁻¹ and 1,056.77 cm⁻¹ represent the stretching vibration of -C-S- in the sulfonic acid group. 755.37 cm⁻¹ and 617.82 cm⁻¹ represent the vibrations of substituent groups on naphthalene rings, reflecting the structure of ARG dye. The infrared spectrum of 24 h degradation solution is the most complex, as shown in Figure 5(b). In addition to the original characteristic peaks, some new characteristic peaks are generated. For example, 3,189.47 cm⁻¹ represents the stretching deformation of -NH₂, and the generation of this new characteristic peak may be caused by the destruction of azo bonds in azo dyes and the formation of amino groups. 2,961.71 cm⁻¹ represents the stretching deformation of -CH₃, and 2,917.74 cm⁻¹ represents the stretching deformation of -CH₂-. The appearance of this functional group represents the ring opening phenomenon of benzene ring or naphthalene ring. At 1,465 cm⁻¹, the stretching vibration of -C = C- is represented. This characteristic peak may be formed by the vibration of benzene ring and naphthalene ring structure in ARG molecular structure and benzene ring or naphthalene ring structure contained in intermediate products. 1,417.00 cm⁻¹ represents the bending vibration of O-H, and 1,264.97 cm⁻¹ represents the stretching vibration of C-N. The production of these new substances represents the functional groups contained in the intermediate degradation products produced after the ARG analytical structure is destroyed, and the substances on the benzene ring substituting group change. As shown in Figure 5(c), the characteristic peaks at 3,357.41 cm⁻¹ and 1,619.07 cm⁻¹ disappear, indicating that the -OH and azo bonds in ARG are broken and ARG is degraded. The disappearance of feature peaks at 3,189.47 cm⁻¹, 1,417.00 cm⁻¹ and 878.21 cm⁻¹ indicates further degradation of intermediates produced during ARG degradation. According to the infrared spectrum of the final degradation solution, the degradation products may contain new functional groups such as benzene ring, naphthalene ring, sulfonic acid group, -CH₂-, -CH₃ and benzene ring substituent group (Lin & Juang 2009; Kagalkar et al. 2011).

As can be seen from Figures 6 and 7, the retention time of ARG solution in mass spectrometry is 7.64 min, and its mass charge ratio is 231.51, which is ARG anion [M-2Na]²⁻. As can be seen from Figure 6, neither the 12 h degradation solution nor the 24 h degradation solution has the retention time of ARG. However, as shown in Figure 7(a), when the retention time is 7.64 min, the 12 h degradation solution contains the mass spectrum peak of ARG, which may be because most of ARG in the 12 h degradation solution has been degraded or the content of organic matter in 12 h is too low. So that its various substances do not show up in the total ion current diagram. As can be seen from Figures 5 and 6, two mass spectrum peaks appeared when the degradation solution was retained for 15.4 min at 24 h, and their mass charge ratios were 417.32 and 403.13, respectively. Figure 8(b) is m/z = 417.32 secondary mass spectrometry, according to the analysis of FTIR, the substance is 5 (acetyl amino) -4-hydroxy-3-amino-2, 7-naphthalene disulfonic acid. M/z = 59.01 is the mass spectrum peak of the ion fragment acetyl amino, and the low mass spectrum peak of other ion fragments may be due to the small content of this substance. Figure 8(c) is m/z = 403.13, which is formed by dehydroxylation of the substance in Figure 8(b), which is 5- (acetyl amino) -3-amino-2, 7-naphthalene disulfonic acid. The other part of azo dye degradation may be aniline, which is then deaminated to form benzene. The benzene ring is relatively stable and forms anion form when H is removed, so it is not detected in the total ion current diagram. According to the analysis of ARG degradation products, it is deduced that the degradation pathway of ARG is as follows: azo bond is broken to form aniline and 5-(acetylamino) -4-hydroxy-3-amino-2, 7-naphthalene-disulfonic acid; aniline is further deaminated to benzene; benzene is degraded to other organic matter by ring opening; 5-acetamino-4-hydroxy-3-amino-2, 7-naphthalenedisulfonic acid was dehydroxylated to form 5- acetamino-3-amino-2, 7-naphthalenedisulfonic acid (Wang et al. 2011; Franciscon et al. 2015).

Corn straw biochar was used as a composite immobilization material, combined with SA to immobilize white rot fungi and treat ARG wastewater. Under the best preparation conditions of immobilized pellets (3% SA concentration, 0.7% corn straw biochar, 5% white rot fungus mycelium suspension, 4% CaCl₂, 36 h immobilization time), the immobilized pellets have rich pore structure and large specific surface area, which is conducive to the degradation of ARG by white rot fungi.

The effects of different conditions on ARG removal were studied by batch experiments. The results of single factor test showed that the optimum treatment conditions of ARG were as follows: the dosage of immobilized small pellet was 100 capsules/100 mL, pH = 5, the rotating speed was 150 r/min, and the initial concentration of dye was 20 mg/L at 30 °C. The results of orthogonal test showed that the initial concentration of dye had the greatest influence on ARG dye. The best combination was: the dosage of immobilized small pellet was 200 capsules/100 mL, pH = 4.5, the rotating speed was 150 r/min, and the initial concentration of dye was 20 mg/L at 30 °C.

According to UV-vis absorption spectrum analysis, FTIR spectrum analysis, LC-MS analysis, the immobilized spheres mainly treated ARG by biodegradation, during which azo bond was broken. ARG was degraded to aniline and 5- (acetylamino) -4-hydroxy-3-amino-2, 7-naphthalene disulfonic acid, aniline was further deaminized to benzene, benzene was degraded to other organic compounds by ring opening. 5- (acetylamino) -4-hydroxy-3-amino-2, 7-naphthalenedisulfonic acid was dehydroxylated to form 5- (acetylamino) -3-amino-2, 7-naphthalenedisulfonic acid. This study shows that the prepared corn straw biochar immobilized pellets can be used as an efficient alternative material for water treatment to remove ARG dye from wastewater.

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