Degradation of aniline: sodium alginate/modified pomelo cellulose double cross-linking system as a bacterial immobilization carrier

Aniline, one of the most important aromatic amine compounds, has been used extensively in the dye, pesticide, and pharmaceutical industries (Ashouri et al. 2021). However, if not stored and used properly, aniline leaks into the environment, causing a series of environmental problems. This can be attributed to the high toxicity of the compound (Tekle-Roettering et al. 2016). This chemical causes significant health hazards as it readily combines with hemoglobin molecules in animals. The bound hemoglobin will lose the ability to transport oxygen (Zhang et al. 2021), which in turn damages the liver and kidneys. Therefore, in 2017, aniline was officially listed as a serious hazardous compound by the International Health Organization.

Therefore, active research is being carried out to develop methods for proper aniline disposal. Many conventional methods for aniline degradation, such as membrane separation, adsorption, and chemical oxidation, have significant drawbacks. These traditional methods are cost-inefficient (Wang et al. 2021), less efficient, and cause secondary pollution (Zhao et al. 2019). Alternatively, biodegradation is considered a promising method for the removal of aniline as the method is cost-effective and environmentally friendly (Chen et al. 2021). Results obtained using the HPLC technique reveal that the biodegradation of aniline involves the degradation of aniline into catechol by bacteria, and the produced catechol is then converted to cis,cis-muconic acid (Cui et al. 2017). Over the past decades, many bacteria, such as Pseudomonas (Tanaka et al. 2009), Acinetobacter (Hou et al. 2018), and Pigmentiphaga daeguensis (Huang et al. 2018), that can degrade aniline have been identified. However, the efficiency of the process of bacterial biodegradation often depends on the survival of microorganisms as the microorganisms often die due to the toxicity of the contaminants (Pinheiro et al. 2022). The process of free bacterial biodegradation is disadvantageous for recycling products in industries, as the separation process is difficult to execute. Therefore, to solve these problems, it is important to develop bacterial immobilization technology (Mehrotra et al. 2021).

As a promising method, the bacterial immobilization technology has attracted the attention of many scholars as it boasts high bacterial density, stability, and good separation characteristics (Beltrán-Flores et al. 2022; Xue et al. 2022). It has been previously reported (Ngah & Fatinathan 2008) that the materials and procedures used for immobilization are crucial for the application of the immobilized cell system (Elisangela et al. 2009; Sharma et al. 2011). Many natural polymer materials, such as chitosan (Denkbaş & Odabaşi 2000), agar, and alginate, have been used for immobilization as they exhibit good biocompatibility. Moreover, these are cheap materials that can be easily processed (Tanaka et al. 2009). Under conditions of immobilization, bacteria are confined to the substrate for better protection and proliferation. Alginate, a linear polysaccharide composed of a-L-mannuronic acid (M unit) and b-D-guluronic acid (G unit), is widely present in a variety of seaweeds, is considered an excellent carrier for bacterial immobilization, as it is non-toxic, can be easily prepared, and obtained from convenient sources. The presence of numerous carboxyl groups on the walls of alginate promotes the reaction of the molecule with other functional groups (Guo et al. 2020). Although alginate is considered the most promising material for cell immobilization, rapid bacterial reproduction, weak transport properties at high bacterial density (Cui et al. 2017), and mechanical fragility of the substrate limit the application of the material. Hence, it is essential to improve the properties of the substrate to achieve high porosity and mechanical strength. Pomelo cellulose (PC) has a unique pipe-like network structure. It is characterized by good biocompatibility, a large specific surface area, and stable chemical properties (Tang et al. 2020). A combination of PC and alginate can be used to integrate the advantages of the two materials. Therefore, alginate can be blended with PC to overcome the drawbacks of alginate and combine the good characteristics of both polymers. When alginate is mixed with cellulose, a strong ionic interaction is generated between the amino groups in cellulose and the carboxyl groups in alginate. This results in the formation of a polyelectrolyte complex (PEC), which imparts good mechanical properties to the support (Shu & Zhu 2002).

This is the first report that describes the method of the formation of a composite fiber ball from the fibers present in the inner layer of grapes. The material was formed following the electrostatic double cross-linking method (Hrenovic et al. 2009). The fibers were cross-linked with sodium alginate, and the characteristics of the obtained material were compared with the characteristics of ordinary sodium alginate gel beads and pomelo fiber/alginate complexes. The rubber pellets exhibit better permeability than the other systems, and the specific surface area of these pellets is larger than the specific surface area of the other systems. This, in turn, imparts protection and growth-promoting properties during bacterial immobilization to the composite fiber-based spheres. The rate of the growth and degradation of bacteria under these conditions far exceeds those recorded in the presence of sodium alginate gel beads. The results reported herein provide a platform for the realization of the application of biomass materials in the field of bacterial immobilization technology.

Commercially available analytical grade chemicals were used. Glyceryltrimethylammonium chloride (GTMAC), dimethyl sulfoxide (DMSO), sodium alginate, NaOH, anhydrous sodium metasilicate, ammonium sulfamate, sodium polyphosphate, sodium bicarbonate, Naf-ethylamine hydrochloride, NH₄Cl, NaCl, MgSO₄·7H₂O, anhydrous sodium acetate, CaCl₂, K₂HPO₄, yeast extract, H₂O₂, H₂SO₄, and ethyl alcohol were purchased from the market.

Deionized water, obtained from self-made RO/EDI equipment, was used for the experiments.

Grapefruit skin fiber is first peeled off of ordinary Shatian pomelo obtained from Yongzhou, Hunan Province (He et al. 2019). The outer layer of the peel containing a large amount of pectin is removed, and the inner layer of the grapefruit peel is placed at a temperature of 45 °C. The oven was dried for 48 h, the inner layer of the peeled water was powdered using a stirrer, and the sample was sieved and dried for 2 h. The obtained product was alkalized to remove impurities. The cellulose to deionized water mass ratio was maintained at 1:25 for 1 h at 25 °C, and the peel fibers were pre-soaked uniformly in sulfuric acid. Following this, it was treated with a solution containing NaOH (7 g/L), Na₂SiO₃ (2%), and sodium polyphosphate (0.25%). The cellulose to sodium hydroxide mass ratio was 1:15, and the high-temperature cooking method was followed at 100 °C for 2 h in a water bath (Liu et al. 2017). The secondary alkali treatment process increases the concentration of the alkalizing agent. The NaOH concentration increases to 11 g/L, and the Na₂SiO₃ content increases to 3% under these conditions. Other experimental conditions were kept the same as the primary alkali treatment condition. Finally, it was bleached with H₂O₂ and then rinsed 5–6 times with deionized water.

The prepared fibers were cationized. During this process, 1,000 mg of water-dispersed dry, pre-treated PC and 50 mg of sodium hydroxide powder were thoroughly mixed using a mortar and pestle at room temperature for 5 min. The solid mixture was then transferred to a polyethylene bag, and this was followed by the addition of solvent (water in various volume ratios or water/DMSO mixtures). A cationizing agent (GTMAC) was added to the previous mixture dropwise, and the sample was kneaded evenly by hand. Subsequently, the reaction mixture was kept in a thermostatic ultrasonic water bath at 65 °C for 4 h. All other conditions were similar to the conditions of the wet process. After 4 h, the product was precipitated using 95% ethanol. It was purified and concentrated following the protocols used in the wet method.

Rhodopseudomonas palustris culture was purchased from Weifang Zhongtian Technology Feed Co., Ltd. It was directly inoculated into the special nutrient solution of photosynthetic bacteria (PSB) through long-term tests. This process was followed as their origin was different (Sun et al. 2012). The liquid culture medium used in this experiment was self-prepared. One liter of the nutrient solution contained 0.1 g of NH₄Cl, 0.5 g of NaCl, 3 g of sodium acetate, 0.1 g of MgSO₄·7H₂O, 0.1 g of CaCl₂, 0.2 g of K₂HPO₄, and 0.5 g of yeast extract. PSB may have community differences in proliferation habits, so the growth factors are regulated. The pH of the nutrient salts proliferating the PSB, growth temperature, light intensity, bacterial inoculum, presence or absence of light, and carbon sources were regulated.

Industrialized PSB may have some intolerance to aniline that is a highly toxic organic compound. Therefore, it is necessary to carry out a domestication process. According to the principle of domestication of PSB (Li et al. 2019), for a certain amount of inoculum, a concentration of approximately 20% of the target degradation concentration is added to the sample at the same time as the nutrient solution. Aniline is sampled every day under suitable conditions to observe the growth of the PSB. When the PSB gradually adapt to the concentration of the aniline nutrient solution, the domesticated bacteria in the nutrient salt are collected and inoculated under conditions of the target degradation concentration (approximately 40%). The method is repeated until it reaches the target concentration (Sheldon 2007). At this time, the bacterial liquid is the domesticated bacterial stock solution, which is used in a subsequent aniline degradation test.

A biological grade sodium alginate is mixed with deionized water in a certain proportion. Following this, it is heated until it dissolves. The mixing process is conducted according to the controlled proportion and the secondary alkalized pomelo fiber. Subsequently, the sample is heated and stirred at 40 °C for 12 h. It is covered with plastic to prevent moisture loss. At the end of the heating process, the heat is turned off, and the temperature of the solution is allowed to come down to room temperature. A certain amount of the bacterial stock is added quickly, and the sample is stirred at high speed for 10 min to distribute the bacteria evenly. Subsequently, the mixed solution is injected into the CaCl₂ solution at a constant rate using a syringe (Shankar & Rhim 2018). After calcification, the sample is washed directly and placed in an oven at 60 °C to obtain the sodium alginate/PC composite gel beads, as shown in Figure 1.

The experiments using PSB were carried out on a shaker containing a triangular bottle of volume 250 mL. In the condition optimization experiment, the bacterial reserve solution (inoculated at 30%) was added to the triangular vial (250 mL), and the solution of aniline at an initial concentration of 500 mg/L was added to it. The samples were placed in a shaker at 150 r/min, and bacterial growth rates, stem cell weight, and aniline concentration were measured to assess the influence of pH, temperature, and other factors on the process of aniline degradation.

The gel beads of sodium alginate/grapefruit cellulose composite were put in 100 mL simulated aniline wastewater (1,000 mg/L), and orthogonal experiments were carried out to determine the optimum content of cellulose, sodium alginate, and CaCl₂. The effects of dissolved oxygen (DO) and light intensity on the aniline degradation process were then assessed under the optimal ratio, and the repeatability of cellulose pellets was also established.

Here, C_t (mg/L) and C₀ (mg/L) represent the residual concentrations of aniline at time t (h) and in the initial, respectively.

The morphology and structure of the pomelo fiber post-treatment and the alginate-cellulose beads were analyzed using a SU8010 scanning electron microscope (SEM) (Hitachi, Japan) at 15 kV. The structural changes of the common sodium alginate gel sphere and the pomeloid fiber sodium alginate composite gel sphere are shown in the image recorded using the Tecnai G2 F30S-Twin transmission electron microscope (TEM) system (Philips-FEI, The Netherlands). The changes in the composition and functional groups of different pomelo fibers following alkalization treatment and NH₃·H₂O grafting were determined using a Nicolet 6700 Fourier transform infrared (FTIR) (Thermo Nicolet, America) spectrometer. The data were recorded in the wavenumber range of 450–4,000 cm⁻¹ (Zhang et al. 2018). The compositional structure of the gel pellets and the composite fiber spheres were changed. The chemical structure of the sample was further verified using the X-ray photoelectron spectroscopy (XPS) technique (PHI-5000C, USA). The surface area and the pore size distribution were analyzed using the Brunauer–Emmet–Teller method (Quantachrome, Autosorb-Iq-MP).

PSB was used for aniline degradation experiments, and it was designed according to the univariate principle. The pH and temperature were changed to obtain the best degradation conditions of aniline.

Under normal circumstances, the degradation efficiency of aniline and the growth of photosynthetic bacteria are one-to-one. The aniline cannot be efficiently degraded as bacteria do not grow efficiently under these conditions. It has been reported that the optimal temperature for the growth of photosynthetic bacteria is approximately 30 °C. Four sets of temperatures (25, 30, 35, and 40 °C) were selected as parameters to study the effect of temperature on the process of degradation of aniline. The experimental results (Figure 8(b)) are consistent with the results reported in the literature. The maximum extent of aniline degradation is achieved at 30 °C, and at 40 °C, the degradation effect decreases slightly. Thus, it can be inferred that temperature affects the activity of the aniline-degrading enzyme.

As shown in Figure 8(c), PSB growth presents a lag period of 0–24 h. This is followed by a logarithmic period of 24–48 h, in which the dry weight of the cell increases significantly. The cells grow rapidly at approximately 48 h. When the degradation time reaches 3 days, the degradation rate of aniline reaches 67.2%. Following this, the degradation rate becomes stable.

The addition of sodium alginate and polyethylene glycol (PEG) increased the viscosity of the liquid and the compactness of the microspheres. This affects the mass transfer of substrates and products and reduces the activity of PSB, eventually reducing the degradation rate. The lower the content of sodium alginate and PEG, the looser the microspheres. The decomposition and destruction of the microspheres are promoted under these conditions.

Pomelo fiber, a kind of biomass fiber produced after two alkalization treatment cycles and cation modification, has a great influence on the bioactivity of alginate gel spheres and the extent of the adsorption of bacteria. In particular, the embedded photosynthetic bacteria transformed into composite gel microspheres, which exerted a strong synergistic effect on mass transfer performance. The gradual increase in the pomelo fiber dosage resulted in an increase in the aniline degradation efficiency (Table 2). The cellulose content was increased from 2 to 3% (Figure 9(a)), and the maximum extent of aniline degradation was recorded under these conditions. Following this, the efficiency gradually decreased. The presence of cellulose can increase the load of bacteria, protect bacteria, and increase the running time of bacteria in simulated wastewater. High cellulose content will reduce the mechanical properties of the pellets, reduce the degradation effect, and is not conducive to the growth of embedded bacteria. When the dosage of sodium alginate was gradually increased, the degradation efficiency first increased (Figure 9(b)). It reached the maximum value when the sodium alginate content was 2%. This can be potentially attributed to the low content of sodium alginate, which results in a weakening of the adhesion strength of the composite fiber microspheres. Eventually, the mechanical properties of the composite fiber ball deteriorate. A large number of internal channels is formed when the concentrations of sodium alginate and calcium ions are increased at this point (Figure 9(c)). A loose structure characterizes the material, and the mass transfer and protection performance of the composite microsphere ball is the best at this point. The best aniline degradation effect is recorded under these conditions. If the pomelo fiber content is too large and the sodium alginate content is too low, the mechanical properties of the microspheres will continue to deteriorate. This will result in the cracking and crushing of the pellets. This will eventually result in the reduction of the degradation effect. However, if the concentration of sodium alginate and CaCl₂ is too high, the density of the fiber microspheres will increase, affecting the process of the exchange of bacterial substances. This will also affect the activity of the aniline degradation enzyme, resulting in a rapid decrease in the aniline degradation rate. In this experiment, there was some correlation between the number of sodium alginate/PC composite gel beads and modified PC content, as shown in Table 3.

As the photosynthesizing bacteria embedded in cellulose microspheres dynamically decompose aniline, cellulose microspheres cannot be used for prolonged periods. We tested the reusability of the prepared fiber microspheres. The optimized preparation conditions of microspheres are presented (DO: 2 mg/L; light intensity: 400 Lx; degradation rate of aniline: 200.0 mg/L). The experiments were conducted with cellulose (3.0%), sodium alginate (2%), and calcium chloride (2.5%), as shown in Figure 12. After continuous use, the degradation rate gradually increased, and the degradation rate was 96.1% after five cycles. The rate decreased to 86.3% after eight cycles. Thus, the material exhibits good stability.

We extracted grapefruit skin fiber and used it to develop the bio-immobilization technology (Jiang et al. 2018). The aniline degradation efficiency of the sodium alginate/PC composite gel beads was higher than that of the simple sodium alginate gel pellet. SEM, FTIR, XPS, and BET techniques were used to reveal the successful extraction of the material and the unique surface structure properties of pomelo peel fibers. Unique biomass transfer characteristics in sodium alginate/PC composite gel beads were also observed. The maximum extent of aniline degradation by bacteria was achieved at 30 °C at pH of 7.5 in the presence of light (4,000 Lx, DO 2.0 mg/L). The sodium alginate content was 2%, the CaCl₂ content was 2%, and the modified PC content was 3%. The mass ratio was 5:1. The results reported herein reveal that plant fibers can be used for bacterial immobilization. Immobilization of natural polymer and organic synthetic polymer can be achieved.

The authors thank the National Science Foundation of China (Grant No. 21736009), the Zhejiang Public Welfare Research Project (Grant No. LGF19B070005), the Basic Public Welfare Research Program of Zhejiang Province (Grant No. 2021C03169), and the Scientific Research Foundation of Zhejiang University of Technology (201910337011) for financial assistance.

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

The authors declare there is no conflict.