Study on Biodegradation Characteristics of Industrial Phenol-Containing Wastewater by Biological Co-Metabolism Technology

: Biological co-metabolism is an economical and efficient technique for treating refractory organic matter, and in recent years, it has been widely used in the treatment of chlorophenol-containing wastewater. It has been found that many conditions affect the bio co-metabolism efficiency, such as the carbon source type, carbon source content, microorganism types, and environmental factors. The carbon source concentration experiment showed that when the dosage ratio of sodium acetate to black aniline powder was 1:2, the degradation rate of black aniline powder was 82%, and the removal rate was 92.9%. When tetrachlorophenol increased from 210 mg/L to 2100 mg/L, the tetrachlorophenol was increased in the effluent, and the microorganism's activity was inhibited. Besides, the sedimentation performance of activated sludge was also damaged. The temperature test showed that the removed 4-chlorophenol was as high as 2100 mg/L at 35 °C, and the apparent 4-chlorophenol residue in the effluent could be detected at 20 °C. Therefore, by appropriately controlling the external operating conditions of the reactor, the co-metabolism of refractory organic such as chlorophenols can be achieved.


Introduction
Chlorophenol wastewater is toxic and poorly biochemical (BOD 5 /COD less than 0.3). The usual treatment techniques for chlorophenol wastewater are physical, chemical, and biological. Among them, the biological method is the more practical treatment method, but the problem is that microorganisms are easily inhibited by toxicity. Biological co-metabolism solves this problem. Co-metabolism is an efficient method to treat toxic wastewater, which has a high application value in treating chlorophenol wastewater. Co-metabolism adds a carbon source to chlorophenol wastewater to improve the wastewater's biochemical properties while strengthening microorganisms' resistance and metabolic function against toxicity. Therefore, co-metabolism has brought new insights to the treatment of toxic and hard-to-degrade wastewater, and many researchers have extensively studied industrial wastewater using co-metabolism methods and achieved good treatment results.

The co-metabolic mechanism
Regarding the principle of co-metabolic reactions, the enzymatic reaction theory has been generally accepted by many researchers. The association between target contaminants, co-metabolized carbon sources, and nonspecific enzymes in the co-metabolic reactions is shown in Figure 1. Target pollutant toxicity inhibits microbial metabolic enzyme capacity. Under the dual induction of carbon source and pollutant, microorganisms use the carbon source and rapidly synthesize non-specific enzymes to degrade the target pollutant and produce the corresponding metabolic intermediate products [1]. The primary metabolites are still toxic and have an inhibitory effect on microbial metabolism. Likewise, the long-term induction of the primary metabolites continues to produce the corresponding degradation enzymes, which further degrade into microbially available small-molecule organic matter. Eventually, the target pollution is entirely used by microorganisms and completed mineralization.
The enzymes associated with chlorophenol cometabolism in microorganisms are dioxygenases and monooxygenases. Oxygenases are used to oxid polycyclic aromatic hydrocarbons by adding oxygen atoms to the carbon-carbon bonds of organic matter to generate carbon-oxygen bonds, which then open the benzene ring through processes such as hydrogenation [2]. Depending on the number of added oxygen atoms, it is classified as double oxygenases (addition of two oxygen atoms) and single oxygenases (addition of one oxygen atom). The oxygenase catalysis mechanism of difficult-to-degrade compounds is shown in Figure 2.

The impact factors of co-metabolism
Although the degradation mechanisms of pollutants at different aerobic levels are different, the key influencing factors of co-metabolism are consistent, including the type of co-metabolized carbon source, dosage, pollutant concentration, and other environmental factors.

Carbon sources type
Many carbon sources are available for co-metabolism, including easily degradable small-molecule organic compounds, refractory large-molecule organic compounds, and easily degradable micro-toxic compounds. Besides, other researchers have found that pollutant analogs can also be used as carbon sources, which mainly originates from the enzyme metabolism theory. Researchers believe that growth analogs can be used as carbon sources for microorganisms and induce microorganisms to produce more specific degradation enzymes and improve the pollutant removal rate.
The co-metabolic efficiency is directly affected by adding different external carbon sources. Xu et al. [4] investigated the degradation mechanism of Iopromide(IOP) by specific genera (I-24) under different carbon source conditions. The results indicated that the IOP removal rate by I-24 was 38.43% when starch was used as the carbon source, but the IOP degradation rate was increased to 76.98% when glucose was used as the carbon source ( Figure 3). The type of carbon source is an important influencing factor on pollutant removal in practical applications, and selecting a suitable carbon source is the key to improving the target pollutants removal rate.

Carbon source concentration
The carbon source dosing is a crucial factor affecting the target pollutant's removal rate. The co-metabolism mechanism is shown in Figure 1. There is metabolic competition between the easily degradable carbon source and the target pollutant for the binding sites of nonspecific enzymes and electron acceptors, which leads to different degrees of reduction in the target pollutant removal rate. Dong et al. [5] found the highest cometabolic removal rate when the dosage ratio of aniline black drug and sodium acetate was 2:1, the measured effluent concentration of aniline drug was 23.21 mg/L, and the effluent COD concentration was 35.6 mg/L, with 82% degradation and 92.9% removal rate. In his study of aniline degradation, Wang et al. [6] also found that aniline removal was best when the COD equivalents of aniline and glucose were dosed at 2:1. Therefore, reasonably controlling the ratio between the carbon source and the target contamination of the dosing is crucial in ensuring co-metabolism efficiency. Besides, studies on carbon source concentration on chlorophenol degradation effect are limited, and a more uniform method for carbon source dosing and control in the process of chlorophenol degradation has yet to be seen.

Pollutant concentration
The degradable pollutant content is an essential indicator of the co-metabolism removal efficiency. Usually, the target pollutant has specific biological toxicity. It has an inhibitory effect on the growth and metabolism of microorganisms, and a higher degradation threshold will damage the microbial organism and may cause irreversible damage to the co-metabolic system. In order to reduce the toxic effect of the target pollutant, it is common to start with low concentrations and gradually increase the influent concentration as the degradable concentration increases [7][8][9][10]. Montalvo et al. [11] increased the 4-chlorophenol concentration from 210 mg/L to 2100 mg/L in a study of its degradation. The results indicated that the 4-chlorophenol concentration in the effluent was increased, the microbial activity was inhibited, and the colloidal cluster structure was severely damaged. Besides, the microbial sedimentation performance deteriorated, and the sludge sedimentation coefficient increased from 86 mL/g to 277 mL/g. It is evident that in the actual wastewater treatment process, the pollutant concentration in the influent should be controlled below the degradation threshold to avoid irreversible damage to microorganisms caused by excessive toxicity.

Microbial types
Currently, microorganisms in the co-metabolic process can be divided into mixed bacteria and single bacteria. The advantage of mixed bacteria is that the pollutant can be completely mineralized, and the metabolites accumulation will not stop the degradation. The pollutant can be degraded to produce various metabolites, fully utilized as carbon and energy sources by mixed bacteria to achieve complete pollutant mineralization. Furthermore, using mixed bacteria would significantly improve the stability and applicability of wastewater treatment rather than being limited to experimental studies. A single bacteria can achieve rapid pollutant degradation, which will generally be better than the case of mixed bacteria, but the toxic metabolic intermediates generated will inhibit the bacteria, and complete mineralization cannot be achieved.
With the extension of domestication, the degradation system will also continue to introduce impurity bacteria, causing reaction stagnation.

Temperature
Co-metabolic reactions are a series of enzymatic reactions, and temperature affects co-metabolic efficiency by influencing enzyme activity. Monsalvo et al. [11] investigated the co-metabolic degradation of phenol and 4-chlorophenol by microorganisms at different temperatures. The results indicated that the degraded 4chlorophenol could be as high as 2100 mg/L at 35°C, while high 4-chlorophenol residue was detected in the SBR effluent under 20°C (Figure 4). Temperature is one of the critical factors affecting the enzymatic reaction.

Dissolved oxygen (DO)
DO is an electron acceptor for aerobic co-metabolic reactions and can directly influence the co-metabolic process by affecting the activity of the oxygenating enzyme. In the anaerobic co-metabolism process, DO is excluded from the degradation system by aeration of nitrogen and other measures to achieve a strictly anaerobic environment. Otherwise, DO will directly damage the anaerobic system and thus reduce the pollutant removal effect. The DO effect can be observed in the 2-DCP degradation experiments ( Figure 5).

pH
Environmental pH affects microbial metabolic activity, and too acidic or too alkaline can inhibit the activity of degradation enzymes. Fan et al. [12] optimized the cometabolic properties of tetrabromobisphenol and found that the degradation of tetrabromobisphenol was less than 20% at pH 3.0 or pH 11.0. In contrast, the degradation rate was higher at pH 7.0-9.0, while the tetrabromobisphenol degradation rate reached 80% at pH 7.0. Furthermore, the pH effect on phenolic degradation needs to be further investigated.

Challenges in chlorophenol cometabolism research
In recent years, domestic and foreign scholars have conducted more studies on chlorophenol co-metabolism, making it possible to treat this toxic wastewater using biological methods. However, it is also found that there need to be more studies on the effects of carbon source type and usage on chlorophenol degradation. Furthermore, chlorophenol co-metabolism studies have used commercial carbon sources. Limited research on developing new carbon sources complicates the practical application of co-metabolism technology, bringing more economic burden to the high energy-consuming wastewater treatment projects. Based on the above research status, there are still some bottlenecks to be solved in the chlorophenol co-metabolism removal process.

Metabolic competition between carbon source and chlorophenols
In chlorophenol degradation, easily degradable organic makes up for the shortage of carbon sources and improves the biochemical properties of wastewater while strengthening the microbial degradation ability of chlorophenol and tolerance to high chlorophenol. However, both carbon sources and chlorophenols are carbon-containing organic substances. Microorganisms are more likely to use easily degradable carbon sources. When both exist in the same system, it is easy to trigger metabolic competition, which will inevitably affect chlorophenol removal. The excessive addition of easily degradable organic matter to microorganisms will inevitably lead to the overgrowth of non-functional bacteria, inhibiting the growth of chlorophenol-degrading bacteria. In addition, excessive carbon sources will significantly reduce the DO in the degradation system, inhibiting oxygenase activity. Therefore, optimizing the carbon source level and the type and add method is the key to solving the competition.

High cost
Currently, most co-metabolized carbon sources are commercial, such as glucose, sodium acetate, and sucrose. These organic are high-quality co-metabolism carbon sources, which can improve the co-metabolism effect and treatment cost. Therefore, searching for economic and efficient alternative carbon sources is essential to improving the applicability of co-metabolism technology.

Difficult in domestication and degradation
Chlorophenols are highly toxic compounds, and it generally takes a long time to cultivate efficient degrading bacteria. In addition, the degradation concentration of chlorophenols by functional bacteria is generally low. Zhao et al. [13] were able to degrade only 1.5 ± 0.5 mg/L of 4-chlorophenol after more than 50 day-domestication. Therefore, further efforts are still needed to shorten the enrichment time of available bacteria and improve the degradable chlorophenols and the system's stability.

Conclusion
Biological co-metabolism is an economical and efficient technique for treating refractory organic matter, and in recent years, it has been widely used in the treatment of chlorophenol-containing wastewater. It has been found that many conditions affect the bio co-metabolism efficiency, such as the carbon source type, carbon source content, microorganism types, and environmental factors. Furthermore, to achieve high biological co-metabolism efficiency, it is necessary to solve the problems of metabolic competition between functional and nonfunctional bacteria and the long microorganisms' domestication period. Meanwhile, seeking reasonable alternative carbon sources to reduce subsequent operating costs is also the key to improving the applicability.