Review of Pyrene Bioremediation Using Mycobacterium Strains in a Different Matrix

Polycyclic aromatic hydrocarbons are compounds with 2 or more benzene rings, and 16 of them have been classified as priority pollutants. Among them, pyrene has been found in higher concentrations than recommended, posing a threat to the ecosystem. Many bacterial strains have been identified as pyrene degraders. Most of them belong to Gram‐positive strains such as Myco‐ bacterium sp. and Rhodococcus sp. These strains were enriched and isolated from several sites con‐ taminated with petroleum products, such as fuel stations. The bioremediation of pyrene via My‐ cobacterium strains is the main objective of this review. The scattered data on the degradation effi‐ ciency, formation of pyrene metabolites, bio‐toxicity of pyrene and its metabolites, and proposed degradation pathways were collected in this work. The study revealed that most of the Mycobacte‐ rium strains were capable of degrading pyrene efficiently. The main metabolites of pyrene were 4,5‐dihydroxy pyrene, phenanthrene‐4,5‐dicarboxylate, phthalic acid, and pyrene‐4,5‐dihydrodiol. Some metabolites  showed  positive  results  for  the Ames mutagenicity  prediction  test,  such  as 1,2‐phenanthrenedicarboxylic  acid,  1‐hydroxypyrene,  4,5‐dihydropyrene, 4‐phenanthrene‐carboxylic  acid,  3,4‐dihydroxyphenanthrene,  monohydroxy  pyrene,  and 9,10‐phenanthrenequinone. However, 4‐phenanthrol showed positive results for experimental and prediction tests. This study may contribute to enhancing the bioremediation of pyrene in a differ‐ ent matrix.


Introduction
The demand for water and energy is increasing, putting additional strain on water and environmental resources. Water scarcity has been identified as a socioeconomic and environmental problem that challenges the world in the twenty-first century, affecting approximately four billion people worldwide at least one month per year [1][2][3]. The continuous release of harmful chemicals such as persistent organic pollutants (POPs) is considered one of the most threatening environmental problems, as mentioned in the Stockholm convention, 2004, Basel convention, 1989, the Rotterdam convention, 1998, Barcelona resolution, 1995, 8 Aarhus Protocol, 1998, and the Arctic environmental protection strategy, 1991 [4]. According to the listed protocols and conventions, harmful chemicals should be eliminated, or their production decreased. POPs are a group of toxic chemicals that stay in the environment long-term and resist natural degradation. There 5, while in 2021, the number of studies was 55, which means that this topic has been gaining researchers' attention. More than 94% of these studies were articles, 2.3%, 2.0%, 0.6%, 0.5%, 0.5, and 2.0% were reviews, conference papers, notes, book chapters, short surveys, and errata, respectively. In this review, the main objective is to collect and organize the scattered information related to the studies that investigated the degradation of pyrene by Mycobacterium strains. The major topics that are investigated in this review are degradation efficiency, pyrene metabolites, bio-toxicity, and the proposed degradation pathways.

Degradation of Pyrene by Mycobacterium sp.
Several bacterial strains have been isolated to use pyrene as a sole carbon and energy source; most of them are Gram-positive, such as Mycobacterium and Rhodococcus [17,18]. Mycobacteria are catalase-positive, non-motile, non-spore-forming, rod-shaped bacteria (0.2-0.6 mm wide and 1.0-10 mm long). The colony morphology of Mycobacteria varies, with some species growing as rough or smooth colonies. Colony color ranges from white to orange or pink [19]. It has been reported that the first isolation of a bacterial strain to mineralize pyrene was in 1988 [20]. Mycobacterium was the most dominant strain to mineralize pyrene [21]. The successful mineralization of pyrene by Mycobacterium strains refers to their ability to produce several functional enzymes capable of metabolizing high molecular weight polycyclic aromatic hydrocarbons, such as pyrene. Dioxygenase is a complex, multi-component enzymatic system containing iron sulfur-containing terminal oxygenase, reductase, and ferredoxin [22]. It has been reported that hydroxylation is the initial biochemical step in the pyrene degradation process. It introduces a couple of oxygen atoms into aromatic pyrene rings [23]. The complete mineralization of pyrene occurs through different enzymatic reactions such as dioxygenase, dihyrogendiol, dehydrogenase, ring cleavage dioxygenase, epoxide hydrolase, alcohol dehydrogenase, acetaldehyde dehydrogenation, and decarboxylation [24]. Figure 2 illustrates the biodegradation of pyrene by Mycobacterium. Many functional genes have been identified in the Mycobacterium strains, such as NidA, NidB, NidAB, NidA3B3, PhdA, PhdB, PdoA, PdoB, and PdoAB. Among Mycobacterium strains, the vanbaalenii PYR-1 strain has many functional genes capable of degrading pyrene and its metabolites. Table 1 includes some of the enzymes produced by different Mycobacterium strains during pyrene degradation. Miller et al. [25] identified NidB and NidA genes that are responsible for producing dioxygenase enzyme when Mycobacterium sp. JLS is used to catabolize pyrene. Zeng et al. [26] reported that the PdoAB gene is responsible for encoding a dioxygenase capable of oxidizing pyrene. Costa et al. [27] observed that PhdA and PhdB are the main genes of the dioxygenase enzyme in the Mycobacterium fortuitum strain. In this review article, more than 40 studies related to pyrene degradation via Mycobacterium strains or consortium culture were collected. In general, Mycobacterium strains showed high degradation efficiency, most of them 80-100%. There are numerous Mycobacterium strains that can degrade pyrene. The phylogenetic tree of Mycobacterium strains is depicted in Figure 3. The Mycobacterium sequences were collected using the NCBI gene bank (Home Nucleotide-www.ncbi.nlm.nih.gov (accessed on 30 December 2021)). The sequences were assembled, aligned, and analyzed with MEGA software version 11.0 Wanapaisan et al. [28] used a consortium culture containing five bacterial strains (Mycobacterium sp. PO1, PO2, Bacillus sp. FW1, Ochrobactrum sp. PW1, and Novosphingobium pentaromativorans PY1). The result showed that 100 mg/L of pyrene was completely eliminated within 6 days of incubation. In addition, the Mycobacterium sp. NJS-1 strain was used to mineralize pyrene on metal-modified montmorillonite. This study revealed that around 93.6% of 15 mg/L of pyrene was degraded within 3 days at neutral pH conditions, and the degradation rate was first-order kinetics 0.62 k/d [34]. Additionally, Zhang et al. [35] applied a consortium of bacterial strains (Micrococcus sp. PHE9 and Mycobacterium sp. NJS-P) to decompose pyrene. About 58% of 100 mg/L of pyrene was removed after 18 days of incubation, and the degradation rate was 3.24 mg/L × day. Sun et al. [36] isolated the Mycobacterium sp. WY10 strain to oxidize 50 mg/L of pyrene in a mineral salt medium. Around 3 10 CFU/mL was inoculated, and the degradation was 83% after 72 h of treatment. Xiaoning Li et al. [37] examined the Mycobacterium sp. NJS-1 strain to treat and remove high molecular weight polycyclic aromatic hydrocarbons, such as pyrene. The author used a mineral medium, and around 1.6 10 CFU/mL was inoculated to degrade 200 mg/L of pyrene; the degradation was 90% of pyrene in the presence of humic acid, while about 10.5% was in the absence of humic acid within 7 days of incubation. The Mycobacterium gilvum CP13 strain was isolated for oxidizing pyrene in a mineral salt medium at alkaline conditions. The bacteria were inoculated at an optical density of 600 nm = 0.5, and 95% of 50 mg/L of pyrene was oxidized after 7 days of degradation treatment [38]. Furthermore, Chen et al. [39] applied biotechnology to treat agricultural and industrial soils contaminated with 16 priority polycyclic aromatic hydrocarbons, including pyrene. The Mycobacterium strain was capable of removing 85% of 100 mg/kg of pyrene during 35 days of treatment in both soils. Also, Terzaghi et al. [40] examined the Mycobacterium gilvum VM552 strain to degrade pyrene suspended on the leaf surface of holm oak (Quercus ilex). The results indicated that after 2 weeks of treatment, the removal was only 17%. Chen et al. [15] attempted to stimulate a microbial degradation approach for soil-containing pyrene. In this study, the active bacterial strains were identified; among them, Mycobacterium strains were the most dominant, and the degradation was 80% of 60 mg/kg within 35 days; the experiment was conducted at pH 8. Sarma and Pakshirajan [41] isolated the Mycobacterium frederiksbergense strain to mineralize pyrene using a batch shake flask reactor. After 200 h of incubation, the pyrene was totally eliminated at neutral pH conditions. Moreover, Peng et al. [17] reported that approximately 81% of 50 mg/kg of soil-containing pyrene was oxidized after 60 days of bioremediation under acidic conditions using the Mycobacterium strain. They pointed out that the NidA gene in Mycobacterium was responsible for generating the dioxygenase enzyme. In addition, the Mycobacterium vanbaalenii PYR-1strain was used in a phosphate-based mineral medium, and 25 μM of pyrene was completely oxidized after 24 h of treatment [18]. Table 2 provides a summary of the studies that used Mycobacterium strains to degrade pyrene in a different medium, pH, optical density, degradation efficiency, incubation time, and initial concentration.
The mass consumption of petroleum products and increase in their demand around the world leads to an increase in the opportunity for pyrene leakage into the environment and increases the opportunity for exposure to pyrene by organisms and humans. Frequent and long-term exposure to pyrene leads to bioaccumulation and biomagnification in the organism cell, which increases the possibility of carcinogenicity and mutagenicity. Many studies have mentioned the negative impacts of pyrene and its metabolites on animals and humans. The toxicity evaluation of pyrene metabolites is important to increase system efficiency. The toxicity assessment of pyrene and its metabolites was carried out using the United States Environmental Protection Agency's software, called Toxicity Estimation Software Tool (TEST) version 5.1. This software is capable of apply-ing mathematical models to predict pollutant toxicity based on Quantitative Structure-Activity Relationship (QSAR) methodology. The data were introduced by inputting the name of each by-product. The lethal concentration of 50% (LC50) (96 h) in fathead minnow and Ames mutagenicity were the considered toxicity for pyrene metabolites using Mycobacterium strain and other biological agents, represented in Table 4. Some metabolites showed positive results for the Ames mutagenicity prediction test, such as 1,2-phenanthrenedicarboxylic acid, 1-hydroxypyrene, 4,5-dihydropyrene, 4-phenanthrene-carboxylic acid, 3,4-Dihydroxyphenanthrene, Monohydroxy pyrene, and 9,10-phenanthrenequinone. However, 4-phenanthrol showed positive results for experimental and prediction tests.

Proposed Biodegradation Pathways
Many bacterial strains have been applied to degrade pyrene in a different medium. Some bacterial strains share the same functional enzymes, which leads to the same degradation pathways, as shown in Table 5. Some genes in the Mycobacterium sp. strain produce enzymes capable of oxidizing pyrene. There are numerous advantages to determining the degradation pathway, including the ability to control the effectiveness of remediation systems, eliminating the influence of degradation on analytical results, and knowledge of degradation pathways for specific compounds can facilitate the assessment of environmental pollution with POPs based on the presence of degradation products. In addition, identifying the degradation pathway is useful for the future development of bioremediation [93,94].

Future Perspectives and Challenges
The current techniques for the biodegradation of pyrene by Mycobacterium strains still need further investigation for future works.
1. A knowledge gap between pyrene oxidation at the field site compared to laboratory conditions needs to be addressed for each product seeking commercial success. 2. The degradation of pyrene by Mycobacterium strains generates many metabolites.
Some of the metabolites and their bio-toxicity have been identified, while most of them need bio-toxicity assessment. 3. The main biodegradation drawback is the limitation of the bioavailability of the target pollutant. Therefore, it is highly recommended to add a biosurfactant to increase the bioavailability. 4. The literature revealed that the biodegradation of pyrene via consortium microbial gives a better result than a single strain. That is referred to diverse enzymes capable of oxidizing pyrene and its metabolites. 5. There are several studies that applied successful synergetic biodegradation systems for pyrene degradation, such as biofuel cells and coupling of the advanced oxidation process and biodegradation system.

Conclusions
This article attempted to provide a review of pyrene bioremediation using Mycobacterium strains in various biodegradation mediums. This study's findings are summarized as follows:  Mycobacterium strains are efficient biological agents to degrade pyrene, that is, referring to their ability to produce many functional enzymes able to metabolite pyrene and its transformation molecules.  Phenantharene-4,5-dicarboxylic acid, dihydroxy pyrene, phenanthrene-4-carboxylic acid, phthalic acid, and pyrene-4,5-dihydrodiol were the most frequent metabolites that were detected when Mycobacterium sp. strains were used for pyrene degradation.  Some metabolites showed positive results for the Ames mutagenicity prediction test, such as 1,2-phenanthrenedicarboxylic acid, 1-hydroxypyrene, 4,5-dihydropyrene, 4-phenanthrene-carboxylic acid, 3,4-Dihydroxyphenanthrene, Monohydroxy pyrene, and 9,10-phenanthrenequinone. However, 4-phenanthrol showed positive results for experimental and prediction tests.