The environment is consistently getting polluted by diverse anthropogenic causes, such as the overconsumption of fossil fuels and biomass. Petroleum is a vast reservoir of thousands of organic compounds, but some chemicals are directly and indirectly harmful to humans, animals, and plants. Without any concern, we are using these toxic organic pollutants in our daily lives, which lead to fatal diseases like cancer, lung and liver dysfunction, skin and brain disorders, kidney failure, reproductive infertility, etc.^1,2

Polycyclic aromatic hydrocarbons (PAHs), composed of multiple benzene rings, like Naphthalene, Anthracene, Phenanthrene, Fluorene, Pyrene, Dibenzothiophene, and several thousand of their derivatives are present in soil, air, and water as a group of organic priority pollutants. They have direct and indirect toxic, genotoxic, mutagenic, or carcinogenic impacts on plants, animals, human health, and microorganisms.^3-5 PAHs bind with environmental pollutants and become more rigid and toxic than before, as a result, they are not easily degradable. Most of the PAHs are hydrophobic, thermostable, and have heterocyclic aromatic ring configurations like fluorene, so they are resistant and highly persistent to the environment.⁶ PAHs are widely used for various purposes such as agrochemicals, dyes, electronics, fluorescent, liquid crystals, pigments, pesticides, pharmaceuticals, resins, roofing tar, thermoset functional plastic, and so on.⁷

The main sources of PAHs are fossil fuels such as petroleum (oil), coal, and gas, plant-biomass, garbage, and narcotic plants like tobacco. PAHs are exerted as byproducts when those organic compounds are combusted incompletely.^8,9 Some PAHs are produced in the reservoir, but some are synthesized or converted from large organic polymers. Seepage and spillage of fossil fuels, industrial waste, stormwater runoff, and volcanic eruptions are also the key sources of fluorene and other PAHs in surface water, and groundwater.^10,11

Polycyclic aromatic hydrocarbons enter humans and other organisms through direct inhalation of carbon engine exhaust, cigarette smoke, and wood smoke, consumption of  grilled or charred meats, contaminated bread, processed and pickled foods, wastewater, and polluted cow milk.⁷ PAHs arrive in the body through skin absorption, or oral administration, then gather in adipose tissue, liver, and kidney through the chylomicron process, and subsequently lead to many known and unknown diseases at cellular and genomic levels.^12,13 PAHs can alter gene regulation and lead to disorders in the future, and even this harmful impact may go to the next generation.²

Physical, chemical, and biological procedures can degrade the toxic PAHs.¹⁴ Anyways, physical, and chemical degradation processes are costly and time-consuming, moreover, both are not suitable for huge, contaminated areas like the sea, landfills, gasworks, and so on. Therefore, the biological process can be better than others. Bioremediation of PAHs refers mainly to microbial degradation by bacteria and fungi and phytoremediation by plants.^15,16

In this study, we described to find how PAHs impact human health and the environment, the general mechanism of their degradation, and potential microbial sources that could biodegrade PAHs remarkably. The objective of this review is to lead to conclusive research in the future.

General properties of PAHs
PAHs are composed of two or more fused benzene cycles, as naphthalene is the smallest polycyclic aromatic hydrocarbon bearing only two benzene rings, and anthracene is composed of three rings. In the PAH structure, the common key elements are Hydrogen, Carbon, and Oxygen, and the structure is stable due to the ring’s resonance ability of pi (π) bonds, however, some other chemical elements or compounds can bind with or between carbon rings to initiate new PAHs, for instance, Dibenzothiophene has a sulfur.¹⁴ Several thousands of PAH derivatives exist on the earth, according to their structures and molecular weights PAHs can be classified into low molecular weight (LMW: <202 g/mol, 2-3 fused benzene rings), middle molecular weight (MMW: <228 g/mol, 4 rings), and higher molecular weight (HMW: >228 g/mol, >4 rings).¹⁷ The physicochemical properties of PAHs differ from one to another based on their molecular weight, nevertheless, almost all PAHs form crystals, and a few are soluble in water, while most of them are soluble in organic solvents like acetone or ethanol.

Fluorene is a white crystal organic compound obtained from coal tar that emits an aromatic smell like naphthalene. It is flammable and has a violet fluorescence. It is normally insoluble in water, but a small amount (1.69 mg/L) is soluble at 25 °C. It is widely used in dyes, polymers, electronic devices, sensors, and photochromic materials, and as a talented blue emitter for organic light-emitting diodes.¹⁸

Toxicity of Fluorene and other PAHs
Fluorene is one of the highly toxic polycyclic aromatic hydrocarbons, broadly scattered in the contaminated water and dry land ecosystems. Its toxicity affects plants, animals, algae, fungi, and even some bacteria; human beings are the most vulnerable victims of it.¹⁹ Hsieh et al. discovered the reproductive, developmental toxicity and immunotoxicity of fluorene and other PAHs through in silico, in vitro, and in vivo methods. They found that polycyclic aromatic compounds (PACs) like fluorene could have diverse toxicity profiles, for instance, genotoxicity or carcinogenicity (in silico toxicity), xenobiotic homeostasis and stress response (in vitro activity), and enriched toxicity in conjunction with the availability of carcinogenicity (in vivo activity).²⁰

The terminal differentiation of mouse embryonic stem cells (mESCs), and embryonic bodies (EBs) was affected by Fluorene-9-bisphenol (BHPF), a derivative of fluorene. The study revealed that BHPF exposure led to loss of self-renewal and pluripotency in mESCs by increasing the expression of the inflammatory gene IL6. Moreover, BHPF altered the terminal differentiation pathway, controlling the expression of 16 genes that related to different cell types, for example, cardiomyocytes, keratinocyte epithelium, lymphatic endothelium, macrophages, monocytes, nephrons proximal tubule cells, neurons, pancreatic beta cells, retinal ganglion cells, and T-cells.²¹

Jia et al. demonstrated that fluorene had strong antiestrogenic adverse effects on female development. They conducted some tests on CD-1 mice and found endocrine-disrupting properties in adolescent mice by multiple toxicological bioassays.²² Benzo[b]fluoranthene (B[b]F), a derivative of fluorene, synthesized glutathione (GSH) in HepG2 cells (liver cells). GSH levels were increased up to 3-fold by B[b]F than control levels, and constant cytotoxicity IC₅₀ > 100 µM after 24, 48, and 72 hours.²³

Fluorene-9-bisphenol also had a toxic effect on freshwater algae, Chlorella vulgaris, which was sensitive to BHPF at a concentration of >0.1 mg/L, and lipid peroxidation was remarkably increased. Besides, the oxidative stress caused by BHPF,  the activities of superoxide dismutase (SOD) were notably declined in algal cells by >0.5 mg/L of BHPF.²⁴

Polycyclic aromatic hydrocarbon contamination is widespread, particularly in countries involved in oil production and processing. Saudi Arabia is one of the petroleum-producing countries, contaminated with several PAHs. Al-Daghri et al. reported that they found at least 6 PAHs, ranging between 54.5% and 90.9% positive in the blood serum of some experienced teenagers (195 children of 17 years and below from 11 locations in Saudi Arabia were examined).²⁵ Another research disclosed that 16 different PAHs were determined from 50 locations in Riyadh City. The concentrations of PAHs in road dust were from 0.01-126 ng/g. Children at high risk for asthma suffered more from PAH exposure dose by ingestion and dermal absorption than adults in the city.²⁶

The abundant presence and toxicity of fluorene and other PAHs in our environment remind us to mitigate them through a harmless and cost-effective process like biodegradation. Numerous studies have identified several bacteria, archaea, fungi, and algae, and their co-cultures, for PAH bioremediation. However, the key procedure of the degradation is poorly understood (Table 1).

Table (1):
Toxicity of PAHs on human health and other species

The biodegradation of PAHs by microorganisms
Microorganisms, mainly bacteria and fungi can adapt themselves to various environmental conditions. They can tolerate many toxic molecules by changing their adaptation efficiency and gaining essential exogenous DNA from the environment. Mohapatra and Phale discovered several species of Proteobacteria, Firmicutes, and Actinobacteria from soil flora, which degrade several polycyclic aromatic hydrocarbons.³³

Another group of researchers reported that Pseudomonas aeruginosa san ai degraded 96%, 50%, and 41% of 20 mg/L of fluorene, phenanthrene, and pyrene respectively, after seven days.³⁴ Desta et al. pointed out that SMT-1 Pseudomonas sp. had a fluorene-degrading 4921-dioxygenase gene confirmed by primer-specific PCR. The 4921-dioxygenase enzyme exhibited the optimum activity at pH 7.5 and 25 °C in Tris-HCl buffer after 1 minute, as indicated by the reaction velocity.¹⁹ Some species of Nocardia showed potential bioremediation on polycyclic aromatic hydrocarbons, phenol, and sodium sulfate; among them, N. farcinica was the most prevalent contributor in PAHs biodegradation.³⁵

A marine fungus named Aspergillus sydowii BOBA1 was studied to degrade spent engine oil and PAHs. The fungus carried several genes such as dioxygenase, decarboxylase, hydrolase, reductase, and peroxidase which were integrated into PAHs and xenobiotic metabolism.³⁶ Another filamentous fungus, Mucor irregularis (strain bpo1), had degraded fluorene efficiently through the Box–Behnken Design (BBD) process with optimum parameters (pH-7, temperature-32.5 °C, substrate concentration 100 mg/L, and dry weight 2 g), and resulted in 81.50% fluorene degradation on 5^th day.³⁷

Struszczyk-wita et al. described that creosote, composed of aromatic hydrocarbons, is normally an undegradable compound. However, Bjerkandera adusta DSM-3375 mycelium contained Mold cells that obtained enzyme was used in the bioremediation of soil contaminated with creosote (2% w/w). The B. adusta degraded 35% of creosote and almost 73%, 79%, and 72% of fluoranthene, pyrene, and fluorene, respectively, after 15 weeks (Table 2).³⁸

Table (2):
Bacterial and fungal species for biodegradation of PAHs

Microbial culture for PAHs biodegradation
According to previous research, bacteria and fungi were collected from contaminated soil, wastewater, sediments, and marine water, and sponges. It seemed that the microbes grown in PAH-contaminated environments can degrade PAHs. The studies targeted to find out the most efficient species of microbes to degrade PAHs. However, the strains of microbes were developed by repeated subcultures on a suitable medium and abiotic parameter or stress (pH, temperature, salt concentration, and metal or compound tolerance). In most cases, we noticed that microbes were cultured in Mineral Salt Medium (MSM) with different PAH doses, moreover, other media like marine agar, nutrient broth, etc. were also used.^44,45 To detect the PAH metabolism by microorganisms, sophisticated chromatographic techniques like High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Thin Layer Chromatography (TLC), Mass Spectrophotometry (MS), and Fourier-Transform Infrared Spectroscopy (FTIS) were used (Table 3), and to execute these experiments some essential chemicals like Anhydrous Na₂SO₄, methanol, n-hexane, and nitrogen gas were also used.^14,19,45

Table (3):
Characteristics of biodegrading microbial species

General pathways of PAH degradation
The mechanism of PAH degradation needs several catabolic reactions in the presence of specific enzymes (Table 4). Some bacteria such as Pseudomonas sp., Rhodococcus sp., Mycobacterium sp., and Staphylococcus sp., for example, start PAH catabolism with ring hydroxylation and oxygenation to activate benzene rings.⁴⁷ The hydroxylated and dihydrodiol intermediates undergo dehydrogenation and yield the central intermediates catechols or protocatechuates. Enzyme, dioxygenase performs an ortho and a meta-cleavage of catechols to muconic acids by completing the tricarboxylic acid (TCA) cycle and finally degrades the PAH into the end-product as carbon dioxide (CO₂).⁴⁸ On the other hand, most fungi cannot use PAHs as their main carbon sources but can co-metabolize PAHs to other products. However, ligninolytic fungi like Aspergillus sp., Cladosporium sp., Cunninghamella sp., and Penicillium sp., have PAHs catabolic ability (2-5 rings). They primarily oxidize PAH by the cytochrome P450 and oxygen to form unstable arene oxide intermediates that are later converted into tarns-dihydrodiols and phenols, and finally to glucosides, glucuronides, xylosides, methoxyls, and sulfates (Figure). Whereas the white-rot fungi produce extracellular ligninolytic enzymes, such as lignin peroxidases, manganese, and laccases that oxidize PAHs in the presence of H₂O₂ to quinones. Finally, the fungi cleave quinones to CO₂.^49,50

Table (4):
The sources and function Enzymes for PAH bioremediation