**2. Objectives**


#### **3. Literature review**

#### **3.1 Aromatic hydrocarbon**

Aromatic compounds are organic molecules that contains one or more aromatic rings. The three major categories of aromatic compounds are polycyclic aromatic hydrocarbons (PAHs), heterocyclic, and substituted aromatics. Polycyclic aromatic hydrocarbon (PAH) has carcinogenic and mutagenic properties that are connected to some chemical properties such as dipole moment, electrophilic potency, intramolecular and subcellular binding, electronegativity or L- and K- region reactivity, hydrophobicity and others.

Whereas heterocyclic hydrocarbon which is also known as heterocycles are compound that contains at least one atom other than carbon and some or all the atoms are joined in rings. Heterocyclic hydrocarbon has high boiling and melting point, low water solubility and low vapor pressure. These characteristics proves that heterocyclic hydrocarbon can stay in the environment for a long period of time [10]. **Figure 1** shows the molecular structure of aromatic heterocyclic organic compound known as carbazole.

Heterocyclic hydrocarbon is by products of incomplete combustion of organic materials such as coal, petroleum, tar and gas [11]. According to Bamforth and Singleton [12], heterocyclic hydrocarbon can be found easily in groundwater, soil and sediments and also the atmosphere. Some areas such San Diego Bay, California,

**Figure 1.** *Molecular structure of carbazole.*

Central Pacific Ocean, intertidal sediments, gas works site soils, sewage sludgecontaminated soils, aquifers and groundwater and atmospheric air pollution have shown presence of high concentration of heterocyclic hydrocarbon. Heterocyclic hydrocarbon also originates from two major sources which are natural and anthropogenic sources. Heterocyclic hydrocarbon does not only occur during partial combustion of organic material but also occur with fossil fuels such as petroleum and coal. Carbazole and dibenzothiophene which are heterocyclic compounds often exist together with PAH and some other aromatic compounds as they are components of crude oil, shale oil and creosote [13].

#### **3.2 Health and environmental concerns**

Heterocyclic hydrocarbon is a major concern in urban and industrial areas as some of them have been classified as carcinogenic, mutagenic and teratogenic [14, 15]. The properties of heterocyclic hydrocarbon are that they are relatively insoluble in water and they are also highly lipophilic. Another found property is that this compound ban be degraded by some bacteria in soil [14, 15]. Whereas in the atmosphere, heterocyclic hydrocarbon can react with different kind of pollutants such as ozone, nitrogen oxides and sulfur dioxide.

Studies have shown that heterocyclic hydrocarbon are strongly bio concentrated or metabolized [16]. Human are exposed to most PAH through their eating diet that consist of marine lives. For heterocyclic hydrocarbon such as carbazole, tobacco smoking and breathing from polluted air are the routes of exposure [17, 18]. Carbazole has also been classified as "benign tumorigen" by Nojiri and Omori [19]. Moreover, heterocyclic hydrocarbons are potential carcinogens that can produce tumors. Benzo(a)pyrene, a common heterocyclic hydrocarbon, is shown to cause lung and skin cancer in laboratory animals. When ingested, heterocyclic hydrocarbons are absorbed very fast into the gastrointestinal tract. This is because of their high lipid solubility [12, 20]. In general, the more number of benzene rings present, the more harmful the heterocyclic hydrocarbon would be.

#### **3.3 Bioremediation**

Bioremediation is defined as a process where microorganisms or their enzymes are used to degrade contaminants to its original condition. Microbial degradation is natural mechanism to demolish hydrocarbon pollutants (and crude oil) from the environment [21, 22]. Research has shown that, in order to induce the ability of certain microbes to degrade or transform toxic or pollutants, bioremediation and biotransformation have been the most successful method chosen [23, 24]. The main objective or aim in bioremediation is to destroy or remove contaminants by using microbes or degrading bacteria and stimulate them with nutrients and other chemicals to aid the degradation process. The presence of suitable microbes and the ideal environmental conditions such as the right amount of nutrient, oxygen and suitable pH and temperature for microbes to grow are some of the factors that also lead to bioremediation [25].

In this process of bioremediation, different microbes will act upon in parallel or sequence in order to degrade the compound. Two approaches are widely used which is in-situ where contaminants are treated at the site and ex-situ approach where contaminants are removed to be treated elsewhere. The ability of variety of microbes to degrade different kinds of pollutants proves that bioremediation is a crucial and important technology that can be used in different conditions [26]. Previous study by Alvarez et al. [27] stated that bioremediation has been proven to be an environmental-friendly technique as the degrading agents are microbes that can be easily decomposed. Therefore, this method appears to fulfill the characteristics of the demanding, growing industry as it is sustainable, easy to implement and cost effective. **Table 1** shows some carbazole degrading bacteria that has been reported in previous literature that are able to contribute to bioremediation.

### **3.4 Degradation of heterocyclic hydrocarbon**

According to Surajit Das from National Institute of Technology, Rourkela Odisha, India, the toxicity, mutagenic properties as well as high carcinogen of PAH in nature has cause a great environmental concern to scientist. Therefore, researchers have collected many marine bacteria that has potential in bioremediation. A research done by Latha and Laithakumari [28] has also shown on how the efficiency of degradation can be increased when a catabolic plasmid from *Pseudomonas putida* that has genotype of hydrocarbon degradation is inserted in a marine bacterium. Some examples of marine bacteria that has been taken and used for bioremediation are *Neptunomonas naphthovorans, Lutibacterium anuloederans*, and *Cycloclasticus spirillensus* [23, 29].

According to research by Nojiri and Omori [19], the structural analog of dioxin and some carbazole-degrading enzymes that plays the same role as dioxin degrading enzymes are the factors that made carbazole known as model compound for bioremediation and led to more study on bacterial degradation of carbazole. There are three main degradation pathways of carbazole that has been identified which are the angular deoxygenation, lateral deoxygenation and hydroxylation pathway of carbazole that can be catalyzed with different enzymes. **Figure 2** shows lateral deoxygenation carbazole at C3 and C4 [9, 30–32].

The common reaction by carbazole degrading bacteria would be hydroxylation as identified by Lobastova et al. [30], who studied on hydroxylation of Aspergillus *flavus* VKM F-1024 by carbazole, where hydroxycarbazole were produced as major product. Nojiri et al. [33] explained on the angular deoxygenation where oxidation


**Table 1.**

*Carbazole degrading bacteria, adapted from Salam et al. [9].*

**Figure 2.**

*Lateral deoxygenation of carbazole at C3 and C4.*

occur at the ring-fused position of carbazole which is induced by a multicomponent enzyme known as carbazole 1,9a- dioxygenase (CARDO) in an angular position. Based on literature, carbazole degradation pathway has been well reported and many types of carbazole degrading bacteria has been identified successfully.
