1.0 INTRODUCTION Compounds with Oxygen, Sulphur, Phosphorus And
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1.0 INTRODUCTION Compounds with oxygen, sulphur, phosphorus and nitrogen substitutions in the aromatic ring are called heterocyclic compounds. The environmental/natural sources of heterocyclic compounds include combustion (coal, biomass, refuse, diesel fuel, tobacco), commercial coal tar and its leachates, fossil fuels, wood-preserving wastes, municipal solid wastes, bleaching of paper pulp, volcanic activities and forest fires, cigarette smoke and automobile emission (Bressler and Fedorak, 2000; Hiraishi, 2003; Lobastova et al., 2004). Anthropogenic heterocyclic aromatic compounds are used as industrial solvents, dyes, explosives, pharmaceuticals and pesticides (Hiraishi, 2003). Interest in the biodegradation of heterocyclic compounds is prompted by the ubiquitous distribution of these pollutants in diverse environment, their persistence and their toxic, mutagenic and carcinogenic properties (Jensen et al., 2003; Lobastova et al., 2004). The presence of nitrogen and sulphur containing heterocycles in crude oil is a serious environmental problem. Combustion of crude oil during the refining process results in their oxidation to NOx and SOx, which react with water in the air, forming acid rain (Kirimura et al., 1999). Hydrocarbon contamination reduces biodiversity of the soil microbiota (Atlas et al., 1991) and imposes selective pressure that only favour limited number of fast-growing hydrocarbon degraders (Bundy et al., 2002). The use of culture-independent techniques premised on 16S rRNA analyses to study microbial diversity in hydrocarbon- contaminated soils allows quantitative assessment of soil microbial community, reveals the presence of viable but not yet culturable hydrocarbon degrading bacteria and eliminate cultivation bias as the DNA template used is extracted directly from the contaminated soils (Kubicek et al., 2003; Jain et al., 2005). 1 1.1 BACKGROUND OF STUDY Petroleum crude oil, a major source of energy in the world is arguably the world‟s most compositionally complex organic mixture in terms of chemically distinct constituents. Petroleum crude oil production and operations such as oil exploration, exploitation, transportation, and distribution with its attendant oil spillage, seepages from oil tankers, release of effluents, and offshore drilling activities is adversely affecting the ecosystems (terrestrial and aquatic) especially in oil producing countries including Nigeria (Atlas, 1991; Cerniglia, 1992). This problem further exacerbate with indiscriminate disposal of spent and used oils at automobile and mechanic workshops, unprecedented use of generators and the release of exhaust into the environment, oil pipelines sabotage and vandalization by oil thieves and restive communities, particularly in the Niger Delta region of Nigeria. Crude oil is a complex mixture consisting of aliphatics, alicyclics, aromatic and heteroaromatic compounds (Benedik et al., 1998). Nitrogen-containing compounds in crude oil are divided into „non-basic‟ (indoles and pyrroles) and the „basic‟ (pyridines and quinolines) molecules (Nojiri and Omori, 2007). The total nitrogen content in crude oil averages about 0.3% of which the non-basic compounds constitute about 70-75% (Benedik et al., 1998). Carbazole is a non-basic nitrogen heterocycle found in coal-tar creosote, fossil fuel and wood-preserving wastes and is recalcitrant, mutagenic and toxic (Nojiri and Omori, 2002). Their combustion in petroleum leads to the formation of nitrogen oxides, emission of which lead to acid rain (Kirimura et al., 1999). Interest in the study of bacterial degradation of carbazole is spurred partly because of the ubiquitous nature of the pollutant and its mutagenic and toxic activities and also because carbazole is a structural analogue of dioxins and carbazole degrading enzymes can partly function as dioxin-degrading enzymes (Nojiri and Omori, 2007). The metabolic propensity of wide array of microorganisms to utilize hydrocarbons such as carbazole as source of carbon and energy necessitate the development of bioremediation techniques 2 where catabolic genes and enzymes of microorganisms are employed for biodegradation of hydrocarbon pollutants. Three major degradation pathways have been reported for carbazole, lateral dioxygenation at carbon positions 3 and 4; monohydroxylation at carbon positions 1, 2 and 3; and angular dioxygenation at carbon positions 1 and 9a (Ouchiyama et al., 1993; Grifoll et al., 1995; Lobastova et al., 2004). In contrast to other two carbazole degradation pathways that lead to production of dead end metabolites and hydroxylated carbazole intermediates, angular dioxygenation lead to complete carbazole mineralization, however, very few bacteria genera with this ability have been reported (Nojiri and Omori, 2002; Nojiri and Omori, 2007). In hydrocarbon-contaminated soils, microbial community structures are influenced by a number of factors such as soil type, concentration and bioavailability of the contaminants, nutrient contents, temperature, oxygen content and pH (Margesin and Schinner, 2001; Greer et al., 2010). Soil microbial diversity assessment using culture-dependent method is fast, cost effective and provides information on the active, heterotrophic component of the population. However, culturable cells represents <1% of the total microbial community in an environment (Hugenholtz, 2002). The use of culture independent method to decipher the microbial diversity of hydrocarbon-contaminated environments provides clues about the type of bacteria able to adapt to such habitats. It also reveal the presence of novel bacteria genera hitherto not reported to contribute to natural attenuation of hydrocarbon pollutants and capture viable but not culturable (VBNC) bacterial groups, which though not captured by cultural methods, play important roles in the decontamination process (Jain et al., 2005; Spiegelman et al., 2005; Rastogi and Sani, 2011). 3 Figure 1.1: Common nitrogen heteroaromatic compounds found in fossil fuels. The nonbasic species are underlined (Benedik et al., 1998). 1.2 STATEMENT OF PROBLEM Carbazole is a nitrogen heterocycle released into the environment with fossil fuels or their products such as creosote. It is recalcitrant, mutagenic and toxic (Nojiri and Omori, 2002). It is a model compound for the study of nitrogen-containing heterocyclic aromatic hydrocarbons. Hazardous carbazole derivatives such as N-methylcarbazole and 7-H- dibenzo[c,g]carbazole found in cigarette smoke and automobile emissions are genotoxic and carcinogenic and are categorized as “IARC Group 2B” carcinogens. In spite of plethora of information on degraders of carbazole from temperate environment, there is virtually no report from African continent as a whole and Nigeria in 4 particular on the ability of autochthonous microorganisms to degrade carbazole. This is important as clean-up strategies are environment specific and local microorganisms with ability to degrade carbazole must be isolated and used as seed for bioremediation. Owing to its relatively low mobility and weak sorption to soil and aquifer materials, there is ubiquitous distribution of carbazole in groundwater, aquatic sediments, seawater, industrial and urban air, subsoil region and bioaccumulation in marine organisms. This poses serious health and environmental concern due to the mutagenic and toxic properties of carbazole. Carbazole degraders from different genera of bacteria have been isolated from diverse climes. Due to the physico-chemical properties of the Nigerian environment, which is quite different from the reported climes, it is imperative to isolate carbazole degraders that can cleave carbazole angularly resulting in its mineralization from our own environment and compare their metabolic potentials with carbazole degraders from temperate environments. Soil contamination with petroleum hydrocarbons imposes significant changes to the structure and functions of the indigenous microflora and favours, over time, the dominance of fast hydrocarbon degraders that can utilize the pollutant as source of carbon and energy. Analysis of microbial diversity of these oil-impacted environments using culture-dependent method does not give a fair representation of the microbial richness of the site and could results in misleading assumptions and conclusions. However, culture-independent method premised on 16S rRNA analyses allows quantitative assessment of soil microbial community, reveals the presence of viable but non-culturable hydrocarbon degrading bacteria and overcome the limitation imposed by cultivation. 1.3 SIGNIFICANCE OF STUDY Carbazole degraders have potential use for bioremediation of carbazole and dioxin- polluted sites. Because of the formation of carbazole through combustion of organic 5 materials in air and as a constituent of crude oil, pollution of the environment is a common occurrence. Thus, knowledge of its complete removal through microbial degradation is significant. Carbazole is a structural analogue of dioxins and carbazole degrading enzymes can partly function as dioxin-degrading enzymes (Nojiri and Omori, 2007). Carbazole degraders obtained in this study can be employed to decontaminate and bioremediate environments polluted with carbazole and other dioxin-related compounds. This is significant due to ubiquitous nature of dioxins and their mutagenic and toxic activities. The use of molecular techniques for detection and identification of novel degrading strains that cannot be captured using classical cultural methods is of paramount importance. Strains