Rapid industrialization and urbanization have resulted in the generation of large quantities of aqueous effluents, many of which contain high levels of toxic heavy metals and xenobiotics that pollute groundwater and soil of affected farmlands. Heavy metals are not biodegradable and as such not removed from the soil but rather accumulate and persist in soil reservoirs, consequently entering the food chain and exerting toxic effects on living organisms. Copper and lead which exert toxic effects even at very low concentrations are common constituents of the Nigerian crude oil and consequently are found in its effluent. Research has shown that removal/recovery of these metals (through bioaccumulation/biosorption by bacteria) is an attractive alternative to traditional physicochemical techniques. Microorganisms tolerant to metals are often isolated from areas of high metal loading, suggesting that metal tolerance or resistance is an adaptive response to excessive metal exposure. In this study, crude oil effluent was analyzed for copper and lead contents and both metals were found to show concentrations higher than the U.S Environmental Protection Agency (EPA) and the Compendium of Environmental Laws for African Countries (CELAC) recommended environmentally accepted standards. Microorganisms were isolated from the effluent and from the effluent-contaminated soil from the site. The largest/most successful colony was subsequently characterized. Through morphological and biochemical tests, it was identified as Bacillus subtilis. Four test groups of mineral salt media containing copper only (Group A), lead only (group B), copper + lead (Group C) and no lead or copper (Group D, control) set at different pHs of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 for each group were used. The organism was standardized and found to contain 6.0 x 108 Bacillus subtilis cells per ml of suspension. Five ml of the organism was inoculated into each experimental medium. The absorbance change (turbidity) of the mineral salt media were measured at 540nm on the 10th, 17th and the 24th days - the evaluation criteria for microorganism growth and adaptation in the used media. The experimental media showing the highest growths for each group was analyzed for residual copper and lead. Also, the bacterial biomass from these media were harvested and analyzed for recovered lead and copper. Results showed that Group D had the highest growth, followed by Group B, Group A and lastly Group C. The organism grew most at pH 7.5 - 8.0. The experimental media that showed the highest growths for each group, when analyzed for residual copper and lead had no trace of metals, implying complete biosorption by the B.subtilis. B.subtilis is therefore recommended for removal of lead at pH 7.5 - 8.0 in crude oil pollution.

Toxic heavy metals in air, soil and water are growing threats to humanity. A number of these heavy metal compounds represent an ongoing eco-toxicological threat (Sag, 2000). Heavy metals have a tendency to bioaccumulate and end up as permanent additions to the environment. For many of the heavy metals, the amounts contributed globally from anthropogenic sources, such as industrial wastes, now exceed those from natural sources (Deans and Dixon, 1992). The disposal of effluent on land has become a regular practice for some industries leading to subsequent pollution of groundwater and farmlands. Copper (Cu), lead (Pb), mercury (Hg), cadmium (Cd) are common heavy metal pollutants at sites in which industrial waste effluents are discharged. One good example of such effluents includes crude oil waste effluent. Crude oil effluent is the water that is mixed with crude oil when it is mined or during refining/processing. Crude oil effluent have been associated with increased concentrations of some heavy metals. Disposal of such effluents over time in the environment may lead to eco-toxicological hazards. This is common where mining and manufacturing operations take place, particularly those established a number of years ago. Copper and lead, which are common constituents of Nigerian crude oil are known to exert toxic effects at low concentrations (Pandey et al 2007). However at very low concentrations, some of these heavy metals such as copper, zinc and boron have been found to be essential in all higher plants and animals. In slightly elevated concentrations, these metals may be taken up by plants and concentrated in certain parts of the plant such as the leaf, stem, and root. When these are consumed by animals, they are further concentrated in them resulting in biomagnification. Consumption of the animal parts in which these metals are concentrated may lead to their significant concentration in human beings (Alloway, 1995) which could be toxic. Consequently, there is a pressing need to remove/recover these. 

1.2      COPPER 
Copper is the first element of group 1B of the periodic table and displays four oxidation states: Cu(0), Cu(I), Cu(II) and Cu(III). Cu(II) or cupric ion is the most important oxidation state of copper generally encountered in water (Cotton and Wilkinson, 1988). Copper does not break down in the environment and when introduced into the environment as Cu2+, it typically binds to inorganic and organic materials contained within water, soil and sediments with varying affinities. As in water, the binding affinities of Cu(II) with inorganic and organic matter in sediments and soil is dependent on pH, the oxidation-reduction potential in the local environment and the presence of competing metal ions and inorganic anions.

1.2.1    Biological Role of Copper
Copper is a trace metal which is essential in all higher plants and animals. A wide range of enzymes exploit copper chemistry to catalyze reactions which include cytochrome oxidase, superoxide dismutase, dopamine ß-hydroxylase, lysyl oxidase and ceruloplasmin. Thus copper ions are essential in cellular respiration, antioxidant defence, neurotransmitter function, connective tissue biosynthesis and cellular iron metabolism. Cytosolic superoxide dismutase (SOD) is an important copper metalloenzyme that protects lipid cell membrane structures from oxidation. This enzyme catalyzes the transformation of free oxygen radicals into hydrogen peroxide, which is later converted to water and molecular oxygen by a cytosolic catalase. In addition to enzymatic roles, proteins take advantage of the redox nature of copper to achieve facile electron transfer reactions and to bind reactive intermediates and avoid their reactivity. Nevertheless, the chemical properties that make copper biologically useful are also potentially toxic.

1.2.2   Environmentally Acceptable Limits for Copper
         The U.S Environmental Protection Agency (EPA) requires that levels of copper in drinking water be less than 1.3mg/l.

         The U.S Maximum Contaminant Level Goal (MCLG) for copper in water is 1.3mg/l.

         Permissible limit for copper in waste water is less than 1mg/l given by Compendium of Environmental Laws for African Countries (CELAC).

         The U.S Department of Agriculture has set the recommended daily allowance for copper at 900µg of copper per day for people above the age of 8.

1.2.3   Molecular Mechanism of Copper Toxicity 
At very low concentrations (1- 1.5µg), copper improves the efficiency of Photosystem II (PS II) apparatus. On the other hand, high concentrations could be toxic, hence the extensive use of copper as fungicide in agricultural practice. Results from other researchers suggest that copper inhibits either the donor or the acceptor side in the PS II. Copper ions oxidize directly the Cyt b559 LP (Low Potential) and HP (High Potential) forms. Using Mossbaner spectroscopy, Cu2+ was shown to influence the valence and spin states of the non-haem iron and the haem iron of Cyt b559. Copper ions oxidized the heme iron into a high spin Fe3+state and enhance the covalency of the bound non-haem iron, keeping the iron in a low spin ferrous state. The new valence and spin states of the non-haem and haem iron reveal the important roles of the quinine-iron complex and cytochrome b559 as regulatory components of the electron transport in PS II.....

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