ISOLATION AND CHARACTERIZATION OF A MICROORGANISM IN CRUDE OIL EFFLUENT AND ITS UTILITY IN METAL ION REMOVAL/RECOVERY

ABSTRACT

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 cellsper 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.

CHAPTER ONE

1.1       INTRODUCTION

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

heavy metals (copper and lead) from industrial effluents (and possibly recycled) before disposal into the environment.

Fig.1   Biogeochemical cycle of metal pollutants in the environment (Adapted from

Hutchinson and Meema, 1987)

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.

1.2.4    Copper Toxicity in Microorganisms

One major mechanism of copper toxicity towards microorganisms is disruption of plasma membrane integrity. Metal-microbe interactions have received considerable attention in recent years. Interest has arisen because of the biotechnological potential of microorganisms for metal removal and/or recovery, the possible transfer of accumulated metals to higher organisms in food chains, and the toxicity of metals towards microbial metabolism  and  growth.  Metal  toxicity towards  microorganisms  is  of  environmental concern because of possible inhibition of essential microbe-assisted processes (biogeochemical cycling). Toxic effects are generally related to the strong coordinating

abilities of heavy conformational modification of cellular macromolecules, displacement of essential ions and disruption of cellular and organelle membrane integrity. At toxic concentrations, copper interacts with cellular nucleic acids and enzyme active sites, although one principal initial site of copper action is considered to be at the plasma membrane. Thus exposure of microorganisms such as fungi and yeasts to elevated Cu concentrations can lead to a rapid decline in membrane integrity which is generally

manifested as leakage of mobile cellular solutes (K+) and cell death. Extensive metal-

induced disruption of membrane integrity inevitably leads to loss of cell viability. Even at relatively small alterations in the physical properties of biological membranes, it can elicit marked changes in the activities of many essential membrane-dependent functions, including transport protein activity, phagocytosis and ion permeability.  The physical properties of a membrane are largely determined by its lipid composition and one important factor is the degree of fatty acid unsaturation. Microbial membrane fatty acid composition is highly variable and is influenced by both environmental and intrinsic factors. Enrichment of S. cerevisiae with polyunsaturated fatty acids such as linoleate and linolenate  was  found  to  markedly  enhance  the  organism’s  susceptibility  to  copper toxicity.

1.2.5    Copper Toxicity in Higher Organisms

Copper toxicity would be the consequence, at least in part, of Fenton or Haber-Weiss reaction in which copper ions catalyze the formation of ROS through its binding to thiol clusters occurring in proteins. Cyt P450 and GSH transferase activities are inhibited by Cu2+ at µM concentrations. Aquatic organisms are more sensitive to copper toxicity than mammals. Copper toxicity in mammals includes a wide range of effects such as liver cirrhosis, necrosis in kidneys and brains, gastrointestinal distress, lesions, low blood pressure and foetal mortality. Observed effect of high copper concentrations on marine creatures  includes  damage  on  gills,  liver,  kidneys  and  the  nervous  system.  Copper

inhibits the enzyme dihydrophil hydratase, an enzyme involved in haemopoiesis. Acute copper poisoning is rare usually from accidental spills or copper nitrate or sulphate. Symptoms include nausea, vomiting, abdominal pain and jaundice.  Also at high copper concentrations in water, copper stain clothes and other items washed in them.

1.3       LEAD

Lead is a heavy low melting, bluish-gray metal naturally in the earth’s crust. However, it is rarely found naturally as metal. It is usually found combined with two or more other elements to form lead compounds. Lead has been used almost since the beginning of civilization. Lead is one of a limited class of elements that can be described as purely toxic. Many other elements including heavy metals such as chromium, manganese, molybdenum, nickel and selenium although toxic at high levels, are actually required as nutrients at lower levels. This is clearly not the case for lead.  In  many decades  of research, no nutritional value or positive biological effect has been shown to result from lead exposure. Neither has any case of lead deficiency ever been noted in the medical literature. Once lead falls onto soil, it sticks strongly to soil particles and remains in the upper layers of the soil. Its extensive use (industrial, petrol/diesel, paints etc.) has resulted in environmental contamination worldwide. Infants and children below the age of 6, as well as pregnant women, are at greatest risk of lead toxicity.

1.3.1   Environmentally Accepted Limits for Lead

The U.S Environmental Protection Agency (EPA) requires that levels of lead in drinking water be less than 0.015mg/l and 0.1mg/l for waste waters (Deans and Dixon, 1992).

1.3.2    Lead Absorption

Lead is absorbed through three major routes:

•    Skin: Lead is absorbed in the skin as alkyl lead compounds, because of lipid solubility (mostly as methyl and  tetraethyl lead).

•   Inhalation: Up to 90% of lead is inhaled depending on particle size.

•   Gastro-Intestinal system (GI):  lead absorption through the GI in adults is 5 to 10%

and 40% in children.

1.3.3     Distribution

Lead is initially carried in red blood cells and distributed to soft tissues (kidney and liver);  it is redistributed to bone, teeth and hair mostly as a phosphate salt. Rates of absorption

and distribution are greatly influenced by dietary intake and body stores of phosphate, calcium and iron relative to lead. Lead distribution in the body is affected in the following ways:

•   High PO42- leads to  Pb2+ storage in bone.

•   High Vitamin D leads to  Pb2+ storage in soft tissues.

•   Low PO42-  leads to the sequestering of Pb2+ in soft tissues.

•   High Ca2+ leads to the sequestering of Pb2+ in soft tissues.

•   Note: Half life of lead ion in blood is 30-60 days and in bone 20-30 years.

1.3.4    Sources of exposure

Sources of lead exposure includes:

•    Paint, pottery, contaminated plant and animal parts which enter the body through the GI system.

•   Metal fumes which enters the body through inhalation.

•   Tetraethyl lead in gasoline which enters the body through the skin.

1.3.5 Mechanisms of Lead Toxicity

Lead exerts toxicity through two major mechanisms;

•    Inhibition of haem biosynthesis (see figure 2). Haem is the essential structural component of haemoglobin, myoglobin and cytochromes.

•    Binds to sulphydryl groups (-SH groups) of proteins leading to the generation of Reactive Oxygen Species (ROS). These affect mostly the Central Nervous System (CNS) and the Renal System.

Fig. 2  Pathway of Haem Biosynthesis, showing points of lead toxicity

(Tiruppathi, 2011) Lead presents a more serious environmental health hazard than copper. The major health effects of lead manifest in three systems:

  1.3.6.   The Haematological System

Lead interferes with the first two steps and the final steps of haem synthesis catalysed by ALA-D synthetase, ALA-D dehydratase, coproporphyrinogen oxidase and haem synthatase (ferochelatase), respectively.

  1.3.7    The Central Nervous System(CNS)

At moderate exposure to lead, there is impairment of the CNS system especially in children, often reflected by inattention, cognitive difficulties and fine motor dysfunction. Heavy exposures may lead to encephalopathy. This is as a result of oxidative stress caused by ROS (Reactive Oxygen Species). Treatment of experimental rats with lead acetate shows that lead can attack synaptic neurotransmission in two ways: by depressing the Ca-KCl-evoked release of ϒ- amino butyric acid (GABA), dopamine and histidine and by a selective stimulation of a spontaneous release (independent of depolarization conditions) of GABA and dopamine but not histidine.

  1.3.8    The Renal System

In the renal system and at high concentrations, lead binds to thiol clusters of proteins thus catalyzing the formation of ROS. These in turn causes functional impairment of the tubular region characterized by mild amino aciduria, glucosuria and hyperphosphaturia. Long term exposure may result in  irreversible nephropathy.

The last stage of haem synthesis, which is the incorporation of iron into protoporphyrin catalysed  by haem  synthetase  (ferochelatase)  takes  place  in  the  inner  matrix  of  the erythroid cell mitochondria in the bone marrow. Lead inhibits the transmitochondrial transfer of iron (Labreche and Pan,1982). This effect results in the accumulation of protoporphyrin in the mitochondria, its incorporation in the globin molecule in place of haem and the subsequent presence of elevated levels of free erythrocyte protoporphyrin (FEP) in the peripheral blood.

1.3.9  Symptoms

(1) Acute exposure leads to nausea, vomiting, thirst, diarrhea/constipation, abdominal pain, haemoglobinuria, oliguria leading to hypovolemic shock

(2) Chronic exposure leads to the following symptoms in the various systems

•  GI: lead colic (nausea, vomiting, abdominal pain)

•  Neuro-Muscular Joints: lead palsy (fatigue, wrist-drop)

•  CNS: lead encephalopathy (headache, vertigo, irritation, insomnia, CNS edema)

1.4 THE USE OF CHEMICALS IN HEAVY METAL REMOVAL

In order to meet the Federal and State guidelines for heavy metal discharge, companies often use chemical precipitation and chelating agents. Example includes TMT (Trimecaptotriazine), Potassium/Sodium thiocarbonate, Sodium dimethyldithiocarbonate (HMP- 2000) etc. Also solvent extraction and ion exchange processes are used.

Problems associated with the use of chemicals in heavy metal removal:

•    Chemical precipitation leads to the production of high toxic metal hydroxide sludge that necessitate highly regulated costly disposal.

•    Ion exchange processes are too expensive due to high cost of synthetic resins.

•    Solvent extraction techniques are not suitable for effluent containing less than 1g/l of targeted heavy metal.

•    The need for large doses of alkaline material to increase and maintain the required pH

range for optimal metal removal.

With these emergent problems associated with the use of chemicals in heavy metal removal, researchers are exploring alternative techniques which will be environmentally friendly, cost effective, more efficient, and highly selective. Thus is primarily the need for this work. Biological methods promise to have high potentials in heavy metal removal through bioremediation processes.

1.5       THE        USE        OF        MICROORGANISMS        IN        HEAVY        METAL REMOVAL/RECOVERY

Bioremediation is an attractive alternative to traditional physicochemical techniques for the remediation of industrial effluents with high heavy metal content before disposal. Of

the   different   biological   methods   bioaccumulation   and   biosorption   have   been demonstrated to possess good potentials to replace conventional chemical methods for the removal of metals (Volesky and Holan, 1995). They appear to have the most potential for environmental biotechnology (Tobin and Roux,1998).

Bioaccumulation is the uptake of toxicants by living cells which is metabolism-dependent i.e. active processes. The toxicant can be transported into the cell and thus accumulate intracellularly, after moving   across the cell membrane and through the cell metabolic cycle (Malik, 2004). Conversely, biosorption can be defined as the passive uptake of toxicants by dead/inactive biological materials. It is due to a number of metabolically- independent processes that essentially take place in the cell wall, where the mechanisms responsible for the pollutant uptake will differ according to the biomass type (Tobin and Roux, 1998; Yee and Fein, 2001).

Biosorbents  for the removal  of metals  mainly come under the following categories: bacteria, fungi, algae, agricultural wastes and other polysaccharide materials. Bacteria may carry determinants of resistance to a number of heavy metals. Bacteria resistance to heavy metals is conferred by specific resistance determinants, which are often, but not always encoded on plasmids or transposons. Potent metal biosorbents under the class of bacteria include genre of Bacillus (Nakajima and Tsuruta, 2004; Tunali et al., 2006), Pseudomonas (Chang et al., 1997; Uslu and Tanyol, 2006) and Streptomyces (Mameri et al., 1999; Selatnia et al., 2004) etc. Many investigators have been able to explain the mechanisms responsible for biosorption which may be one or combination of ion exchange, complexation, coordination, adsorption, electrostatic interaction, chelation and microprecipitation (Veglio and Beolchini, 1997; Volesky and Schiewer, 1999).  The main chemical groups in biomass which are able to partake in biosorption are electronegative groups such as hydroxyl or sulphydryl groups, anionic groups such as carboxyl or phosphate  groups  and   nitrogen-containing  groups  such  as  amino  groups.   Being negatively charged and abundantly available on the bacterial cell wall, these functional groups play important roles in biosoptive processes.

1.5.1    Microorganisms in Metal Absorption

1.5.1.1 Biosorption by fungi

Among microorganisms, fungal biomass offers the advantage of having a high percentage of cell wall material which shows excellent metal binding properties. Polysaccharides, in association with lipids and proteins, represent the main constituent of fungal cell wall. In filamentous fungi outer cell wall layers mainly contain neutral polysaccharides (glucans and mannans),while the inner layers contain more of glucosamines (chitin and chitosan) in amicrofibrillar structure. Ligands within these matrices include carboxylate, amine phosphate, hydroxyl, sulphydryl and other functional groups. Proteins are also found to be          associated    with    metal    binding    (Thakur,    2007).    Rhizopus,    Aspergillus, Streptoverticillum and Saccharomyces are important fungi used for metal biosorption.

1.5.1.2 Biosorption by algae and moss

Photoautotrophs marine algae have bulk availability of their biomass from water bodies. Special polysaccharides present in the algae cell wall contain potential metal ion binding sites. The number and kind of binding sites depend on the chemical composition of the cell wall. In Pheophycean members, algin is present and contributes significantly to metal binding. It has been suggested that the polysaccharides of cell wall could provide amino and carboxyl group as well as the sulphate. The amino, carboxyl groups and the nitrogen and oxygen based moieties could also form coordinated bond with metal ion. Metal ion could  also  be  electrostatically  bonded  to  unprotonated  carboxyl  oxygen,    sulphate covalent bonding between divalent cation and algae cell wall proteins has also been reported (Thakur, 2007). Mechanisms such as entrapment of metal both in the form of insoluble micro deposits in the inter and intra-fibrillar capillaries and paracrystalline regions of polysaccharides and the binding to other biopolymers (RNA, Polyphosphates) can contribute to the metal binding. The photoautotrophs eukaryotic algae cell wall are mainly  cellulosic  and  the  potential  metal  binding  groups  are  carboxylate,  amine, imidazole, phosphate, sulphydryl, sulfate and hydroxyl. These amine and imidazoles are positively charged when protonated and build negatively charged metal complexes. The

amino and carboxyl groups, nitrogen and oxygen of the peptide bonds are also available for coordination bonding with metal ions such as lead (II), copper (II) and chromium (IV).

1.5.2    History of Bacterial Bisorption

Early 1980 witnessed the advent of the discovery and utilization of some microorganisms to accumulate metallic elements. Numerous research reports have been published from toxicological points of view, but these were concerned with the accumulation due to the active metabolism of living cells, the effects of metal on the metabolic activities of the microbial cell and the consequences of accumulation on the food chain (Volesky, 1987). However,  further  research  has  revealed  that  inactive/dead  microbial  biomass  can passively  bind  metal  ions  via  various  physicochemical  mechanisms.  With  this  new finding, research on biosorption became active,with numerous biosorbents of different origins being proposed for the removal of metals/dyes.

Researchers have understood and explained that biosorption depends not only on the type or chemical composition of the biomass, but also on the external physicochemical factors and solution chemistry. Many investigators have been able to explain the mechanisms responsible for biosorption, which may be one or combination of ion exchange, complexation, coordination, adsorption, electrostatic interaction, chelation and microprecipitation (Vegliò and Beolchini, 1997; Volesky and Schiewer, 1999).

1.5.3    Bacterial Structure and Bisorption Mechanism

1.5.3.1 Bacterial structure

Bacteria are a major group of unicellular living organisms belonging to the prokaryotes, which are ubiquitous in soil and water, and as symbionts of other organisms. Bacteria can be found in a wide variety of shapes, which include cocci (such as Streptococcus), rods (such as Bacillus), spiral (such as Rhodospirillum) and filamentous (such as phaerotilus). Eubacteria have a relatively simple cell structure, which lack cell nuclei, but possess cell walls (Salton, 1964). The bacterial cell wall provides structural integrity to the cell, but differs from that of all other organisms due to the presence of peptidoglycan (poly- N- acetylglucosamine and N-acetylmuramic acid), which is located immediately outside of the cytoplasmic membrane (Rogers et al., 1980). Peptidoglycan is responsible for the

rigidity of  the  bacterial  cell  wall,  and  determines  the  cell  shape  (Kolenbrander  and Ensign, 1968). It is also relatively porous and considered as an impermeability barrier to small substrates. The cell walls of all bacteria are not identical. In fact, the cell wall composition is one of the most important factors in the analysis and differentiation of bacterial species. Accordingly, two general types of bacteria exist, of which Gram- positive bacteria (Fig. 3a) are comprised of a thick peptidoglycan layer (Beveridge, 1981; Dijkstra and Keck, 1996) connected by amino acid bridges. Imbedded in the Gram- positive cell wall are polyalcohols, known as teichoic acids, some of which are lipid- linked to form lipoteichoic acids. Because lipoteichoic acids are covalently linked to lipids within the cytoplasmic membrane, they are responsible for linking peptidoglycan to the cytoplasmic membrane. The cross-linked peptidoglycan molecules form a network, which covers the cell like a grid. Teichoic acids give the Gram-positive cell wall an overall  negative  charge,  due  to  the  presence  of  phosphodiester  bonds  between  the teichoic acid monomers (Sonnerbfeld et al., 1985). In general, 90% of the Gram-positive cell wall is comprised of peptidoglycan.

On the contrary, the cell wall of Gram-negative bacteria (Fig. 3b) is much thinner, and composed of only 10–20% peptidoglycan ( Beveridge, 1999). In addition, the cell wall contains an   outer membrane composed of phospholipids and lipopolysaccharides (Sheu and Freese, 1973). The highly charged nature of lipopolysaccharides  confers an overall negative charge on the Gram-negative cell wall. Sherbert (1978) showed that the anionic functional groups present in the peptidoglycan, teichoic acids and teichuronic acids of Gram-positive bacteria, and the peptidoglycan, phospholipids, and lipopolysaccharides   of   Gram-negative   bacteria   were   the   components   primarily responsible for the anionic character and metal-binding capability of the cell wall. Extracellular polysaccharides are also capable of binding metals (McLean et al., 1992). However, their availability depends on the bacterial species and growth conditions and they can easily be removed by simple mechanical disruption or chemical washing (Yee and Fein, 2001).

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