With the growth of industry, there has been a considerable increase in the discharge of industrial waste to the environment, chiefly soil and water, which has led to the accumulation of heavy metals, especially in urban areas. Slow depletion of heavy metals also takes place through leaching, plant uptake, erosion and deflation. The indiscriminate release of heavy metals into the soil and waters is a major health concern worldwide, as they cannot be broken down to non-toxic forms and therefore have long-lasting effects on the ecosystem. Many of them are toxic even at very low concentrations; arsenic, cadmium, chromium, copper, lead, mercury, nickel, selenium, silver, zinc etc. are not only cytotoxic but also carcinogenic and mutagenic in nature . Some metals are required by plants in very small amounts for their growth and optimum performance. However, the increasing concentration of several metals in soil and waters due to industrial revolution has created an alarming situation for human life and aquatic biota. This is evident from various reports citing harmful effects of heavy metals on human health (Table 1).
In order to make the environment healthier for human beings, contaminated water bodies and land need to be rectified to make them free from heavy metals and trace elements. There are several techniques to remove these heavy metals, including chemical precipitation, oxidation or reduction, filtration, ion-exchange, reverse osmosis, membrane technology, evaporation and electrochemical treatment. But most of these techniques become ineffective when the concentrations of heavy metals are less than 100 mg/L . Most heavy metal salts are water-soluble and get dissolved in wastewater, which means they cannot be separated by physical separation methods . Additionally, physico-chemical methods are ineffective or expensive when the concentration of heavy metals is very low. Alternately, biological methods like biosorption and/or bioaccumulation for removal of heavy metals may be an attractive alternative to physico-chemical methods . Use of microorganisms and plants for remediation purposes is thus a possible solution for heavy metal pollution since it includes sustainable remediation technologies to rectify and re-establish the natural condition of soil. However, introduction of heavy metals into the soil causes considerable modification of the microbial community, despite their vital importance for the growth of microorganisms at relatively low concentrations . The modification of the microbial make up is mainly brought about by exerting an inhibitory action through blockage of essential functional groups, displacement of essential metal ions or modification of active conformations of biological molecules [6,7]. The response of microbial communities to heavy metals depends on the concentration and availability of heavy metals and is a complex process which is controlled by multiple factors, such as type of metal, the nature of the medium, and microbial species .
Table 1. Toxic effect of some heavy metals on human health.
|Heavy Metal||EPA Regulatory Limit (ppm) ||Toxic Effects||Ref.|
|Ag||0.10||Exposure may cause skin and other body tissues to turn gray or blue-gray, breathing problems, lung and throat irritation and stomach pain.|||
|As||0.01||Affects essential cellular processes such asoxidative phosphorylation and ATP synthesis|||
|Ba||2.0||Cause cardiac arrhythmias, respiratory failure, gastrointestinal dysfunction, muscle twitching and elevated blood pressure|||
|Cd||5.0||Carcinogenic, mutagenic, endocrine disruptor, lung damage and fragile bones, affects calcium regulation in biological systems||[1,13]|
|Cu||1.3||Brain and kidney damage, elevated levels result in liver cirrhosis and chronic anemia, stomach and intestine irritation||[1,14]|
|Hg||2.0||Autoimmune diseases, depression, drowsiness, fatigue, hair loss, insomnia, loss of memory, restlessness, disturbance of vision, tremors, temper outbursts, brain damage, lung and kidney failure||[15,16,17]|
|Ni||0.2 (WHO permissible limit)||Allergic skin diseases such as itching, cancer of the lungs, nose, sinuses, throat through continuous inhalation, immunotoxic, neurotoxic, genotoxic, affects fertility, hair loss||[1,18,19,20]|
|Pb||15||Excess exposure in children causes impaired development, reduced intelligence, short-term memory loss, disabilities in learning and coordination problems, risk of cardiovascular disease||[1,14,21]|
|Se||50||Dietary exposure of around 300 µg/day affects endocrine function, impairment of natural killer cells activity, hepatotoxicity and gastrointestinal disturbaces|||
|Zn||0.5||Dizziness, fatigue etc.|||
Bioremediation is an innovative and promising technology available for removal of heavy metals and recovery of the heavy metals in polluted water and lands. Since microorganisms have developed various strategies for their survival in heavy metal-polluted habitats, these organisms are known to develop and adopt different detoxifying mechanisms such as biosorption, bioaccumulation, biotransformation and biomineralization, which can be exploited for bioremediation either ex situ or in situ [24,25,26,27]. A global survey to examine the use of bioremediation technologies for addressing the environmental problems was carried out by Elekwachi et al. . They found that despite aspirations from respondents to apply bioremediation techniques, it should not become the current practice. Developed economies made higher use of low-cost in situ bioremediation technologies such as monitored natural attenuation, while their developing counterparts appeared to focus on occasionally more expensive ex situ technologies. Despite significant investment and widespread availability of online resources, their limited use underlines the need to explore improved training and development of more user-friendly resources. There are many reports about biodegradation and bioremediation strategies being utilized by bacteria or plant species [29,30,31,32], but so far none of these investigations suggest possible drivers in the global use of the said techniques . Among the preferred methods for treatment of contaminated areas, 51% of the respondents preferred environment friendly approaches, including microbial remediation (35%) and phytoremediation (16%) [33,34].
Microorganisms uptake heavy metals actively (bioaccumulation) and/or passively (adsorption) . The microbial cell walls, which mainly consist of polysaccharides, lipids and proteins, offer many functional groups that can bind heavy metal ions, and these include carboxylate, hydroxyl, amino and phosphate groups . Among various microbe-mediated methods, the biosorption process seems to be more feasible for large scale application compared to the bioaccumulation process, because microbes will require addition of nutrients for their active uptake of heavy metals, which increases the biological oxygen demand or chemical oxygen demand in the waste. Further, it is very difficult to maintain a healthy population of microorganisms due to heavy metal toxicity and other environmental factors [37,38]. Fungi of the genera Penicillium, Aspergillus and Rhizopus have been studied extensively as potential microbial agents for the removal of heavy metals from aqueous solutions [39,40]. Xiao et al. reported a novel technology for obtaining highly efficient biosorbents from endophytes, a hyperaccumulator, which is more convenient than the traditional method of obtaining biosorbents . Sun et al. evaluated the genetic diversity of endophytic bacteria from the copper-tolerant species of Elshotzia apliendens and Commelina communis, reporting increased dry weights of roots and aboveground tissues compared to uninoculated plants . Further, they also reported significant amounts of (ranging from 63% to 125%) Cu content in inoculated plants compared to uninoculated ones.
In view of such reports on the use of microorganisms and plants for removal of heavy metals from contaminated sites, the present review focuses on recent developments in bioremediation techniques. Additionally, new approaches such as the designer plant approach and rhizosphere modification to achieve the goal of bioremediation in a cheaper and safer way are also discussed.
Environmental degradation is assuming an alarming rate worldwide. This problem might not be unconnected with the rapid industrialization, population growth and technological advancement being experienced in most parts of the world today. A long list of potential hazardous substances is released on a daily basis which contributes to the global pollution burden. As part of this ‘list’, heavy metals result from industrial wastes; electronic wastes etc, and pose serious threat to both man and animals if not properly abated.
Indiscriminate disposal of engine oil into gutters, water drains, open vacant plots and farms is a common practice in Nigeria especially by motor mechanics (Okonokhua et al, 2007). Heavy metals such as Vanadium, Lead, Aluminium, Nickel and Iron usually below detectable limits in unused lubricating oil have been reported by Whisman et al, (1971) to give high values (ppm) in used oil. These metals may be retained in soils in the form of oxides, hydroxides, carbonates, exchangeable cations, and/or bound to organic matter in soil (Yong et al., 1992). There is a growing global concern because of the numerous health risks to animals and humans following exposure.
The term heavy metal refers to any metallic chemical element that has a relatively high density and is toxic or poisonous at low concentrations. Examples of heavy metals that are harmful to humans include Mercury, Lead, and Arsenic. Chronic exposure to these metals can have serious health consequences. Humans are exposed to heavy metals through inhalation of air pollutants, consumption of contaminated drinking water, exposure to contaminated soils or industrial waste, or consumption of contaminated food grown on contaminated land. Food sources such as vegetables, grains, fruits, fish and shellfish can become contaminated by accumulating metals from surrounding soil and water. Heavy metal exposure causes serious health effects, including reduced growth and development, cancer, organ damage, nervous system damage, and in extreme cases, death. Exposure to some metals, such as Mercury and Lead, may also cause development of autoimmunity, in which a person's immune system attacks its own cells. This can lead to joint diseases such as rheumatoid arthritis, and diseases of the kidneys, circulatory system, and nervous system. Metals are particularly toxic to the sensitive, rapidly developing systems of foetuses, infants, and young children. Some metals, such as lead and mercury, easily cross the placenta and damage the foetal brain. Childhood exposure to some metals can result in learning difficulties, memory impairment, damage to the nervous system, and behavioural problems such as aggressiveness and hyperactivity. At higher doses, heavy metals can cause irreversible brain damage. Children may receive higher doses of metals from food than adults, since they consume more food for their body weight than adults (Roane, 1999).
Environmental contamination by heavy metals is consequently the most critical environmental problem as it poses significant impacts to the health of humans as well as the numerous ecosystems that support it. The contaminants infiltrate deep into underground, pollute ground water and surface water. There is a probability of entering the human food web through plants and aquatic animals that bioaccumulate them, consequently transferring them from one food chain to another in a process known as biomagnification.
The effectiveness of commonly employed methods of treating heavy metals polluted wastes, including among others, precipitation and ion exchange remains limited, (Kratochril et al., 1997), particularly for dilute metal wastes (Corder and Reeves, 1994).
Over the last decade, biosorption developed as an alternative treatment method (Volesky, 1990; Williams et al., 1998). This technology involves the accumulation of heavy metals by biological material either by metabolically mediated methods or by purely physico-chemical means (Sakaguchi and Nakijama, 1991) unlike physical and chemical treatments, biosorption is relatively cheap.
However, this technology relies on active microbial presence and metabolism which in some environmental matrices is inhibited by nutrient availability and bioavailability as there exist a direct relationship between nutrient supply and microbial growth.
This study seeks to assess and evaluate the efficacy of providing a low cost, environmentally friendly and socially acceptable means of remediating some selected heavy metals from spent oil contaminated soil using microbial consortia from poultry litter. This study is justified because of the common problem associated with conventional treatment namely; the difficulties encountered in treating the solid waste subsequently generated might not exist since biological treatment concentrates the wastes into smaller volumes which are subsequently easier to dispose of appropriately (Bhude et al., 1996).
2. Microorganisms and Heavy Metals
Microorganisms have evolved various mechanisms of metal resistance and scientists have tried to exploit genetic/metabolic basis of all such mechanisms for the production of superior strains. Silver et al (2001) reported that although chromosomal genes may be involved, bacterial resistance to heavy metals is often conferred by products of genes simulated on plasmids rendering genetic manipulation for strain improvement easy and feasible.
A possible way to confer resistance to heavy metals on microorganisms is to progressively increase the concentration of the toxicants microbes are exposed to and observe microbial metabolism and proliferation. Scientists have explored this attribute to ascertain if tolerance of higher concentrations can subsequently aid in biodegradation of such toxicants. Donmez and Aksu (2001) tried to adapt strains of Candida species (isolated from sewage) to Copper and Nickel by serial subcultures in Copper and Nickel supplemented growth medium. Adapted cells could grow well in presence of higher metal concentrations while non adapted ones perished. Moreover the specific metal uptake capacity and metal removal (%) by adapted cells were higher than the non-adapted cells at all the concentrations tested. Although the authors could not elucidate the mechanism of adaptation, they implied constitutive synthesis of methalothionein (other copper binding proteins or changes in the genetic makeup) as one of the factors in adaptation. Baillet et al(1997) adapted Thiobacillusferroxidans strain via successive exposure to higher concentrations of Cadmium. The biomass (living but not growing) of adapted strain had cadmium uptake capacity of 0.31 g/g dry weight as compared to 0.21 g/g dry weight in case of non adapted cells.
Other non conventional techniques reported by Soares et al (2002) are the comparison of flocculent strain and non flocculent strain of S. cerevisiae for metal bioremediation. They inferred that flocculent strain S646-1b accumulated more copper than non flocculent S646-8D strain in the first 10 minutes of contact with the metal. The authors attributed this to the presence of additional metal binding on cell surface of flocculent strains.
3. Statement of Problem
Thereare relatively large amounts of hydrocarbons in spent engine oil including highly toxic Polycyclic Aromatic Hydrocarbons (Wang et al., 2000). Ekundayo et al.,(1989) have shown that a marked change in the properties occur in soils polluted with petroleum hydrocarbons affecting the physical, chemical and microbial properties of the soil. Oil pollution leads to a buildup of essential organic nutrients such as carbon, phosphorus, calcium and magnesium and non essential Lead, Zinc, Iron, Copper and Cobalt. These elements if in excess will lead to translocation of plant tissues. Although some heavy metals at low concentrations are essential micro nutrients for plants, at high concentrations they could cause high metabolic disorder and growth inhibition for most plant species. This coupled with the potential of some plants to bio accumulate heavy metals make the presence of spent oil in soil worrisome. Soil contamination with engine oil that is rampant in the Nigerian environment has adverse effects for seasonal crops such as tomato (Lycopersicumesculentum) and maize (Zeamays). (Okonokhua et al., 2007).
4. Significance of the Study
Since contamination of soil and groundwater by the indiscriminate discharge of petroleum products has become a significant problem today, a number of technologies have been investigated to remedy the situation. Treatment processes have incorporated chemical, physical and biological methods or a combination of them. Treatment options include excavation and landfill disposal, incineration, surfactant application and a host of others that are expensive, environmentally unfriendly and only transfer the contaminants from one place to another. Bioremediation is inexpensive, naturally and socially acceptable and environmentally friendly. The technology under study will help minimize the prohibitive cost associated with soil remediation, prevent significant soil texture alteration, prevent the transfer of pollutants from one medium to another and ensure a healthier technique for remediating heavy metals.
5. Materials and Methods
The experiment was carried out in a private laboratory in Benin City, Edo State, Nigeria. Top soil (0.7cm-15cm), was obtained from a mechanic site along Federal Government Girls College Road Benin City and transported to the laboratory in a plastic container. Poultry Litter was obtained from the University of Benin poultry farm within the Ugbowo Campus in Benin City, Edo State, Nigeria and air dried in the laboratory. The dried poultry droppings were carefully sorted, sampled for the presence of microorganisms, ground to semi-powder and used as organic fertilizer to support the growth of microorganisms and remediation of heavy metals. Air dried spent engine soil samples were measured into four plastic buckets used as test cells (1 kg each) and mixed thoroughly with poultry manure in a soil to manure ratio of 80% weight of soil to 20% weight of manure, 70% weight of soil to 30% weight of manure and 60% weight of soil to 40% weight of manure and labelled as PL 20%+SEOCS (Poultry Litter + Spent Engine Oil Contaminated Soil), PL 30%+SEOCS and PL 40%+SEOCS and CONTROL respectively. Each bucket was watered as necessary to achieve optimum moisture requirement for microbial growth. Leachate was re-introduced into the test cells before sampling every week.
6. Methods6.1. Process Description
Soil samples were carefully sorted out to remove impurities and weighed (20 kg). Soil samples were characterized for their physico-chemical and microbial parameters using standard methods.6.2. Soil pH
Soil pH was determined using the method of Bates (1954).6.3. Soil Moisture Content
Soil moisture content was determined by evaporation on Whatman filter paper No. 1 at 103C in an electrical oven.6.4. Total Heterotrophic Bacterial and Fungal Counts
Samples were enumerated by making ten-fold dilutions of the soil samples from 101 to 103. Aliquot 0.1ml of the 10-3 dilution was transfer plated in nutrient agar amended with nystatin (0.5-1 µg/ml) for isolation of bacteria while potato dextrose agar amended with streptomycin (0.02-1 µg/ml) was used for the isolation of fungi. The plates were prepared and inoculated in duplicates. The inoculated nutrient agar plates were incubated at 37C for 24 hours while the potato dextrose agar plates were incubated at 28C for 72 hours. After incubation, the colonies of the isolates were counted and expressed in CFU/g.; isolated colonies were further purified by sub-culturing and identified using bio-chemical tests and microscopy.6.5. Isolation of Degrading Microorganisms
The culture medium used for the isolation of hydrocarbon degrading bacteria was mineral salt agar which is the enrichment medium for the isolation of hydrocarbon degrading bacteria. Aliquot of 0.1 ml of the 10-3 dilution was plated in the medium and the plates were incubated at 30C for 5 days. Discrete colonies that developed were counted and expressed in CFU/g.6.6. Identification of Isolates
Each isolate was examined for its size, shape, margin, consistency, elevation, pigmentation, Gram reaction and cell morphology. The isolates were characterized as described by Holt et al., (1999). Biochemical tests which were carried out included production of catalase, indole and oxidase enzymes. Spore production and oxidation/ fermentation of sugars were also carried out.6.7. Heavy Metals Analysis
Heavy metals analysis was carried out using the wet oxidation method for soils (APHA, 2000); soil samples dried at room temperature was sieved using 2mm mesh sieve. About 2.5 g of the soil was weighed into a 300m3 conical flask. 1 ml of concentrated HF under a fume hood was added. Heating was continued until dense white fumes appeared. Finally, it was heated strongly (medium to high heat) for half a minute. It was allowed to cool, and the 40-50 ml of distilled water was added. The solution was cooled and filtered completely with a wash bottle into a 100 ml pyrex volumetric flask. It was made up to the mark with distilled and filtered with Whatman No. 42 filter paper. The soil extracts were aspirated into the air acetylene flame of Varian 220 (fast sequential) Atomic Absorption Spectrophotomer (AAS)6.8. Experimental design
Three different quantities of poultry litter were measured into separate test cells and thoroughly mixed. The quantities were in ratio of 20: 80, 30: 70 and 40: 60 respectively. Microbial growth/density as well as heavy metal concentration was considered as experimental responses. Heavy metal concentration was measured on a weekly basis for 10 weeks. Untreated soil (spent engine oil contaminated soil without poultry litter) was analysed as control.6.9. Statistical analysis
All statistical parameters (ANOVA at 95% probability level of significance) were carried out using Statistical Package for Social Sciences and Microsoft Excel 2007.
7. Results and Discussion7.1. pH
The results of the hydrogen ion analysis are presented in Figure 1.
pH for untreated soil at baseline week (week 0) was 6.9 while it was 7.20 for poultry litter used as nutrient additive. The results obtained at the end of the experiment indicate a range of 8.1 (Week 1 PL 20% + SEOCS) and 8.4 (week 7 PL 20% + SEOCS). These results show that pH in treatment categories was slightly higher than the control throughout the experiment although this difference was insignificant.
Biological activities are regulated by enzymes that operate within a fairly stringent range of pH. When the extremes of this range are exceeded then microbial growth and physiology is significantly distorted. Boonchan, (2000) and Joanne et al.,(2008) reported that optimum pH for bioremediation is between 6.0 and 8.9. They opined that changes from initial levels of pH could be as a result of the release of acidic and alkaline intermediates and final products during biodegradation of hydrocarbons which has an effect on the pH. The findings of this research indicate that pH level fell within a suitable range to support microbial growth in all the treatment categories and control.7.2. Soil Moisture Content
Table 1 shows the percentage water content by mass in the treatment categories.
In this study, percentage initial moisture content was 2.0%. Watering during the research phase improved moisture content and analysis revealed that the lowest average percentage moisture content by mass was 17.8% in PL 30% + SEOCS, while the highest average percentage moisture content by mass was 18.4% in PL 40% + SEOCS. In control samples, moisture content was 18.9%. The values in the treatment categories had no significant differences with the control (P<0.05).
Microorganisms require water for microbial growth and for diffusion of nutrients and by-products during the degradation process (Malik, 2004). If the soil is too dry, many rnicroorganisrns will die. If water content of the soil is too high, oxygen transfer to microorganisms will be resisted by the flooded soil and the rate of the hydrocarbon degradation will be reduced. The optimum soil water content for bioremediation is dependent on the soil type. Generally, the optimum activity occurs when the soil moisture is 15-40% of the field capacity, also termed the water holding capacity which is defined as "the amount of the water remaining within the soil after gravitational water has drained away" or the percentage of water in a soil when it was (Baker, 1994). When moisture content is lower than 10% of the holding capacity, the bioactivity becomes marginal (Testa and Winegardner, 1991). Water was added to the entire experimental group so as to achieve optimum moisture content of at least 17%. Based on the results observed, moisture content was optimum in all the treatment categories as well as control during the period of experiment.7.3. Biotic count
Table 2 presents results of Total Microbial Count.
In this study, identification of microorganisms in poultry litter and contaminated soil revealed the following heterotrophic microbial species; Pseudomonas, Enterococcus, Micrococcus, Corynebacterium, Arthobacter, Klebsiella, Acinetobacter, Bacillus, Penicillum, Sachoromyces, Mould and Trichoderma. The microbial density and diversity prevalent in a contaminated medium play important roles in degradation of pollutants.
The extent of bioremediation depends on microbial diversity present which can survive in highly concentrated areas. However, reports abound (Yong et al, 1992; Volesky, 1990; Bhude et al., 1996 and Baillet et al., 1997) suggesting that microbial performance in bioremediation is hindered by such factors as nutrient availability, temperature, pH, pollutant concentration, moisture content, oxygen content as well as soil properties of impacted soils. These finding agrees with some studies (Malik, 2003, Bade et al, 2012) which reported that Bacillus, Acinetobacter, Pseudomonas and Arthobacter have great potentials for accumulating and removing some heavy metals. This finding also corroborates the study by Akpokona (2011) who observed an increase in uptake of Chromium and TPH degradation in brackish water impacted with crude oil when NPK and poultry litter were used as nutrient additives to stimulate microbial growth.
8. Heavy Metals Degradation8.1. Arsenic (mg/kg)
The results of arsenic analysis are presented in Figure 2. In this study, Arsenic (mg/kg) in the control sample reduced from 2.19 mg/kg at the start of the experiment to 1.91 mg/kg and 2.13 mg/kg (2.78%) on the final sampling week. In PL 20%+SEOCS, the concentration reduced from 1.8 mg/kg to 1.08 mg/kg on the third week and 1.32 mg/kg (26.6%) on the final week of the experiment. In PL30%+SEOCS the initial concentration was 1.78 mg.kg and it dropped to 1.11 mg/kg and eventually to 1.2 mg/kg (32.5%) on the final sampling week. In PL 40%+SEOCS the initial concentration of 1.87 mg/kg dropped to 1.1 mg/kg (41.17%) on the final week of the experiment. The peaks observed in the plots might be as a result of experimental errors. ANOVA shows that there was a significant difference between the treatment categories and the control (P<0.05). There was also a significant difference between week 0 and week 10 (P<0.05).
Arsenic is naturally present at high levels in the soil in a number of countries and can be further increased by anthropogenic activities. This heavy metal is a known carcinogen and causes numerous other serious health problems (Santhosh, 2008). It is highly toxic in its inorganic form and exposure to arsenic is usually through drinking contaminated water, using contaminated water in food preparation and irrigation of food crops, industrial processes, eating contaminated food and tobacco smoking. Katerina et al(2008) reported the removal of arsenic from contaminated soil using bioremediation. This research corroborates the fact that arsenic can be reduced using a biological treatment scheme. The decrease of arsenic might be as a result of the presence of Bacillus species in the soil samples that have high adsorptive capacity due to high peptidoglycan and techoic acid content in their cell walls which enables an ion exchange reaction.8.2. Barium (mg/kg)
The results of barium analysis are presented in Figure 3. In this study, Barium (mg/kg) in the control sample reduced from 1,290 mg/kg in the first week to 1202 mg/kg (6.28%) in the final week of the experiment. In PL 20%+SEOCS barium (mg/kg) reduced from 1,130 mg/kg to 724 mg/kg (35.19%) in the final sampling week. In PL 30%+SEOCS, initial concentration was 790 mg/kg and this value reduced to 702 mg/kg (11.1%) while in PL 40%+SEOCS the initial concentration of 503 mg/kg reduced to 180 mg/kg (64.21%). The peaks observed in the plot might be due to experimental errors. ANOVA shows there was no significant difference between PL 30%+SEOCS and control (P>0.05) while there was a significant difference between PL 20%+SEOCS, PL 40%+SEOCS and control. There was also a significant difference (P<0.05) between week 0 and week 10.
Barium causes respiratory effects such as benign pneumonoconiosis, silicosis (Seaton et al, 1986) and pulmonary lesions (Tarasenko et al, 1977). This metal has also been incriminated in cardiovascular and gastrointestinal effects with abdominal cramps, nausea, vomiting as acute symptoms of exposure to barium carbonate powder (Shankle and Keane 1988). Other significant health effects include renal failure, body weight loss; metabolic effects such as decrease in plasma potassium concentration as well as reproductive impairment especially disturbances in spermatogenesis. The reduction in the concentration of barium over time during this experiment might not be unconnected with the presence Pseudomonas Sp. which produces rhamnolipids, a surfactant that shows specificity for certain metals such as barium and cadmium. This report is in agreement with the findings of Sand et al.,(1992) who studied the leaching potential of Leptospirillumferoxidans and reported the significance of surfactants in the remediation of barium and other heavy metals.8.3. Cadmium (mg/kg)
Figure 4 shows the results for cadmium (mg/kg) during the research period. In the study, cadmium (mg/kg) dropped from 0.6 mg/kg to 0.45 mg/kg (24%) in control, in the treatment categories; cadmium mg/kg had an initial concentration of 0.32 mg/kg in PL 20%+SEOCS and fluctuated throughout the research duration and eventually was 0.75 mg/kg on the final week of sampling. In PL 30%+SEOCS the initial concentration of 0.13 mg/kg dropped to 0.08 mg/kg (38%), while in PL 40%+SEOCS the initial concentration of 0.15 mg/kg dropped to 0.1 mg/kg (33.3%). The peaks observed were probably due to experimental errors. Analysis of variance shows that cadmium reduction in the treatment categories was not significantly different from the control and although there were reductions observed during the research duration, they were however not significant (P>0.05).
The United States Environmental Protection Agency (2001) states that acute effects of exposure to high levels of cadmium in humans may result in adverse effects on the lungs such as bronchial and pulmonary irritation. The metal is considered to have a high acute toxicity based on short term animal tests on rats. The Agency also details chronic effects to long term exposure to cadmium to include kidney diseases, including protenuria, effects on the lungs including bronchioloitis and emphysema, adverse effects are also reported on the liver, bone, immune systems, blood and nervous systems. Human exposure to cadmium can be from consumption of contaminated water or consumption of plants grown on cadmium polluted sites. Studies on the remediation of cadmium conducted by Ike et al, (2007) report a technique of successfully remediating cadmium contaminated soil using symbiosis between leguminous plants and recombinant rhizobia, Bagot et al (2005) selected bacteria, (Bacillus), fungus and actinomycetes for bioremediation of cadmium contaminated agricultural soils. Their results indicate that Bacillus and actinomycetes successfully reduced cadmium. The slight reduction of cadmium concentration in this study suggests a necessity for optimization of the biochemical process of cellular sequestration and growth of Bacillus Sp.8.4. Chromium (mg/kg)
Figure 5 shows the results for chromium.
In this study, there was a gradual reduction of chromium in the treatment categories. Chromium (mg/kg) in the control sample was 19.5mg/kg intitially, this concentration was 13.4mg/kg in the seventh week and ended at 15.6mg/kg (20%) on the final week of the experiment. In the PL20%+SEOCS the initial concentration of 18.9mg/kg reduced to 13.9mg/kg (26%) on the final sampling week. In PL30%+SEOCS the initial concentration of 15.5mg/kg reduced to 6.5mg/kg (58.06%) while in PL40%+SEOCS, the initial concentration of 14.6mg/kg reduced to 7.8mg/kg (46.57%). Analysis of Variance indicates that there was no significant difference (P>0.05) between PL20%+SEOCS and control while PL30%+SEOCS and PL40%+SEOCS were significantly different from the control. There was a significant reduction in the concentration of chromium over time (P<0.05).
The Occupational Safety and Health Administration’s fact sheet (2006) reports that adverse health effects associated with chromium exposure include occupational asthma, eye irritation and damage, perforated ear drums, respiratory irritation, kidney damage, liver damage, pulmonary congestion and edema, upper abdominal pain, nose irritation and damage, respiratory cancer, skin irritation and erosion, allergic skin reaction and contact dermatitis. Human exposure to environmental chromium is usually from handling chromium contaminated medium and contact with contaminated sites either through dermal exposure or through ingestion of contaminated drinking water.
For Chromium, bioremediation relies on the reduction of soluble and mobile hexavalent chromium, Cr (VI), to its reduced form, Cr (III). Chromium reduction may occur if the growth of microbes is stimulated via the addition of a reduced carbon source, such as lactate, acetate, or molasses. The product, Cr (III), is often assumed to precipitate as relatively insoluble hydroxide solid.
Studies on specific bioremediation of chromium by Jeyasingh (2005) using optimization of operating parameters under laboratory conditions revealed a reduction aerobically and anaerobically by indigenous microorganisms that were isolated from a previously contaminated site. After studying the mechanism of hexavalent chromium detoxification by microorganisms and bioremediation application potential, Cheung et al(2007), reported that microbial reduction of chromium is commonly catalyzed by soluble enzymes with Pseudomonas spp and E. coli capable of secreting chromium reductases. They also reported that through the utilization of membrane associated reductases, Pseudomonas maltophilia and Bacillus megaterium could detoxify chromium. The presence of Pseudomonas Sp., Bacillus Sp. and E. coli and subsequent reduction in the concentration of chromium during the experiment is in agreement with the findings of these researchers.8.5. Cobalt (mg/kg)
Results of cobalt degradation are presented in Figure 6. In this study, cobalt (mg/kg) in the control sample was 2.9 mg/kg on the first week of sampling; it reduced to 2.73 mg/kg (5.86%) on the final sampling week. In the treatment categories, PL 20%+SEOCS had initial cobalt concentration of 2.4 mg/kg and this concentration reduced to 1.12 mg/kg (53.3%). Cobalt concentration in PL 30%+SEOCS was 2.3 mg/kg initially and it reduced to 1.01 mg/kg (5.60%) on the final sampling week. In PL 40%+SEOCS the initial concentration of 2.1 mg/kg reduced to 0.81 mg/kg (61.42%) on the final sampling week. Analysis of Variance indicates that reductions in all the treatment categories were significantly different from the control. Overall reduction throughout the duration of the research was significant (P<0.05).
Swennel et al, (1993) reported adverse haematological effects of exposure to cobalt, Jordan et al, (1990) reported neurological effects including memory loss, Bucher et al., (1990) reported reproductive effects in animals while Mur et al., (1987) studied carcinogenecity of cobalt and reported that an increase in deaths due to lung cancer was found in workers exposed to cobalt in a plant refining cobalt and sodium. Studies on microbial remediation of trace cobalt conducted by Gogada (2008) using E. coli,expressing NiCoT genes revealed the viability of E. coli strains in the remediation of trace cobalt. Santhosh studied the biosorption of heavy metals by Paenibacillus polymyxa and reported that the heavy metal cobalt was reduced to up to 90% primarily due to the binding of the metal to the cell walls through the extra cellular polymeric substances and precipitation of sulphides. This study idenfied the presence of E.coli and the possibility of biosorption to the cell surfaces and subsequent reduction in the concentraion of cobalt. These results agree with the findings of Gogada (2008) and Santhosh (2008).8.6. Lead (mg/kg)
Results of Lead degradation are presented in Figure 7. In this study, lead (mg/kg) in the control sample was 11.1 mg/kg on the first week, it dropped slightly to 9.98 mg/kg on the 8th week and eventually ended at 10.8 mg/kg (2.70%) on the 10th week. In the treatment categories, PL 20%+SEOCS had initial concentration of 6.14 mg/kg and reduced to 5.2 mg/kg (15.3%). In PL 30%+SEOCS initial concentration of lead was 7.23 mg/kg, it reduced to 5.49 mg/kg (24.06%) on the final sampling week. In PL 40%+SEOCS, initial Lead (mg/kg) concentration was 8.1 mg/kg, it reduced to 5.3 mg/kg (34.5%) on the final sampling week. There was a significant reduction in the concentration of Lead (P<0.05) when compared with the control.
The United States Environmental Protection Agency, (USEPA, 2001) reports that Lead can be harmful to humans when ingested or inhaled, partciularly to children under the age of six. The most prominent adverse health effect of Lead is the impairment of neurological development. The agency further states that exposure routes can be through contaminated water soil, paint chips or dust causing poor muscle coordination, nerve damage to the sense organs and nerves controlling the body, increased blood pressure, hearing and vision impairment, reproductive problems (e.g. decreased sperm count), retarded fetal development even at relatively low exposure levels. In children Lead poison can cause damage to the brain and nervous system, behavioural problems, anemia, liver and kidney damage, hearing loss, hyperactivity, developmental delays and in etxreme cases death.
Lead also has the potential to be bioaccumulated rising from low concentrations to potentially high concentrations that have detrimental effects on human health.
Chatterjee et al, (2012) reports that various lead resistance mechanisms employed by lead resistant bacteria includes efflux mechanism, extracellular sequestration, biosorption, precipitation, alteration in cell morphology, enhanced siderophore production and intracellular lead bioaccumulation. Adsorption, ion exchange, precipitation, and complexation with organic matter are other factors that contribute to the high adsorption rate of Lead in soil (Evanko and Dzombak, 1997).
Boricha and Fulekar, (2009) reported that potent metal biosorbents under the class of bacteria include the genera of Bacillus, Pseudomonas, Streptomyces and P. aeroginosa they found that P. plecoglossicida as a novel organism for the bioremediation of cypermethrin while P. aeruginosa, Bacillus sp., Streptomyces sp., P. fluorescens were effective against lead.
Macaskie et al. (2000) found that sulfate-reducing bacteria successfully treated the metal leachates generated by sulfuric-acid producing Thiobacillus sp.
Puranik and Puranik (1997) reported that amongst the various microorganisms, fungal biomasses were very effective due to presence of high percentage of cell wall material, which may have the excellent metal binding capacity. Many fungi and yeast have excellent biosorption potential includes the genera of Rhizopus, Aspergillus, Streptoverticullum and Sacchromyces.
The article Metal Bioremediation Through Growing Cells demonstrated that many bacterial and fungal strains can assist with lead removal. Organisms like Pseudomonasmarginalis, Plectonemaboryanum, and Desulfosporiosinus orients can all assist with lead uptake (Malik, 2003).
Other findings corroborate the fact that many grown bacterial and fungal strains can assist with lead removal, research today is focusing on the precipitation of metal sulfides utilizing sulfate-reducing bacteria. The Mine Waste Technology Program of the United States Environmental Protection Agency and Department of Energy uses sulfate-reducing bacteria for bioremediation purposes. They also use passive treatment systems like compost for remediation. Although this is currently found not to be an effective treatment because the precipitates are not guaranteed to stay in their precipitated forms (Malik, 2003).
However there exist the challenge to find fungal or bacteria biofilms that can work in many different conditions and keep the metals from leaching into the environment. It is important for researchers to discover ways to tackle this challenge; otherwise industries will continue to be skeptical about using bioremediation at metal-contaminated sites. (Beyenal and Lewandowski, 2004).
This study showed a significant reduction in Lead concentration, the presence of Bacillussp, and Pseudomonas sp in the sampled soil and subsequent growth and proliferation aided heavy metal concentration reduction by adherence to the extracellular protein structure and intracellular protein sequestration respectively. Sacchoromyces cerevisiae’s presence might have further aided reduction through the adherence of Lead onto the cell wall and membrane and also internally in the cytoplasm. These mechanisms of microbial accumulation of heavy metals have been documented previously by Roane (1999), Sub et al., (1998) and Torres et al, (1998).
The research findings indicate that bioremediation using growing microorganisms present in contaminated soil and animal wastes can reduce the concentration of heavy metals in soil. The presence of consortia of microorganisms improved the metals scavenging properties and ensured a continuity in the process irrrespective of the external environmental fluctuations. Ability to grow and utilize cells sourced from potential waste streams also promises an economically viable option. The exploitation of locally available support materials and cheap carbon/nutrient sources for the cells can further improve the economic imperative for heavy metals remediation.
However, choosing the right consortia must depend on initial heavy metal concentrations and should be carefully carried out so as to ensure some microorganisms metabolism do not inhibit the growth of others.
If there is an improvement in laboratory analysis to identify the exact mechanisms of uptake, the bioaccumulative ‘store houses’ of the cells and the genetic materials/properties responsible for biodegradation, then the gap between successful reproduction of the process at commercial scale as well as ability of these strains to continually take in heavy metals would have been bridged.
The authors are profoundly grateful to Emmanuel Ejoke of Success Laboratory, Benin City Nigeria for his efforts towards the success of this research. Daramola Charles Temitayo, Ebelechukwu Uba, Osasere Christopher, Barry Okoro, Abale Daniel, Obitte Benneth and Agbanelo Clarine are imensely appreciated as well.
|||APHA. (1998). Standard methods for the examination of water and wastewater, 20th edition. American Public Health Association, Baltimore.|
|||Bade, R. Sanghwa, O and Won, S. (2012). Assessment of metal bioavailability in smelter contaminated soil before and after lime ammendment. Journal of Ecotoxicology AndEnvironmental Safety. Elsevier. Pp 1-9.|
|||Baillet, F, Magnin, J.P, Cheruy, A, and Ozil P. (1997). Cadmium tolerance and uptake by a Thiobacillus ferrooxidans biomass. Environ Technol; 18: 631-8.|
|||Baker, A.J.M., Reeves, R.D. and Hajar, (2006). Heavy metal accumulation and tolerance in British populations of the metallophyte thlaspi caemulescens J and C presl (Bassicaceae) Wiley online library. P. 19.|
|||Beyenal H, Lewandowski Z. (2004). Dynamics of lead immobilization in sulfate reducing biofilms. Water Research. 38 (11): 2726-2736.|
|||Bhide, J.V, Dhakephalkar, P.K. and Paknikar, K.M (1996) Microbiological process for the removal of Cr (VI) from chromate-bearing cooling tower effluent. Biotechnol. Lett., 18 (1996), pp. 667-672.|
|||Boricha, H., and Fulekar, M.F., (2009) “Pseudomonas plecoglossicida as a novel organism for the bioremediation of cypermethrin.” Biology and Medicine, 1. 1-10.|
|||Bucher, J.R., Elwell, M.R., Thompson, M.B., Chou, B.J., Renne, R., and Ragan, H.A. (1990). Inhalation toxicity studies of cobalt sulphate in F344/N rats and B6C3F1 mice. Fundamental and Applied Toxicology, 15: 357-372.|
|||Chatterjee, M, Canario, J.,Sarkar, S.K, Brancho, A. Bhattacharya, K and S. Saha, (2009) Mercury enrichments in core sediments in Sunderban mangroves, northeastern part of Bay of Bengal and their ecotoxicological significance, Environmental Geology, 2009, 57: 1125-1134.|
|||Cheung, K.C., Poon, B.H.T., Lan, C, Y, and Wong,M.H., (20030. Assessment of metal and nutrient concentrations in river water and sediment collected from the cities in the Pearl River Delta, South China. Chemosphere, 52 (9): 1431-1440.|
|||Corder, S.L., Reeves, M. (1994). Biosorption of nickel in complex aqueous waste streams by Cyanobacteria. Appl. Biochem. Biotechnol. 45/46: 847-859.|
|||Donmez G, Aksu Z. (1999). The effect of copper (II) ions on growth and bioaccumulation properties of some yeasts. Process Biochem. 35: 135-42.|
|||Ekundayo, J A; Aisueni, N; Benka-Coker, M.O. (1989). The Effects of drilling fluids in some waste and burrow pits in western operational areas of Shell Petroleum Development Company of Nigeria Limited on the soil and water quality of the areas. Environmental Consultancy Service Group, Consultancy Services Unit, University of Benin, Benin City, Nigeria.|
|||Evanko, C.R. and Dzombak, D. (1997). Remediation of metals contaminated soils and ground water. Ground water remediation technologies analysis center, Pittsburg USA. Pp. 1-48.|
|||Fourest, E., Canal, C. and Roux, J.C. (1994). Improvement of heavy metal biosorption by mycelial dead biomasses (Rhizopus arrhizus, Mucor miehei and Penicillium chrysogenum): pH control and cationic activation. FEMS Microbiol Rev 14: 325-32.|
|||Katarina, C., Darina, S., Vladimir, K., Miloslava, P., Jana, H., Andrea Puškrov, B., Ferianc, P., (2004). Identification and characterization of eight cadmium resistant bacterial isolates from a cadmium-contaminated sewage sludge, Biologia. Bratislava. 59: 817-827.|
|||Kratochvil, D. and Volesky, B. (1997). Biosorption of heavy metals. Water ResourcesJournal. Pp. 1-24.|
|||Macaskie, L.E., Empson, R.M., Cheetham, A.K., Grey, C.P. and Skarnulis, A.J., (2005) Uranium bioaccumulation by a Citrobacter sp. as a result of enzymically mediated growth of polycrystalline. Science, 257. 782-784. 2005.|
|||Malik A, (2000). Studies on biodesulfurization of coal. PhD thesis. India: Indian Institute of Technology Delhi; 2000.|
|||Malik A, Dastidar MG, Roychoudhury PK. (2001). Biodesulfurization of coal: effect of pulse feeding and leachate recycle. Enzyme Microb Technol: 28: 49-56.|
|||Malik A, Kakii K. (2003). Intergeneric coaggregations among Oligotropha carboxidovorans and Acinetobacter species present in activated sludge. FEMS Microbiol Lett 224: 23-8.|
|||Malik A, Kakii K. (2003 b) Pair-dependent coaggregation behavior of non-flocculating sludge bacteria. Biotechnol Lett;25: 981-6.|
|||Malik A, Sakamoto M, Kakii K. (2002) Coaggregation of Microbacterium esteraromaticum S51 with other strains of non-flocculating sludge bacteria. IWA’s Water EnvironManage Ser: 737-48.|
|||Malik A, Sakamoto M, Ono T, Kakii K. (2003) Coaggregation between Acinetobacter johnsonii S35 and Microbacterium esteraromaticum strains isolated from sewage activated sludge. J. Biosci. Bioeng.91 (1): 10-5.|
|||Okonokhua, B.O., Ikhajiagbe, B., Anoliefo, G.O., Emede, (2007). The Effects of Spent Engine Oil on Soil Properties and Growth of Maize (Zea mays L.), Journal of Applied Sciences and Environmental Management 11 (3): 147-152.|
|||Puranik, P.R. and Paknikar, K.M.,“Biosorption of lead and zinc from solutions using Streptoverticillium cinnamoneum waste biomass.” Journal of Biotechnology, 55. 113-124.1997.|
|||Roane, T.M. (1999): Lead resistance in two isolates from heavy metal contaminated soils. Microbial Ecology 37, 218-224.|
|||Sakaguchi, T and A. Nakajima (1991) Accumulation of heavy metals such as uranium and thorium by microorganisms In: R.W. Smith and M. Misra (Editors), Mineral Bioprocessing. The Minerals, Metals and Materials Society.|
|||Sand, W, Rohde, K, Sobotke, B, Zenneck C. (1992) Evaluation of Leptospirillum ferrooxidans for leaching. Applied and Environmental Microbiology, 58 (1): 85-92.|
|||Santosh, K.S, Chatterjee, M. and Asokkumar, B. (2009) An assessment of mercury loading in core sediments of Sunderban mangrove wetland, India (a preliminary report), Bulletin of Environmental Contamination and Toxicology, 2009, 81: 105-112.|
|||Silver, S., Phung, L.T., Lo J.F, and Gupta, A. (2001) Toxic metal resistances: molecular biology and the potential for bioremediation. In: Nuzhat A, Qureshi FM, Khan O, Khan Y, editors. Industrial and environmental biotechnology. Wymondham, UK: Horizon Scientific Press. p. 33-41.|
|||Soares, E.V., Geoffrey, D.C., Duarte, F, and Soares (2002). HMVM. Use of Sacchromyces cerevisiae for Cu2 + removal from solution: the advantages of using a flocculent strain. Biotechnol Lett;24: 663-6.|
|||Swanell, R.P.J., Lee, K., and McDonagh, M (1996): Field evaluation of marine oil spill bioremediation. Microb. Rev. 342-365.|
|||Tarasenko, M, O. Promin, and A. Silayev. (1977). Barium compounds as industrial poisons (an experimental study). J. Hyg. Epidem. Microbiol. Immunol. 21: 361-373.|
|||Testa, S.M. and Winegardner, D.L. (1991). Aquifer Restoration and Soil Remediation Alternatives. In: Restoration of Petroleum contaminated Aquifers, Lewis Publishers Inc. MI, USA, pp. 153-190.|
|||Torres, M., J. Goldberg and T.E. Jensen, (1998). Heavy metal uptake by polyphosphate bodies in living and killed cells of Plectonema boryanum (Cyanophyceae). Microbios, 96: 141-147.|
|||USEPA (2001). Mine Waste Technology Program. Activity III, Project 16: integrated passive biological treatment process demonstration. Annual Report. http: //www.epa.gov/ORD/NRMRL/std/mtb/mwtp2001/index.html; 2001.|
|||Volesky, B. (1990). Removal and recovery of heavy metals by biosorption. In: Volesky B, editor. Biosorption of Heavy Metals. Boca.Ration, FL: CRC Press. pp. 7-43.|
|||Wang, H., Kimberley, M.O., Schlegelmilch, M., 2001. Biosolids derived nitrogen mineralization and transformation in forest soils. J. Environ. Qual. 32, 1851-1856.|
|||Wang, Q R; Liu X M; Cui, Y S; Dong, Y T and Christie, P (2002). Responses of legume and non-legume crop species to heavy metals in soils with multiple metal contamination. Journal of Environmental Science and Health. Part A, Toxic/Hazardous Substances and Environmental Engineering, 37: 611-621.|
|||Whisman, M. L., Goetzinger, J. W. and Cotton, F. O. (1971). Radiotracer study of turbine aircraft fuel stability. Air Force Aero Propulsion Laboratory. United States Bureau of Mines. United States of America. P. 126.|
|||Williams, M., E.B. Rastetter, D.N. Fernandes, M.L. Goulden, S.C. Wofsy, G.R. Shaver, J.M. Melillo, J.W. Munger, S.-M. Fan and K.J. Nadelhoffer. (1996). Modelling the soil-plant-atmosphere continuum in a Quercus-Acer stand at Harvard Forest: the regulation of stomatal conductance by light, nitrogen and soil/plant hydraulic properties. Plant Cell Environ. 19: 911-927.|
|||Yong, R.N., Mohamed, A.M.O. and Warkentin, B.P. (1992). Principles of contaminant transport in soils. Elsevier. 322 P.|