Revolutionizing Soil Remediation: Exploring the Frontiers of Bioremediation through the Performance Evaluation of Vernonia Galamensis and Vernonia Amydalina Spices in Hydrocarbon-Contaminated Soil

2.1. Review of Previous Works in Bio-Remediation

Since the 1970s, bioaugmentation, or the addition of oil degrading microorganisms to supplement the indigenous populations, has been proposed as an alternate strategy for the bioremediation of oil contaminated environments. The rationale for this approach is that indigenous microbial populations may not be capable of degrading the wide range of potential substrates present in complex mixtures such as petroleum (Leahy and Colwell, 1990) or that they may be in a stressed state as a result of the recent exposure to the spill. Other conditions under which bioaugmentation may be considered are when the indigenous hydrocarbon-degrading population is low, the speed of decontamination is the primary factor, and when seeding may reduce the lag period to start the bioremediation process (Forsyth et al., 1995). For this approach to be successful in the field, the seed microorganisms must be able to degrade most petroleum components, maintain genetic stability and viability during storage, survive in foreign and hostile environments, effectively compete with indigenous microorganisms, and move through the pores of the sediment to the contaminants (Atlas, 1977; Goldstein et al., 1985). Methods involving the addition of selected oil-degrading microorganisms into spilled oil have been patented and marketed since early 1970s (Azarowick, 1973; Linn, 1971; and Mohan et al., 1975). However, before the Exxon Valdez spill in 1989, little information on the performance of commercial bioaugmentation products was available in the peer-reviewed literature. Atlas and Bartha (1973) conducted one of the first laboratory tests on the effectiveness of commercial mixed bacterial cultures. Two commercial petroleum-degrading bacterial inocula, Ekolo-Gest (also marketed as Petrobac, National Chem. Corp.) and DBC bacteria (Gerald Bauer Corp.), were tested using shake flasks to compare the degradation of Sweden crude oil. The study found that none of the commercial mixtures was superior to the indigenous microorganisms in coastal marine waters.

One of first field trials on oil bioremediation using a microbial product in a marine environment was reported by Lee and Levy (1987). The study involved seeding a mixed culture of marine oil-degrading bacteria (strains of Pseudomonas aeruginosa, Pseudomonas stutzeri, and Bacillus subtilis grown on bran) in a Scotian Shelf Condensate (SSC) contaminated sandy beach.

The extent of biodegradation was measured by the decline in the n-C17/pristane ratio in this study. The results showed that the n C17/pristane ratio in the seeded plots did decrease slightly.

However, due to high inter-and intra-plot variability, no significant difference in the rate of oil loss was observed among the treatments. This study also observed that the number of oildegrading bacteria did not increase until 10 to 15 days after the addition of oil. However, the addition of the microbial product did not reduce this lag period, suggesting that the toxic volatile components in the oil, which evaporated mostly during the first week, was the main cause of the lag period.

Since the application of nutrient amendments for the clean-up of the Exxon Valdez spill in 1989, bioremediation has received increased attention, and several field tests and applications of bio augmentation have been reported. Venosa et al. (1992) conducted a field test in Prince William Sound following the Exxon Valdez spill to investigate the effectiveness of two commercial microbial products vis-à-vis natural attenuation and nutrient addition alone. These products were selected based on a previous laboratory study (Venosa et al., 1991). This field trial failed to demonstrate enhanced oil biodegradation by these products. No bio stimulation occurred in the nutrient control plots either. There were no significant differences between any of the treatment and control plots during the 27-day trial period. However, the site where the project took place (Disk Island) was characterized as having highly weathered (degraded) oil and very calm waters, so dissolved oxygen may have been limiting, thus precluding effective biodegradation by any means.

One approach in overcoming the competition problem was proposed by Rosenberg et al. (1992). They developed a product that combined a polymerized urea formaldehyde fertilizer, which they called F-1, with a selected oil-degrading culture capable of using this fertilizer as a nitrogen source. Thus, the culture had a selective advantage over the indigenous population unable to utilize F-1 as nutrient source. A field trial conducted at an Israeli beach showed that this approach seemed to be successful in enhancing oil biodegradation. However, conclusions were confounded by the lack of adequate controls in the study (Swannell et al., 1996; Venosa, 1998). To evaluate the effectiveness of two commercial bio augmentation products in an estuarine environment, a field trial was carried out in a Texas coastal wetland by a research group from Texas A&M University (Simon et al., 1999; Townsend et al., 1999). The two products were selected based on a previous laboratory efficacy test, in which four out of twelve products showed an enhancement of oil biodegradation with significantly higher degradation rates of alkanes and aromatics when compared to a nutrient control (Aldrett et al., 1997). The 21- plot site, named San Jacinto Wetland Research Facility (SJWRF) has been used for a series of studies on oil spills and their countermeasures. In this study, four treatment strategies were examined: an oiled control, biostimulation with inorganic nutrient addition (diammonium phosphate), and commercial bioaugmentation with 2 different products. Arabian medium crude oil was selected in this test and the 21 plots each measuring 5 x 5 m were arranged in a balanced, incomplete block experimental design. Oil constituents were determined using gas chromatography/mass spectrometry (GC/MS) and were normalized to 17α(H), 21β(H)-hopane to reduce the effects of sample heterogeneity and physical losses. The results showed that the addition of microbial products could not significantly enhance oil biodegradation rates. No differences were observed between treatments when comparing the first order biodegradation rate coefficients for the total target saturates, total target aromatics, and individual hydrocarbon target analysts. The authors also pointed out that one of the products (BP8) “did show consistently higher biodegradation rates, though the rates were not significantly different from the control.” Because this microbial product was applied with vendor supplied inorganic nutrients (Townsend et al., 1999), it is difficult to conclude whether the “consistently but insignificantly” higher rates resulted from the additions of the microbial components or the nutrient components. The fact that neither addition of bioaugmentation agents nor application of inorganic nutrients significantly enhanced oil biodegradation suggested that other factors, such as oxygen, could have been limiting oil degradation in that environment. Studies comparing the performance of bioaugmentation and biostimulation have suggested that nutrient addition alone had a greater effect on oil biodegradation than did the addition of microbial products when oxygen supply was not limited (Jobson et al., 1974; Lee etal., 1997; Venosa et al., 1996). This is probably because the hydrocarbon-degrading population is rarely a limiting factor as compared to the nutrients since the size of the hydrocarbondegrading bacterial population usually increases rapidly in response to oil contamination. One of the first comprehensive field tests evaluating various bioremediation approaches to enhance oil biodegradation was carried out in a soil environment in northwest area of Canada in early 1970s (Jobson et al., 1974). A randomized block design was used to examine the effects of four treatments (control, inorganic fertilizer application, addition of a microbial culture alone , and combined fertilizer and microbial culture addition) over a 308-day time period. The microbial culture was grown in the laboratory and consisted of several genera of oil-degrading bacteria (Flavobacterium and Cytophoga sp., Pseudomonas sp., Xanthomonas sp., Alcaligenes sp., and Arthrobacter sp.). The study showed that the nutrient application resulted in a significant stimulation of bacterial numbers and in the degradation rate of n-alkane components of the crude oil. The application of the microbial agent, however, resulted in only a slightly enhanced degradation rate of n-alkane components of chain lengths C20 to C25.

A field study conducted on a sandy beach in Delaware also showed that addition of a microbial inoculum did not enhance oil biodegradation more than addition of inorganic nutrients alone (Venosa et al., 1996). A randomized block design was used in this study to assess the effects of three treatments: a no-nutrient control (natural attenuation), addition of water-soluble nutrients, and addition of water-soluble nutrients supplemented with a natural microbial inoculum from the site. No significant differences were observed between plots treated with nutrients alone and plots treated with nutrients and the indigenous inoculum, suggesting that supplementation of the natural population with indigenous cultures from the same site still did not result in further enhancement over simple nutrient addition on marine shorelines. The authors also indicated that this conclusion could be extended to include exogenous microbial inocula or commercial microbial agents because “if indigenous cultures do not accelerate the degradation rates, organisms enriched from different environments, grown in the laboratory, and not acclimated to a particular climatic or geographic location should be even less able to compete with the natural population.”

Lee et al. (1997) conducted a 129-day field trial to compare the effect of four treatments on biodegradation of weathered Venture Condensate on a sandy beach in Nova Scotia, Canada. The four treatments (control, inorganic nutrient addition, a commercial bioremediation product, and addition of inorganic nutrients along with bioremediation product) as well as an uncoiled control were replicated in a complete block design using 20 enclosures or plots. C2-chrysene was used as the normalizing biomarker due to the low concentration of hopane in the condensate.

PRP (PetrolRem, Inc.) was selected to be the representative commercial bioremediation agent in this study. This product is no longer listed in the current NCP Product Schedule. According to Lee et al. (1997), PRP contains mineral nutrients and nonpathogenic bacteria within spherical particles made from plant derived natural products (beeswax) and exhibits both bioaugmentation and biostimulation properties. The agricultural fertilizer used in this study was a mixture of granular forms of ammonium nitrate (N:P:K: 33-0-0) and triple super phosphate (N:P:K: 0-46-0).

2.2. Limitations and Environmental Issues of Bioremediation in the Niger Delta

The delta covers 20,000 km² within wetlands of 70,000 km² formed primarily by sediment deposition. Home to 20 million people and 40 different ethnic groups, this floodplain makes up 7.5% of Nigeria’s total land mass. It is the largest wetland and maintains the third-largest drainage basin in Africa. The Delta’s environment can be broken down into four ecological zones: coastal barrier islands, mangrove swamp forests, freshwater swamps, and lowland rainforests.

This incredibly well-endowed ecosystem contains one of the highest concentrations of biodiversity on the planet, in addition to supporting abundant flora and fauna, arable terrain that can sustain a wide variety of crops, lumber or agricultural trees, and more species of freshwater fish than any ecosystem in West Africa. The region could experience a loss of 40% of its inhabitable terrain in the next thirty years as a result of extensive dam construction in the region. The carelessness of the oil industry has also precipitated this situation, which can perhaps be best encapsulated by a 1983 report issued by the NNPC, long before popular unrest surfaced:

Since then, there has been the slow poisoning of the waters of this country and the destruction of vegetation and agricultural land by oil spills which occur during petroleum operations. But since the inception of the oil industry in Nigeria, more than twenty-five years ago, there has been no concerned and effective effort on the part of the government, let alone the oil operators, to control environmental problems associated with the industry. (Bogumil, 2014).

Reports on the extent of the oil spills vary. The Department of Petroleum Resources (DPR) estimated 1.89 million barrels of petroleum were spilled into the Niger Delta between 1976 and 1996 out of a total of 2.4 million barrels (Vidal, 2010) spilled in 4,835 incidents. (The Daily Independent,2010) (approximately 220 thousand cubic metres). A UNDP report states that there have been a total of 6,817 oil spills between 1976 and 2001, which account for a loss of three million barrels of oil, of which more than 70% was not recovered.(UNDP,2006) 69% of these spills occurred off-shore, a quarter was in swamps and 6% spilled on land.

The Nigerian National Petroleum Corporation places the quantity of petroleum jettisoned into the environment yearly at 2,300 cubic metres with an average of 300 individual spills annually (Bronwen,2007). However, because this amount does not take into account “minor” spills, the World Bank argues that the true quantity of petroleum spilled into the environment could be as much as ten times the officially claimed amount. (Moffat and Linden, 2009). The largest individual spills include the blowout of a Texaco offshore station which in 1980 dumped an estimated 400,000 barrels (64,000 m3) of crude oil into the Gulf of Guinea and Royal Dutch Shell’s Forcados Terminal tank failure which produced a spillage estimated at 580,000 barrels (92,000 m3). (Nwilo,2001) In 2010 Baird reported that between 9 million and 13 million barrels have been spilled in the Niger Delta since 1958. (Baird, 2010) One source even calculates that the total amount of petroleum in barrels spilled between 1960 and 1997 is upwards of 100 million barrels (16,000,000 m3). (Bronwen,2007)

During bioremediation activities, it has been discovered that some of the constituents remains in the soil after the spills are treated. Studies by several scholars show the effects as thus described in the analysis done by Mauro and Wynne.

The study showed that an average of 11.0% of the n-alkanes remained in the oiled control plots, and only 0.1% of the oil remained in the enclosures treated with inorganic nutrients alone; 5.4% of the alkanes were found in the plots treated with inorganic nutrients and PRP, and 25.3% remained in the plots treated with PRP alone. The results indicate that periodic addition of inorganic nutrients was the most effective strategy for enhancing oil degradation and that the full potential of the bioremediation product was limited by nutrient availability. This field trial demonstrated that adding the bioremediation product did not perform better in terms of enhancing alkane degradation than applying inorganic agricultural fertilizers alone.

Several other possible reasons for the failure of inocula in degrading contaminants in nature were summarized by Goldstein et al. (1985), which include: (1) the concentration of the contaminant may be too low to support the growth of the inoculated species, (2) the natural environment may contain substances inhibiting growth or activity of the inocula, (3) the growth rate of the inoculated species may be limited by predation such as protozoa, (4) the added species may use other substrates in nature rather than the targeted contaminants, and (5) the seeded microorganisms may be unable to move through the pores of the sediment to the contaminants.

A few field trials did claim success in demonstrating the effectiveness of oil bioaugmentation, such as using Alpha BioSeaTM (Alpha Environmental, Inc.) to treat the Angolan Palanca crude oil spilled from Mega Borg off Texas coast (Mauro and Wynne, 1990; Swannell et al., 1996) and using TerraZymeTM (Oppenheimer Biotechnology) in enhancing biodegradation of a heavy oil spilled from Nakhodka in Japan (Tsutsumi et al., 2000). However, the success of these studies was based on either visual observation (i.e., the Mega Borg study) or digital photographic image analysis (i.e., the Nakhodka study). No comprehensive monitoring program was used to verify the oil was indeed removed through enhanced biodegradation. The two products basically contain the same bacterial cultures and nutrients (Hozumi et al., 2000). The observed visual effects might have been due to physical or chemical processes such as surfactant action associated with the products (Swannell et al., 1996) or sinking.

All these peer-reviewed journal articles show that even though the addition of microorganisms may be able to enhance oil biodegradation in the laboratory, the effectiveness of bio augmentation has not been convincingly demonstrated in the field. Actually, most field studies indicated that bio augmentation is not effective in enhancing oil biodegradation in inland, estuarine, and marine environments. It appears that in most environments, indigenous oil degrading microorganisms are more than sufficient to carry out oil biodegradation if nutrient levels and other adverse environmental conditions do not limit them.

2.3. Current Bioremediation Technologies for Crude Oil Contaminated Sites

Bioremediation is a technology that exploits the abilities of microorganisms and other natural habitat of the biosphere to improve environmental quality for all species, including man. The development of innovative bioremediation technology as a functional tool in clean-up of crude oil polluted environment has depended so much on the basic knowledge of the physiology and ecology of the natural bacterial populations found in such polluted sites. Many advances in biochemistry and molecular biology are now applied in various bioremediation efforts (Olson and Tsai, 1992; Bouwer, 1992). According to some investigators (Barbee, et al., 1996; Ritter and Scarborough, 1995), bioremediation does not always result in complete mineralization of organic compounds. Many of these compounds are naturally transformed to metabolites of unknown persistence and toxicity. Therefore, some basic steps that may be necessary for a successful bioremediation project will include compliance analysis, site characterization, method selection / feasibility studies, remediation proper and end for project analysis (Bonaventura, et al., 1995). Compliance analysis requires examination of the contaminated site in the light of the governing regulation and the action plan. Examination of the site will lead to its characterization and this is a very challenging and difficult aspect of a bioremediation efforts. Knowledge of soil parameters such as cation exchange capacity, relevant nutrient availability, acidity (soil pH), aeration or oxygen level, hydraulic properties etc are paramount and this requires the assistance of specialists in these areas. The last stage of any bioremediation project should include bioassay of the treated site. This confirms complete or near complete removal of the PHC contaminant. According to Lovely (2003), combining models (including mathematical models) that can predict the activity of microorganisms involved in bioremediation with existing geochemical and hydrological models should transform bioremediation technology.

Some necessary process variables involved in bioremediation of petroleum hydrocarbon polluted environments that need to be known include the characteristics of the polluting crude oil, its biodegradability and the characteristics of the polluted site (physical and chemical) Logistic problems with respect to accessibility to the polluted site (e.g., swamps) must be known, together with the impact of the clean-up operation. The last point is very important because it is known from several studies that in some natural detoxification processes, cellular mechanisms of hydrocarbon compound metabolism can create compounds or metabolites that are more toxic than the parent hydrocarbons, especially when the end products are not only carbon dioxide and water. The situation is even complicated by the fact that biochemical reactions rarely proceed by a single pathway. Hence one of the greatest difficulties in assessing the success of bioremediation of crude oil-contaminated environment is having knowledge of the fate of the metabolites after uncontained in situ treatment (Jenkin and Sanders, 1992).

In full-scale bioremediation technologies of crude oil polluted ecosystems, many rate-limiting factors are known (Atlas, 1991; Prince, 1992), and they include presence of other toxic compounds other than crude oil pollutant, the level of available oxygen and nutrients (particularly nitrogen and phosphorus), temperature and pH. Other factors are moisture content or water availability, biodiversity of hydrocarbon clastic and co-metabolising bacteria at the site. The adsorptive capacity of the hydrocarbons to the soil and sediment, and rate of mixing and mass transfer are also important factors. In terrestrial ecosystem, spilled oil adsorbs to the soil particles, forming a cohesive, toxic mixture that is deleterious to the indigenous microorganisms. These events or soil characteristics reduce or increase the bioavailability of petroleum hydrocarbons, the inherent toxicity and hence biodegradability. These factors are responsible for the long delays in the mineralization of the petroleum hydrocarbons (PHC) by the indigenous or applied microbial populations. Effective metabolism of crude oil requires adequate oxygen supply as electron acceptor. Under low oxygen tension as in the mangrove ecosystem, the use of biologically active absorbent (Gregorio, 1996) to fix the oil and effect medium term biodegradation is desired. It should be noted that the extent of crude oil impact on the soil equally depends on the concentration spilled, ease of dissociation from the soil matrix, particle size of the soil, porosity, or permeability. To facilitate bioremediation requires methods that can dissociate the PHC and create conditions for mass transfer process (Onwurah, 2000).

Bioremediation of crude oil contaminated environment may require some engineering process, so as to facilitate recovery efforts. Engineering may include construction of booms, trenches, and barriers for contaminant containment, boreholes, bio-cells and using engineered microbial systems. Increasing bioavailability of the PHC can be achieved by physically processing the crude oil-polluted soil or sediment by excavation, pulverising and mixing. The above processes maximize aeration and surface area for microbial activity. Some specific bioremediation processes that may require engineering are summarized below.

2.3.1. In Situ Land Treatment

The simplest method of bioremediation of oil polluted soil is in situ land treatment. This technology utilizes standard farming procedures such as plugging the oil-polluted soil with a tractor, periodical irrigation and aeration. This technology embraces the use of aerobic microorganisms to degrade the PHC and other derivatives to carbon dioxide and water, or other less toxic intermediates. Experience has shown that when land-farming technology is properly executed for PHC contaminated soil, non-volatile components of petroleum and other related products are rapidly immobilized, so may not be leached out. This technology may involve nutrient enrichment in the form of fertilizer application or further manipulation of site conditions such as inoculations with selected or adopted microbial population, mixing and aeration of the soil surface, pH adjustment and irrigation. Using this technology an enhancement in the decontamination of 50cm topsoil of an area previously polluted with crude oil was achieved (Compeau, et al., 1991). Possible enhanced soil fertility recovery for such oil polluted agriculture soil has been demonstrated in soil microcosm experiments where germination and growth of sorghum grains were improved after treatment with adapted Azotobacter inoculum (Onwurah, 1999a).

2.3.2. Composting Technology

Composting technology is becoming important in the treatment of oil polluted coastal area. It involves the mechanized mixing of contaminated soil or sediment with compost-containing hydrocarbonoclastic bacteria, under aerobic and warm conditions. Through the addition of corn slash (post-harvest leaves and stems), microbial nitrogen fixation has been co-optimized with petroleum hydrocarbon degradation (Paerl, et al., 1996).

2.3.3. Bioreactors

A bioreactor is essentially an engineered system in which biochemical transformation of materials is promoted by optimizing the activity of microorganisms, or by “in vitro” cellular components of the microbial cells (enzymes). Bioreactors for the remediation of oil-polluted soil utilize an aqueous slurry phase system. Slurry bioreactor is considered as one of the fastest bioremediation technologies because contaminants can be effectively transported to the microbial cells. Some limiting factors affecting the slurry phase bioreactor process during decontamination of oil-contaminated soil and how they can be controlled are listed in Table 2.1. An attractive alternative to the slurry bioreactors for treating oil-contaminated soils are the rotating drum bioreactors since they can handle soils with high concentrations of petroleum hydrocarbons (Gray et al., 1994; Banerjee et al., 1995). The fluid phase enhances transport of nutrients and “solublized” or dispersed PHC contaminants to the degrading bacteria. With a bioreactor, temperature, pH and other parameters are optimized for degradation. The rotating drum bioreactor incorporated with blade impellers inside was demonstrated to be effective in decontaminating hydrocarbon-polluted soil (Hupe et al., 1995). The contaminated soil must be excavated, mixed with water and introduced into the reactor. Generally, the rate-limiting factors in any bioreactor system used for crude oil degradation are, the degree of PCH solubilisation through bio-surfactant production and the level or concentration of active biomass of hydrocarbon clastic bacteria maintained in the system (Stroo, 1992). Degradation products in bioreactors are easily monitored and input regulated. Bioreactors are however intrinsically more expensive than in situ or land treatment technologies because they are specialized.

2.3.4. Biodegradation

Biodegradation, especially by microbes, is one of the primary mechanisms of ultimate removal of petroleum hydrocarbons from polluted environments (Atlas, 1988; NRC, 1985). The acceleration of this natural process is the objective of bioremediation efforts. Seeding a contaminated environment with strains of bacteria that are tolerant and capable of degrading a high percentage of the contaminating petroleum hydrocarbons, and thus supplementing the natural resident microbial population has proven to be useful in bioremediation. The relative success of such adapted (oxotic) bacteria when added to crude oil polluted site will depend on a number of factors including competitive interactions with the native bacteria, their rate of growth in the system as well as their tolerance to the physio-chemical environment (Leahy and Colwell, 1990). The advent of high-throughput methods for DNA sequencing and analysis of genomes as well as modelling of microbial processes have revolutionized environmental biotechnology (Lovely, 2003). Genetically altered or engineered microorganisms

2.4. Sources and Effects of polluted soils in the Niger Delta

2.4.1. Sources

Oil spills are a common event in Nigeria [Baird, 2010]. Half of all spills occur due to pipeline and tanker accidents (50%), other causes include sabotage (28%) and oil production operations (21%), with 1% of the spills being accounted for by inadequate or non-functional production equipment. Corrosion of pipelines and tankers is the rupturing or leaking of old production infrastructures that often do not receive inspection and maintenance. (Nwilo, 2001)

A reason that corrosion accounts for such a high percentage of all spills is that as a result of the small size of the oilfields in the Niger Delta, there is an extensive network of pipelines between the fields, as well as numerous small networks of flow lines—the narrow diameter pipes that carry oil from wellheads to flow stations—allowing many opportunities for leaks. In onshore areas most pipelines and flow lines are laid above ground. Pipelines, which have an estimate life span of about fifteen years, are old and susceptible to corrosion. Many of the pipelines are as old as twenty to twenty-five years. (Bronwen, 1999)

Shell admits that “most of the facilities were constructed between the 1960s and early 1980s to the then prevailing standards. SPDC (Shell Petroleum and Development Company) would not build them that way today.”(SPDC, 1999) Sabotage is performed primarily through what is known as “bunkering”, whereby the saboteur attempts to tap the pipeline. In the process of extraction sometimes the pipeline is damaged or destroyed. Oil extracted in this manner can often be sold.

Sabotage and theft through oil siphoning has become a major issue in the Niger River Delta states as well, contributing to further environmental degradation. (Anderson, 2005) Damaged lines may go unnoticed for days, and repair of the damaged pipes takes even longer. Oil siphoning has become a big business, with the stolen oil quickly making its way onto the black market.(Bronwen, 1999).

While the popularity of selling stolen oil increases, the number of deaths are increasing. In late December 2006 more than 200 people were killed in the Lagos region of Nigeria in an oil line explosion. (CNN, 2016).

2.4.2. Effects of Oil Spillage on the Environment

Oil spillage has a major impact on the ecosystem into which it is released and may constitute ecocide. Immense tracts of the mangrove forests, which are especially susceptible to oil (mainly because it is stored in the soil and re-released annually during inundations), have been destroyed. An estimated 5 to 10% of Nigerian mangrove ecosystems have been wiped out either by settlement or oil. The rainforest which previously occupied some 7,400 km² of land has disappeared as well.

Spills in populated areas often spread out over a wide area, destroying crops and aquacultures through contamination of the groundwater and soils. The consumption of dissolved oxygen by bacteria feeding on the spilled hydrocarbons also contributes to the death of fish. In agricultural communities, often a year’s supply of food can be destroyed instantaneously. Because of the careless nature of oil operations in the Delta, the environment is growing increasingly uninhabitable.

People in the affected areas complain about health issues including breathing problems and skin lesions; many have lost basic human rights such as health, access to food, clean water, and an ability to work. [Baird, 2010] other socio-related effects are as follows:

• a. Human Health Risk

As much as it is sad to say or admit, a lot of people have suffered as a result of oil spillage in many different ways. Particularly people that live near the banks of Niger Delta are prone to these risks than any other. Oil definitely poisons the water and considering that people living there are mostly farmers that depend on it for their daily livelihood, it ends up affecting them health wise. As much as Oil brings many benefits, the risk it poses to human life is devastating.

• b. Damage to The Ecosystem

The impacts of oil pollution on marine ecosystem can be categorized into long term and short term effects. Suffocation cause by oil spills and oil poisoning are among the first group.Oil spills reduce oxygen absorption of the water, causing oxygen dissolution under oil spills to be even less than the deep sea levels. The oil penetrates and opens up the structure of the plumage of birds, reducing its insulating ability, and so making the birds more vulnerable to temperature fluctuations and much less buoyant in the water. It also impairs birds’ flight abilities, making it difficult or impossible to forage and escape from predators.

• c. Health Hazard to The Aquatic Animals

We all understand what the marine nature offers to our society. Oil spillage causes so much damage to the aquatic animals. The spillage poisons the water and eventually leads to death of the animals that come to contact with the area affected by it.

• d. Risk to Food Security

With poverty looming in some parts of the country, the oil spillage is making the situation even more critical. The spillages that occur on lands cause damage to the plants, it poisons the soil and leads to poor farm produce which should be among the major source of food stuff in the country.

• e. Loss of Aquatic Plants

There are several plants that play very important role in the water bodies. The Oil spillages sometimes get to the bottom and cause more harm to the aqua life. Some of the plants act as food to other species of aquatic animals. This generally leads to other poisoning that affects the entire ecosystem.

• f. Depletion of Fish Population

Fish has a special place in the nature’s food chain especially to humans. Considering the nutritional value of fish to the humans, playing a role in their depletion in a wasteful manner is the most unfortunate thing. Fishing is also one of the most profitable industries; taking good care not to destroy them has much more benefits to the society.

• g. A Danger to The Wildlife in General

Wildlife is one of the major tourist attractions of the country, the wildlife should always be protected at all cost. Unfortunately, oil spillage plays a big role in destroying the wildlife. As the oil spillage poisons the water, most of the wildlife is affected as they all depend on it.

• h. Pollutes The Air Leading to Other Illness.

It is known that oil has chemical gas that can get mixed up with the air we breathe, there are several cases reported that link to these chemicals. This effect can be fatal to individuals who are exposed to these chemicals.

• i. Acid Rain

Acid rain is also another factor as the rain water becomes corrosive and also pollutes the remaining water sources. This also affects the plants and soil in general. Acidic soil affects the plants negatively; plants don’t get enough nutrients leading to poor produce.

• j. Poverty

Poverty is a state that exists to be fought at all times, it can only be achieved with concentration. The major problem is that the oil spillage diverts money that would have been used to help eradicate poverty to other use like treating the affected areas.

It is unfortunate to say that most of these spillages which are sometimes termed as accidents are due to human error. Companies in such matters should always treat this matter as very important and make an important step to deal with it. It is important to ensure that the future of the next generation is safe guarded, we should all take this as a challenge and deal with this issue on a personal level or else it will come to haunt us in the near future.

2.5. Mechanisms for Phytoremediation of Hydrocarbons

Phytoremediation is the use of plants and their associated microorganisms to remove, sequester or degrade contaminants in soil (Cunningham et al., 1996). Phytoremediation is a proposed technique to remediate both inorganic contaminants like heavy metals (Brown et aI., 1994; Pilon-Smits et al., 1999), and organic like pesticides (Siciliano and Germida, 1998b) and petroleum hydrocarbons (Aprill and Sims, 1990). Plants useful for remediation of inorganics are species that hyperaccumulate the element of concern (Banuelos et al., 1997). The plants are eventually harvested, removing the metals from the soil; this is called phytoextraction (Ebbs et al., 1997). Some plants can also volatilize (i.e., transfer the contaminant to the atmosphere) heavy metals (Zieve and Peterson, 1984, Pilon-Smits and Pilon, 2002).There are three mechanisms by which organic contaminant phytoremediation can occur: degradation, stabilization, and volatilization (Sims and Overcash, 1983; Cunningham et al., 1996; Siciliano and Germida, 1998b). While stabilization or volatilization is acceptable in some situations, degradation of the contaminant into nontoxic compounds is the most desirable outcome.

2.5.1. Degradation

Degradation occurs when hydrocarbons are broken down into simpler and usually less toxic compounds (Eweis et al., 1998). Plants and microorganisms in isolation, and in association, degrade hydrocarbons. However, the ability to degrade hydrocarbons is much less common in plants than in microorganisms.

2.5.2. Stabilization

Stabilization occurs when a plant reduces the bioavailability of a contaminant; the contaminant is not, however, degraded. This method of phytoremediation is useful in situations where prevention of ground water contamination is desired, or where the contaminant is not mobile or toxic to humans (Cunningham et al., 1996).

2.1.3. Volatilization

In some cases, chemicals are neither degraded nor stabilized; they are volatilized into the atmosphere. Plants absorb some chemicals through their roots and then release them into the atmosphere through stomata (Wiltse et al., 1998). Naphthalene volatilization is enhanced by the presence of bell rhodesgrass (Chloris Gayana Kunth). Nitrobenzene volatilization is a major route of chemical loss when soybean, barley (Hordeum vulgare L.), hybrid poplar (Populus x robusta Schneid.) and honeysuckle (Lonicera tatarica L.) are grown in soil contaminated with this chemical. About 10% of diesel range hydrocarbons (C11- C16) in soil are volatilized over 150 days when planted with grasses. Diesel volatilization over 360 days is 580/0 when white clover (Trifolium repens L.) and ryegrass (Lolium perenne L.) are grown together. Larger hydrocarbons may be less likely to volatilize: less than 2% of benzo(a) Yrene (a five-ring hydrocarbon) loss from silty loam soil planted with tall fescue is due to volatilization. Concerns regarding volatilization arise when the chemical is potentially dangerous in its gaseous form. (Robson, 2003).

3.3.2. TO CONDUCT THE MICROBIAL ANALYSIS, THE FOLLOWING MATERIALS ARE REQUIRED:

• Conical flask: Used for holding and mixing liquids during the analysis.
• One 5 ml syringe: Used for precise measurement and transfer of liquids.
• One 1 ml syringe: Used for more accurate and smaller volume measurements.
• Distilled water: Used as a solvent or diluent for various solutions.
• Nutrient agar: A solid growth medium used for culturing and isolating microorganisms.
• Vernonia leaf extract from the four species of bitter leaf (Vernonia amygdalina and Vernonia galamensis bitter leaf): The extract obtained from the Vernonia leaves, which will be used for microbial analysis.
• 30 Petri dishes: Used as containers for the agar medium and microbial cultures.
• Masking tape: Used for labeling and identification purposes.
• Cotton wool: Used for sterilization and plugging of test tubes
• Aluminium foil: Used for covering the Petri dishes to protect the cultures.
• Test tubes: Used for preparation and storage of samples or media.
• Nutrient broth: A liquid growth medium used for cultivating microorganisms.
• Autoclave: A device used for sterilizing equipment and media through high-pressure steam.
• Weighing balance: Used for accurately measuring the weight of substances. These materials are essential for conducting the microbial analysis and ensuring the proper cultivation, identification, and characterization of microorganisms present in the Vernonia leaf extracts.

THE PROCEDURES FOR THE MICROBIAL ANALYSIS ARE AS FOLLOWS:

• Wash the conical flask, test tubes, and petri dishes with distilled water. This step ensures cleanliness and reduces the risk of contamination during the analysis.
• Prepare the nutrient agar culture: a. Dissolve 28 grams of nutrient agar in 1 liter of distilled water to prepare the standard culture. This forms the base for the nutrient agar medium. b. Using a spatula, carefully place the powdered nutrient agar on the foil. c. In a conical flask, dissolve 3 grams of the weighed nutrient agar powder in 100 ml of distilled water. d. Cover the conical flask with cotton wool to prevent contamination. After completing these steps, you can proceed with further analysis and experimentation. It is important to maintain a sterile environment throughout the process to ensure accurate results and prevent unwanted microbial growth.

TO PREPARE THE NUTRIENT BROTH CULTURE, FOLLOW THESE STEPS:

• Place a small piece of aluminum foil on the digital weighing balance and zero the scale to ensure accurate measurements.
• Using a spatula, carefully place the pellets of nutrient broth on the foil until the weighing balance reads 5 grams. This ensures the correct amount of nutrient broth for the culture.
• In a conical flask, dissolve the weighed 5 grams of nutrient broth in 500 ml of distilled water. Stir or swirl the flask gently to aid in the dissolution process. By following these steps, you will have prepared the nutrient broth culture. This liquid medium is commonly used for cultivating and growing a wide range of microorganisms. It provides necessary nutrients to support microbial growth and can be used for various purposes in microbial analysis, such as culturing and studying specific bacteria or fungi strains.
• Using the 1 ml syringe, collect 20 milliliters of the nutrient broth water
• Carefully transfer the collected nutrient broth water into 6 test tubes, distributing approximately 20 milliliters in each test tube.
• Cover each test tube with cotton wool. This acts as a barrier to prevent contamination from external microorganisms.
• 4 Wrap aluminum foil around each test tube to further protect them and maintain their sterility.
• Once the test tubes are properly covered and wrapped, they are ready for sterilization using an autoclave. Autoclaving is a process that effectively kills microorganisms and ensures the sterility of the samples. Follow the manufacturer’s instructions or standard laboratory protocols for autoclaving to properly sterilize the test tubes. By following these steps, you will have prepared the nutrient broth culture in the test tubes, ensuring their sterility and readiness for further analysis or experimentation.
• 1 ml syringe: Used for precise measurement and transfer of liquids during the dilution process.
• 6 sterilized test tubes with nutrient broth: These test tubes containing nutrient broth are pre-sterilized and ready for use in the dilution process. They provide a growth medium for the microorganisms present in the Vernonia leaf extract.
• Vernonia leaf extract: The Vernonia leaf extract obtained previously, which will be diluted for further analysis. 4.
• Bunsen burner: A gas burner used for sterilization purposes. It provides a high-temperature flame to sterilize the syringe and other equipment by passing them through the flame. These materials are essential for performing a serial dilution, a technique used to decrease the concentration of a substance, in this case, the Vernonia leaf extract, to a level suitable for microbial analysis. The dilution process allows for the isolation and enumeration of microorganisms present in the extract by reducing their initial concentration.

TO PERFORM A TENFOLD SERIAL DILUTION, FOLLOW THESE STEPS:

• Collect 1 ml of Vernonia leaf extract from each different species and place it in a separate test tube containing nutrient broth. This step ensures that each species extract is diluted individually.
• Shake each test tube vigorously to ensure thorough mixing of the Vernonia leaf extract with the nutrient broth.
• From the first test tube, take 5 ml of the mixture and transfer it to a new test tube.
• Shake the second test tube vigorously to mix the 5 ml of the mixture with the nutrient broth.
• From the second test tube, take 1 ml of the mixture and transfer it to another new test tube.
• Repeat this process by taking 1 ml from the previous test tube and adding it to a new test tube until you obtain about 20 test tubes that have been serially diluted.

By following these steps, you will have successfully performed a tenfold serial dilution of the Vernonia leaf extract. Each subsequent dilution reduces the concentration of the extract, allowing for the isolation and enumeration of microorganisms present in the initial sample. This process provides a range of diluted samples for further analysis and microbial counting.

TO CONTINUE WITH THE PREPARATION OF THE CULTURE, FOLLOW THESE STEPS:

• Pour approximately 20 ml of nutrient agar into each sterilized petri dish. Make sure to distribute the agar evenly across the dish.
• Invert the petri dishes so that the nutrient agar hangs from the top of each dish. This is done to allow the agar to solidify and adhere to the bottom surface of the dish.
• Wait until the nutrient agar has completely solidified and sticks to the top of the petri dishes. This ensures a stable surface for the subsequent steps.
• Take 1 ml from each of the serially diluted broth cultures and place it in a separate petri dish. This step involves transferring the diluted broth culture onto the solidified nutrient agar.
• Carefully spread the 1 ml of diluted broth culture across the surface of each petri dish using a sterile spreader or an appropriate technique, ensuring an even distribution.
• Once the diluted broth culture has been spread, cover the petri dishes and place them into an incubator or a temperature-controlled environment where the temperature is maintained at 37°C (not 370°C, as that would be extremely high). By following these steps, you will have prepared the culture by inoculating the serially diluted broth cultures onto nutrient agar plates. The incubation at 37°C promotes the growth of microorganisms, allowing for their visual observation and further analysis.

For the preparation of plant material and plant extracts, a total of four different extraction solvents were employed, namely cold ethanol, hot ethanol (at a temperature of 80°C), cold water, and hot water (also at a temperature of 80°C). These solvents were utilized for the extraction of the desired constituents from the plants.

FOR THE HOT ETHANOLIC AND HOT AQUEOUS EXTRACTION METHODS, 50 grams of finely ground powder from V. amygdalina and V. galamensis were separately placed in two conical flasks. Subsequently, 200 ml of absolute ethanol was added to the ground V. amygdalina, while distilled water was added to the ground V. galamensis. The mixture in each flask was then heated to a temperature of 80°C using a water bath and allowed to stay at this temperature for 1 hour. Afterward, the mixture was cooled down, passed through a muslin cloth, and filtered using a Whatman No.1 filter paper with a diameter of 110 mm

The filtrate obtained from the extraction process was subjected to evaporation at a controlled temperature of 45°C until it completely dried out. The resulting residue was carefully stored inside aluminum foil to maintain its integrity. Later on, the residue was reconstituted in 95% ethanol to create a stock solution with a concentration of 250 mg/ml, which was stored at a temperature of 4°C.

4.1.1. Swampy Soil Bio Remedial Analysis

In the study, swampy soil samples were collected and mixed with Bonny Light crude oil to simulate the conditions of the Ogini pollution scenario. The soil samples were measured before and after the crude oil was mixed to assess the impact of the contamination. Additionally, the study focused on measuring the response factors that are central to our investigation. Table 4.1 presents the initial and final conditions of these response factors. These recorded measurements provide valuable insights into the changes that occurred as a result of the crude oil contamination. By comparing the initial and final conditions, researchers can analyze the extent of the impact on the response factors and evaluate the effectiveness of the remediation methods employed. This data plays a crucial role in understanding the effects of the contamination and the potential efficacy of the remedial measures implemented in the study.

Table - Initial and Final readings of the Response factors for swampy soil

pH and HC readings for samples before contaminant
Initial Content sample	pH	HC	Pb (ug/ml)	Zn (ug/ml)	Cr(ug/ml)
Sandy Loam Soil, SLSi	6.76	2.59	0.018	0.022	0.015
pH and HC readings for samples after contaminant
Final Content sample	pH	HC	Pb (ug/ml)	Zn (ug/ml)	Cr(ug/ml)
swamp soil, SSf	6.81	4.7	1.24	0.921	1.107

4.1.3. Sandy-Loamy Soil Bio Remedial Analysis

To replicate the conditions found in the Ogoni land, sandy-loamy soil samples were collected and mixed with Bonny Light crude oil. The pH levels, hydrocarbon contents, and metal concentrations were carefully measured by comparing the initial and final values before and after the addition of crude oil. Table 4.3 presents the recorded initial and final conditions of the response factors.

Table 4. 3- Initial and Final readings of the Response factors for clay soil.

pH and HC readings for samples before contaminant
Initial Content sample	pH	HC	Pb (ug/ml)	Zn (ug/ml)	Cr(ug/ml)
Sandy Loam Soil, SLSi	6.76	2.59	0.018	0.022	0.015
pH and HC readings for samples after contaminant
Final Content sample	pH	HC	Pb (ug/ml)	Zn (ug/ml)	Cr(ug/ml)
Sandy Loam Soil, SLSf	6.75	4.67	1.22	0.923	1.103

Table 4. 3- Initial and Final readings of the Response factors for clay soil.

pH and HC readings for samples before contaminant
Initial Content sample	pH	HC	Pb (ug/ml)	Zn (ug/ml)	Cr(ug/ml)
Sandy Loam Soil, SLSi	6.76	2.59	0.018	0.022	0.015
pH and HC readings for samples after contaminant
Final Content sample	pH	HC	Pb (ug/ml)	Zn (ug/ml)	Cr(ug/ml)
Sandy Loam Soil, SLSf	6.75	4.67	1.22	0.923	1.103

4.2-. Modal- Prediction Analysis

Now that we have ascertained the performance of the leaf extracts, it is customary that we try to develop a model that illustrates the individual remedial activity in the soil from the data generated. The metals and the hydrocarbon contents stands as the measurable responses from the remediation process with independent factors like the grams of vernonia species and the days taken. Since from the analysis done, the wet blended method showed more promising remediation effects. It is best we use this approach and perform the models on this methodology.

Performing a multiple regression analysis, the use of the least square method will be employed using the Minitab software.

4.2.1. Model for Swamp Soil

Using the least square method explained earlier, the multiple regression for the remediation process can be explained below by using the data in Appendix ….:

(A). IN THE REGRESSION ANALYSIS FOR VERNONIA GALAMENSIS, THE EQUATION TO PREDICT THE HC (HYDROCARBON) CONCENTRATION IS AS FOLLOWS:

HC = 11.0 + 0.00416 Time + 0.00107 Mass - 1.63 * pH

The coefficient values for each predictor variable are as follows:

• Constant: 11.040
• Time: 0.0041616 with a standard error (SE) of 0.0003246
• Mass: 0.0010702 with a standard error (SE) of 0.0003751
• pH: -1.627 with a standard error (SE) of 1.359.

The T-values and P-values indicate the significance of each predictor variable:

• Time: T = 12.82, P < 0.000 (highly significant)
• Mass: T = 2.85, P = 0.008 (significant)
• pH: T = -1.20, P = 0.242 (not significant

Regression: The model includes 3 degrees of freedom (DF) and the sum of squares (SS) for regression is 0.100302. The mean sum of squares (MS) is 0.033434. The F-value is 91.04, indicating that the regression model is statistically significant. The p-value is 0.000, further supporting the significance of the regression model.

Residual Error: The residual error, which represents the variability not explained by the regression model, has 26 degrees of freedom. The sum of squares for the residual error is 0.009549, and the mean sum of squares is 0.000367.

Total: The total sum of squares is 0.109851, with a total of 29 degrees of freedom