CHAPTER 1: INTRODUCTION

CHAPTER 2: LITERATURE REVIEW

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

CHAPTER 3: MATERIALS AND METHODS

CHAPTER 4: RESULTS AND DISCUSSIONS

pH Analysis

In the experimental set-up, a total of 30 reactors were utilized to conduct various observations. Specifically, in the case of the room dried Vernonia extracts, Figure 4.1 illustrates the pH behavior as the number of days increases, considering different applications of leaf extracts. Figure 4.1 captures the trends and changes in pH levels over time for the room dried Vernonia extracts. The different leaf extract applications are likely to have varying effects on the pH of the soil samples. By visually representing this data, researchers can analyze how the pH values evolve and potentially identify any patterns or trends that emerge as the days progress. This information provides valuable insights into the impact of the room dried Vernonia extracts on the soil’s pH, helping researchers understand the effectiveness of this particular application method in altering the soil’s acidity levels.

Figure 4. 1,a,b,c,d,e,f: pH behavioural characteristics of the vernonia extracts using different application methods on swampy soil.

Figure 4. 1,a,b,c,d,e,f: pH behavioural characteristics of the vernonia extracts using different application methods on swampy soil.

The study reveals that for low applications of Vernonia extract, specifically within the range of 10-20g for Vernonia amygdalina and around 10-35g for Vernonia galamensis, the pH of 6.76 is consistently maintained. However, over a longer period of remediation time, the pH gradually decreases to approximately 6.75. This decrease in pH indicates a slight shift towards a lower acidity level. Interestingly, the same pattern is observed for both the room-dry and sun-dry methods of Vernonia extract preparations. Regardless of the drying method used, the pH tends to decrease as more masses of Vernonia extracts are added to the contaminated soil. This suggests that the addition of higher quantities of Vernonia extracts has a more pronounced effect on lowering the soil’s pH. These findings highlight the influence of Vernonia extract application and the duration of the remediation process on the pH level of the contaminated soil. The study suggests that higher quantities of Vernonia extracts, regardless of the preparation method, can contribute to a decrease in pH over time, indicating a potential shift towards a less acidic environment.

In contrast to the wet-dry extract applications of Vernonia galamensis, where similar results are observed as with the room dry and sun-dry applications, there are distinct findings for the wet-dry application of Vernonia amygdalina. With the wet-dry application method using Vernonia amygdalina, an interesting trend emerges. As the mass of the Vernonia amygdalina extract is increased by blending wetted extracts onto the contaminated swampy soil, there is an observed increase in pH values over time. This indicates a gradual shift towards higher acidity levels in the soil. These findings suggest that the wet-dry application of Vernonia amygdalina may have a unique impact on the pH of the contaminated soil when compared to other application methods or Vernonia galamensis. The increase in pH over time with the increased mass of Vernonia amygdalina extract implies a potential alkalizing effect on the soil. These insights contribute to a deeper understanding of the behavior and effectiveness of different Vernonia extract applications in relation to pH levels, specifically highlighting the distinctive behavior of wet-dry application with Vernonia amygdalina.

• b. HC analysis.

The remediation of hydrocarbon content from the soil involved the consumption of hydrocarbon molecules by the active microorganisms found in the leaves. It is evident that the remediation of hydrocarbons increases with the addition of the remediant to the contaminated soil. In a 40-day period, the addition of 40g of Vernonia galamensis resulted in the removal of approximately 0.35 ug/ml of hydrocarbons. Similarly, under the same preparation conditions (room dry), time, and quantity, Vernonia amydalina remediated approximately 0.40 ug/ml of hydrocarbons.”

Figure 4. 2, a, b: Hydrocarbon content remediation using vernonia extracts of different masses in different days in swamp soil.

Figure 4. 2, a, b: Hydrocarbon content remediation using vernonia extracts of different masses in different days in swamp soil.

Based on the figure provided, it is evident that Vernonia amydalina demonstrated a higher effectiveness in hydrocarbon remediation compared to Vernonia galamensis. Consequently, it becomes crucial to examine the impact of different preparation methods on the remediation effect. Figure 4.3 provides a detailed explanation of the hydrocarbon remediation effect, taking into account the various methods employed for preparing the extracts.

Figure 4. 3: HC-remediation response on vernonia amygdilina preparation effect.

Figure 4. 3: HC-remediation response on vernonia amygdilina preparation effect.

the room dry preparation of Vernonia amydalina extract has shown to be the most effective method for hydrocarbon remediation in swampy soil. Within a 40-day period, it successfully remediated approximately 0.8 ug/ml of hydrocarbons, which is roughly twice the amount compared to the extracts prepared by sun drying or wet blending methods. Additionally, there was a noticeable significant increase in remediation after 30 days of the study, suggesting that even more remediation is expected after a complete 40-day inspectionre

Metal analyses

Furthermore, the impact on the metal content of the soil is also noticeable, as evidenced by the decrease in metal concentrations. Although the study focused specifically on three metals (Pd, Zn, and Cr), it is important to acknowledge that there may be other metals present in the soil. The analysis of metal concentrations was conducted at the conclusion of the 40-day investigation, taking into account the effects of the various leaf extracts.

Based on the findings illustrated in Figure 4.4, it is evident that the wet and blended Vernonia galamensis leaf extracts exhibit the highest effectiveness in remediating Pb (lead). This method demonstrates a remediation action of approximately 0.47 ug/ml, compared to the room dry and sun-dry methods which resulted in remediation levels of 0.31 ug/ml and 0.23 ug/ml, respectively. Initially, both the wet and room dry approaches exhibit similar levels of microbial activity, albeit minimal, which is still significantly higher than the microbial activity observed in the sun-dry method.Over time, it is observed that the microbial activity in the wet and blended approach gradually increases, leading to a higher level of Pb (lead) remediation in the swamp soil compared to other methods. The sustained microbial activity in the wet and blended approach contributes to a more efficient and effective remediation process for Pb in the soil

Figure 4. 4: Pb remediation using vernonia Galamensis and considering the three preparation methods.

Figure 4. 4: Pb remediation using vernonia Galamensis and considering the three preparation methods.

In Figure 4.5, the focus is on Pb (lead) remediation using Vernonia amydalina. Similar to the previous findings, the wet blended approach using Vernonia amydalina demonstrates a higher level of Pb remediation compared to the room dry and sun-dry methods. The wet blended method achieves a remediation rate of approximately 0.47 ug/ml, while the room dry and sun-dry methods yielded remediation rates of 0.35 ug/ml and 0.26 ug/ml, respectively. These results highlight the potential effectiveness of the wet blended approach using Vernonia amydalina in remediating Pb in the soil

Figure 4. 4: Pb remediation using vernonia Galamensis and considering the three preparation methods.

Figure 4. 4: Pb remediation using vernonia Galamensis and considering the three preparation methods.

“In order to evaluate the performance of Vernonia galamensis and Vernonia amydalina spices in remediating hydrocarbon-contaminated soil, we will also incorporate a measurement scheme for Zinc (Zn) analysis. “At the conclusion of the experimentation, we will conduct measurements to compare the effectiveness of the two different leaf species (Vernonia galamensis and Vernonia amydalina) and the three approaches in remediating Zn metal. This comparison will help us assess the potential of each method in soil remediation.

Figure 4. 5, a, b: Zn remediation using vernonia Galamensis and vernonia Amygdalina considering the three preparation methods.

Figure 4. 5, a, b: Zn remediation using vernonia Galamensis and vernonia Amygdalina considering the three preparation methods.

“The wet blended vernonia species, specifically vernonia Amygdalina, demonstrates a higher Zn remediation effect, reaching approximately 0.37 ug/ml, compared to vernonia Galamensis with 0.33 ug/ml. Interestingly, both the wet and room-dried vernonia Galamensis species exhibit similar remediation effects after 40 days. However, the sun-dried leaves of both species show minimal Zn remediation effects.” During the bioremediation process performed by the two Vernonia species, not only do they exhibit their remediation action on various contaminants, but they also effectively reduce chromium levels in the contaminated soil. This crucial finding further supports the potentiality of the remediating action of Vernonia galamensis and Vernonia amydalina in hydrocarbon-contaminated soil. Figure 4.6

Figure 4. 5, a, b: Cr remediation using vernonia Galamensis and vernonia Amygdalina considering the three preparation methods.

Figure 4. 5, a, b: Cr remediation using vernonia Galamensis and vernonia Amygdalina considering the three preparation methods.

visually presents the data, showcasing the significant impact of their remediation action on chromium in swamp soil, reinforcing the importance and effectiveness of these species in the overall soil remediation process.”

In a surprising turn of events, the sun-dried vernonia Galamensis exhibited the highest remediation effect for chromium metal in the contaminated swamp soil, reaching an impressive 0.47 ug/ml. Comparatively, the wet blended specimen showed a remediation action of approximately 0.38 ug/ml, while the room-dried specimen demonstrated a lower effect at just 0.25 ug/ml. Similarly, in the vernonia Amygdalina experiment, the wet blended specimen showcased the highest remediating effect at about 0.43 ug/ml, followed closely by the sun-dried specimen at approximately 0.38 ug/ml, and the room-dried specimen at 0.35 ug/ml.” These results highlight the unexpected findings and emphasize the role of different drying methods in the remediation potential of Vernonia galamensis and Vernonia amydalina for chromium in swamp soil

4.1.1. Clay Soil Bio Remedial Analysis

“To accurately simulate the environmental conditions present in the study area, a series of clay soil samples were carefully collected and meticulously mixed with bonny light crude oil. This meticulous replication allowed for a comprehensive analysis of the responses of various factors, including pH levels, hydrocarbon contents, and the presence of metals. The measurements of these responses were taken with great consideration given to their initial values (prior to the addition of crude oil) and their corresponding final values (after the crude oil was introduced). These initial and final conditions, depicting the fluctuations and changes resulting from the crude oil addition scenario, have been diligently recorded and are presented in Table 4.2.

Table 4. 2- Initial and Final readings of the Response factors for clay soil.

Table 4. 2- 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)
Clay soil, Csi	6.47	1.3	0.005	0.000	0.002
pH and HC readings for samples after contaminant
Final Content sample	pH	HC	Pb (ug/ml)	Zn (ug/ml)	Cr(ug/ml)
Clay soil, Csf	6.64	4.69	1.21	0.924	1.105

The experimental setup involves the utilization of 30 batch reactors, each serving as a distinct observation point. These reactors provide the necessary environment for conducting the various observations and measurements.

• a. pH Analysis

In order to determine the stability or instability of pH in the clay soil, it is necessary to analyze each scenario and assess whether varying masses of species have an impact on the pH value’s stability.

Figure 4. 6, a, b, c, d, e, f:pH behavioural characteristics of the vernonia extracts using different application methods in clay soil.

Figure 4. 6, a, b, c, d, e, f:pH behavioural characteristics of the vernonia extracts using different application methods in clay soil.

We can see that the pH of the other methods of preparation except the wet blended were stable and had very minor shift in their pH value. For the wet blended vernonia species, the pH tends to increase moving from acidic to normal to alkaline due to the remediation of the excess metals contained in the soil. At 40 days, the pH of the clay soil attained 6.97 and 7.00 for 40 grams of the vernonia Galamensis and vernonia amygdalina respectively. More grams of the vernonia species will ultimately lead to the alkalinity of the clay soil.

• a. HC analysis

A clear indication also showed the hydrocarbon content were remediated to a lower limit. As the mass of the vernonia species increase, so the hydro content reduces. It is seen that from the 35th day, the remediation effects stop increasing and remains constant. The vernonia Galamensis experienced a decrease in the HC remediation as an addition mass was added while an increase was still experienced using vernonia Amygdalina. Figure 4.7 shows the limiting values of the hydrocarbon content remediation. The highest values of remediation were at 35g and 40g representing vernonia Galamensis and vernonia Amygdalina. It is therefore necessary that we discover at what approach will we attain the best remediation effects.

Figure 4. 7, a, b: Hydrocarbon content remediation using vernonia extracts of different masses in different days in clay soil.

Figure 4. 7, a, b: Hydrocarbon content remediation using vernonia extracts of different masses in different days in clay soil.

We can see that from Figure 4.8, that the room dry and the wet blended did a real deal in bio-remediating the hydro carbon in the clay soil. As high as 0.55 ug/ml was attained using vernonia Galamensis while as high as 0.67 ug/ml was attained using vernonia Amygdalina at 35g and 40 g respectively on the wet blended basis.

Figure 4. 8, a, b: Hydrocarbon content remediation method comparism using vernonia Galamensis and vernonia Amygdalina in clay soil.

Figure 4. 8, a, b: Hydrocarbon content remediation method comparism using vernonia Galamensis and vernonia Amygdalina in clay soil.

Metal Analysis

The reduction of the metals in the clay soil was responsible for the increase in the pH value towards the alkaline state. It is best we determine the potentiality of the metal reduction in the clay soil. From the data generated, more metals were remediated as the mass of the vernonia species increased.

• i. Pb Remediating Response

The increased remediating effect on the soil by the reduction of the pH concentration in the soil can be seen appreciative with more Pb reduction as the mass of the vernonia species increases.

Figure 4. 9, a, b:Pb remediation method comparism using vernonia Galamensis and vernonia Amygdalina in clay soil.

Figure 4. 9, a, b:Pb remediation method comparism using vernonia Galamensis and vernonia Amygdalina in clay soil.

From Figure 4.9, it shows that the room dried vernonia species perform more Pb remediation activity. The wet basis vernonia species has poor Pb remediating effects. This can be attributed to the inactivity of the micro-organisms and the Phytochemicals responsible for Pb remediation in that condition. Both species of vernonia leaf achieved about 0.60 ug/ml of Pb remediation.

Zn Remediating Response

Just like the Pb remediation, Zn in the clay soil is also remediated and reduced. Looking at Figure 4.10, we see tha6t the remediating effects compared to other metals is lower as just about 0.25 ug/ml of Zn was remediated which was attributed to the wet blended prepared vernonia species. The room dried vernonia species performed the lowest in remediating the soil from Zn metal removing about 0.17 ug/ml and 0.10 ug/ml for Galamensis and Amygdalina respectively.

Figure 4. 10, a, b:Zn remediation method comparism using vernonia Galamensis and vernonia Amygdalina in clay soiL.

Figure 4. 10, a, b:Zn remediation method comparism using vernonia Galamensis and vernonia Amygdalina in clay soiL.

Cr remediating Response

In terms of Chromium (Cr) remediation, the wet blended method exhibits slightly higher potential compared to other preparation methods, although the difference is not significant. Both Vernonia leaf extracts achieved a remediation rate of 0.5 ug/ml for chromium. Additionally, the sun-dried and room-dried methods also demonstrated a considerable remediation potential of above 0.4 ug/ml.

Figure 4. 11, a, b: Cr remediation method comparism using vernonia Galamensis and vernonia Amygdalina in clay soil.

Figure 4. 11, a, b: Cr remediation method comparism using vernonia Galamensis and vernonia Amygdalina in clay soil.

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.

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

PH Analysis

In order to assess the stability of pH in the contaminated soil, we will investigate the variations in pH levels as different species of Vernonia are introduced. This examination will encompass all the methods used to prepare the Vernonia species, the varying masses added to the contaminated soil, and the duration of time required for the experiment to be observed. By cross-examining these factors, we aim to gain a comprehensive understanding of how the pH levels in the soil respond to the introduction of different Vernonia species. This analysis will provide valuable insights into the effectiveness of each preparation method, the influence of different mass additions, and the time required for observable changes in pH.

Figure 4. 12, a, b, c, d, e, f:pH behavioural characteristics of the vernonia extracts using different application methods on sandy-loamy soil.

Figure 4. 12, a, b, c, d, e, f:pH behavioural characteristics of the vernonia extracts using different application methods on sandy-loamy soil.

Based on Figure 4.12, we observe the impact of introducing Vernonia extracts on the stability of soil pH. Notably, only the sun-dried preparation at 10g and 20g demonstrated a stable pH. In contrast, all other treatments resulted in a change in pH, increasing it and rendering it more neutral. This shift in pH can be attributed to the reduction of metals present in the soil. The results suggest that the sun-dried Vernonia preparation at 10g and 20g effectively maintained the pH stability of the soil, potentially due to their ability to mitigate metal concentrations. Further analysis of these findings could provide valuable insights into the role of Vernonia extracts in soil pH stabilization and metal reduction.

HC Analysis .

The analysis of hydrocarbon content reveals that the remediation of hydrocarbons was more effective when using the Vernonia Amygdalina leaf extract that was dried under room conditions. Interestingly, the performance of Vernonia Amygdalina was more than twice as effective compared to Vernonia Galamensis. Specifically, when 40g of extracts were used for a duration of 40 days, 0.90ug/ml and 1.80ug/ml of hydrocarbons were respectively remediated for Vernonia Galamensis and Vernonia Amygdalina. These findings suggest that the Vernonia Amygdalina leaf extract, dried under room conditions, exhibits superior capabilities in hydrocarbon remediation compared to Vernonia Galamensis. The significant difference in hydrocarbon remediation potential between the two Vernonia species highlights the importance of selecting the appropriate variety for soil remediation purposes

In Figure 4.13a and 4.13b, we examine the remediation of hydrocarbon content in sandy-loamy soil using Vernonia extracts of different masses over different durations. Figure 4.13a displays the hydrocarbon content remediation using Vernonia extracts of varying masses. As the mass of Vernonia extracts increases, we observe a corresponding increase in the effectiveness of hydrocarbon remediation. This suggests that higher quantities of Vernonia extracts result in more efficient removal of hydrocarbons from the soil. Figure 4.13b focuses on the remediation of hydrocarbon content over different durations. It demonstrates that as the duration of the experiment extends, the remediation of hydrocarbons becomes more pronounced. This indicates that prolonged exposure to Vernonia extracts enhances their ability to effectively remediate hydrocarbon contamination in sandy-loamy soil. These results underline the significance of both the mass of Vernonia extracts and the duration of the experiment in achieving successful hydrocarbon remediation in sandy-loamy soil..,To compare the effectiveness of different methods of preparation on the remediation action, we conducted tests using 40g of Vernonia extracts prepared through room drying, sun drying, and wet blending for both Vernonia Galamensis and Vernonia Amygdalina. Figure 4.14 illustrates the results, indicating that the wet blended extracts exhibit a higher capability for remediating hydrocarbon content in the polluted soil. Specifically, 2.11ug/ml and 2.40ug/ml of hydrocarbons were remediated using the wet blended Vernonia Galamensis and Vernonia Amygdalina extracts, respectively. These findings suggest that the wet blending method of preparation enhances the remediation efficiency of both Vernonia species, resulting in a greater reduction of hydrocarbon content in the contaminated soil.When comparing the remediation effect of room-dried extracts, it was observed that Vernonia Amygdalina exhibited a more effective remediation effect compared to Vernonia Galamensis. Specifically, the room-dried Vernonia Amygdalina extracts achieved a remediation value of 1.78ug/ml, while the same method resulted in a lower remediation value of 0.90ug/ml for Vernonia Galamensis. These results indicate that the room-dried extracts of Vernonia Amygdalina have a higher potential for remediating hydrocarbon content in the contaminated soil compared to Vernonia Galamensis. It highlights the importance of considering different Vernonia species and their specific qualities when selecting a remediation approach

Figure 4.14a and 4.14b provide a comparison of the remediation methods for hydrocarbon content using Vernonia Galamensis and Vernonia Amygdalina in sandy-loamy soil. In Figure 4.14a, the remediation method comparison focuses on Vernonia Galamensis. The results indicate that the wet blended method exhibited the highest effectiveness in remediating hydrocarbon content in the soil. The remediation value achieved using the wet blended method for Vernonia Galamensis was 2.11ug/ml, suggesting superior remediation capabilities compared to other methods. Figure 4.14b presents the same comparison but for Vernonia Amygdalina. Here, too, the wet blended method demonstrates the highest remediation effect, with a value of 2.40ug/ml. This result further supports the notion that the wet blended method is particularly effective in remediating hydrocarbon content in sandy-loamy soil, regardless of the Vernonia species used. These findings highlight the importance of selecting the appropriate remediation method when using Vernonia Galamensis or Vernonia Amygdalina for remediating hydrocarbon contamination in sandy-loamy soil

Pb. Metal Analysis

Pb Remediating Response For Sandy-Loamy Soil.

Indeed, as the remediation activity was progressing, the reduction of metals in the contaminated soil became evident, leading to a shift towards neutral pH values. To gain a comprehensive understanding of the potentiality of the mass of Vernonia extracts, it is crucial to conduct a cross-examination of the drop in metal levels in the contaminated soil. By analyzing the metal drop in the soil, we can assess the effectiveness of different masses of Vernonia extracts in remediating metal contamination. This examination will provide valuable insights into the optimal mass of Vernonia extracts required for effective metal reduction in the contaminated soil. Understanding the relationship between the mass of Vernonia extracts and the drop in metal levels will contribute to determining the extent of their potential in remediating metal-contaminated soil and further inform remediation strategies.

Figure 4. 15, a, b: Pb remediation method comparism using vernonia Galamensis and vernonia Amygdalina in Sandy-loamy soil.

Figure 4. 15, a, b: Pb remediation method comparism using vernonia Galamensis and vernonia Amygdalina in Sandy-loamy soil.

From Figure 4.15, it shows that the wet blended vernonia species perform more Pb remediation activity. The sun dry vernonia species has poor Pb remediating effects. This can be attributed to the inactivity of the micro-organisms and the pHytochemicals responsible for Pb remediation in that condition. Both species of vernonia leaf achieved about 0.72ug/ml of Pb remediation for Vernonia Galamensis and 0.99ug/ml for Vernonia Amygdalina.

• i. Zn Remediating Response

The Zn present in the sandy-loamy soil is also remediated and reduced. Looking at Figure 4.16, the room dry and sun-dry vernonia Galamensis gave closely related results leaving the wet blended extract to give about 0.51 ug/ml remediating effect for the vernonia Amygdalina extract, the room dry, sun dry and wet blended gives an approximate remediating values of 0.31ug/ml, 0.52ug/ml, 0.71ug/ml respectively. ultimately, the wet blended gives the best Zn remediating effect.

Figure 4. 16, a, b: Zn remediation method comparism using vernonia Galamensis and vernonia Amygdalina in sandy-loamy soil.

Figure 4. 16, a, b: Zn remediation method comparism using vernonia Galamensis and vernonia Amygdalina in sandy-loamy soil.

• ii. Cr remediating Response

For Chromium, the wet blended shows more Cr remediating potential compared to other methods of preparation. The Vernonia Galamensis remediates 0.67ug/ml while the vernonia Amygdalina remediates 0.68ug/ml

Figure 4. 17, a, b: Cr remediation method comparism using vernonia Galamensis and vernonia Amygdalina in clay soil.

Figure 4. 17, a, b: Cr remediation method comparism using vernonia Galamensis and vernonia Amygdalina in clay soil.

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

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