Introduction
The growing population of the world, along with an increasing acceptance of an industrialized lifestyle, has inexorably increased the anthropogenic impact on the biosphere, petroleum exploration and exploitation being the major contributor (Chu and Karr, 2017). Petroleum, like all other fossil fuels, is made up of complex chemical structures known as hydrocarbons. At high dosages, hydrocarbons found in crude oil and petroleum products are extremely toxic to many species, including humans. Due to the global economy’s reliance on petroleum products, these toxins are released into populated areas and ecosystems all over the world (Ojumu et al., 2003). The long-term persistence of petroleum contamination depends on the amount and quality of the hydrocarbon mixture, as well as the characteristics of the impacted environment (Ojo, 2005). The petroleum sector has provided economic benefits to many countries but resulted in pollution that has brought environmental and socio-economic difficulties.
Oil-producing areas are frequently in danger because the soil is destroyed and rendered infertile as a result of oil spills and other concerns related to oil exploration or transportation, preventing crop development. Erhenhi and Ikhajiagbe (2012) have observed that oil has a multitude of phytotoxic effects on plant growth and development, thus resulting in massive agricultural and economic losses in contaminated areas. Other researchers (Vwioko and Fashemi, 2005; Anoliefo et al., 2017; Ikhajiagbe et al., 2017) have reported that petroleum hydrocarbons are hazardous to both plants and mammals. The presence of oil in the soil is a major environmental concern as it creates unfavorable conditions for plant growth, most commonly due to insufficient soil aeration. This could be attributable to oil displacing air from pore spaces and an increase in oxygen demand caused by the activities of oil-decomposing bacteria, which affects normal diffusion processes (Gudin and Syratt, 1975).
Nowadays, physical and chemical technologies are mostly used for cleanup in petroleum hydrocarbon management procedures. The most common cleaning methods are neither straightforward nor environmentally friendly (Ashraf et al., 2014). For example, chemical sorbents and dispersants are considered fail-safe since they introduce more dangerous substances into the environment (Ashraf et al., 2014). Therefore, the bioremediation technology must be relied upon. In bioremediation, biological organisms, especially bacteria, are used in environmental cleanup, such as B. subtilis, Micrococcus varians, and P. aeruginosa (Anoliefo et al., 2006; Ikhajiagbe et al., 2012; Ikhajiagbe and Anoliefo, 2012a, 2012b). These bacteria, as well as many others that use hydrocarbons, help in soil recovery. Bioaugmentation with materials such as animal waste is used to improve the activities of hydrocarbon utilizers and soil properties that are necessary for the intrinsic rehabilitation of hydrocarbon-polluted soils (Ikhajiagbe and Anoliefo, 2012a, 2012b).
Microorganisms have been proven to be cost-effective and ecologically acceptable in the decomposition of petroleum hydrocarbons and their components in the environment (Agarry et al., 2013; Yakubu, 2007).The ability of the soil microbial community to breakdown petroleum pollutants is determined by its structure and diversity. Previous studies have reported that bioaugmentation of contaminated marine and terrestrial environments showed superior treatment efficiency (Tang et al., 2010; Kadali et al., 2012). However, some studies have also reported that bioaugmentation did not result in a significant increase in bioremediation and that the hydrocarbon-degrading bacteria did not show any degradation activity in some cases (Yu et al., 2005). Therefore, the use of microbes and soil amendment materials in bioremediation is becoming increasingly popular (Nwinyi and Akinmulewo, 2019). During the bioremediation process, soil amendments or additions such as sawdust, plant waste materials, plant debris, manure, and fertilizers are used to enhance the activity of microorganisms. Soil amendment improvesthe physical properties of the soil, such as water retention, permeability, water infiltration, drainage, aeration, and structure (Davis and Wilson, 2005). Moreover, most of the animal wastes such as dung contain a diverse spectrum of microorganisms and other expelled components that are crucial for soil amendment. This research aimed to explore how chicken and pig manure alters the microbial composition and bioremediation performance of diesel-contaminated soil.
Materials and methods
Study area
Soil, poultry droppings, and pig dung were obtained from the field, poultry, and pig farm, respectively, in the Nigeria Institute for Oil Palm Research (NIFOR)in Edo State. This research institute (latitude 06°33′N and longitude 05°37́′N) is located in the rainforest zone of Nigeria. The rainfall ranges from 1500 to 2135 mm. The mean temperature ranges between 31 and 21°C.
Sample collection
Soil samples were collected at depths of 0–15 cm, and poultry droppings and pig dung were air-driedand sieved with a 2-mm sieve. Diesel was bought from the FAGCoop Filling Station in the University of Benin campus, Nigeria, and transported to NIFOR where the experiment was set up.
Experimental design
First, 1250 ml of diesel was added to 5000 g of soil and mixed thoroughly. The diesel-contaminated soil was subjected to the addition of the poultry droppings – pig dung mixture in three ratios: 1 : 1, 1 : 2, and 2 : 1 (Table 1). The treatments were conducted in five replicates. The soil samples were kept for 4 weeks in plastic containers in a well-ventilated screen house located at the Department of Plant Biology and Biotechnology, University of Benin. The setup was constantly wetted with 250 ml of distilled water every other day. Each of the experimental setups had a control, in which diesel was added but not the poultry droppings–pig dung mixture.
Isolation of the total bacteria and fungi
The pour plate method adopted from Adams et al. (2014) and Cheesbrough (2006) was used to isolate bacteria and fungi from the samples (soil, poultry droppings, and pig dung). The samples were air-dried and sieved through a 2-mm mesh to remove debris. Then, they were diluted by transferring 1 g of the samples to 9 ml of sterile distilled water in sterile glass containers as blank. The glass containers were shaken for 5 min, and the samples were taken as l0−1 dilution factor; then, 10 ml was transferred from the 10−1 dilution into another 9 ml blank to obtain a dilution factor of 10−2, and the same transfer process was repeated twice to obtain a dilution factor of 10−4. For each of the samples, 1 ml of the 10−4serially diluted portion was inoculated onto nutrient agar plates for bacterial count determination and potato dextrose agar plates for fungal count determination. The plates were inoculated at room temperature for 24 and 72 h, respectively, for bacterial and fungal growth. After incubation, colonies were counted, and the colony-forming units per gram (CFU/g) of the soil samples was determined. Nystatin at a final concentration of 0.05 mg/l (Morton Grove Pharmaceuticals, Inc., USA) was added to the nutritional agar to prevent fungal growth during bacterial isolation, whereas 0.05 mg/l of chloramphenicol (Rambaxy Nigeria Limited, Lagos) was added to the potato dextrose agar to prevent bacterial growth during fungal isolation. The Petri dishes were covered, inverted, and incubated for 48 h at 37°C for bacterial isolation and 96 h at 28°C for fungal isolation. Isolation and characterization of bacterial isolates were carried out using the methods of Cowan and Steel (1974) and Cheesebrough (2006).
Fungal isolates were characterized by determining their colonial morphology on plates, texture, and surface appearance. A microscopic examination of the isolates was carried out to determine the nature of the mycelium and the type of the fruiting body. The fungi atlas was used to identify the microorganisms (Adams et al., 2014; Cheesbrough, 2006).
Isolation and identification of hydrocarbon-utilizing bacteria and fungi
For the isolation and identification of hydrocarbonutilizing bacterial (HUB) and fungal isolates in the diesel-contaminated soil samples, the experiments were set up using 2% (v/v) of diesel as a carbon source in the basal mineral salt medium. The composition of the basal mineral salt medium used in this study was as follows (g/l): NaNO3 (2.0), NaCl (0.8), KCl (0.8), CaCl2 · 2H2O (0.1), KH2PO4 (2.0), Na2HPO4 · 12H2O (2.0), MgSO4 (0.2), and FeSO4 · 7H2O (0.001).
The initial pH was adjusted to 6.8. The mineral salt agar was sterilized at 121°C for 15 min at 15 psi and allowed to cool to about 47°C. Filter paper moistened with diesel was placed on the lid of each plate. The plates were inverted, and their edges were sealed with masking tape to increase the vapor pressure of the hydrocarbons, which provides carbon as the sole energy source. The viable counts of hydrocarbon-degrading bacteria and fungi in the samples were taken, and their colonies were purified by streaking them on fresh nutrient agar and inoculating them on PDA, respectively. Then, the colonies were stored in slants at 4°C prior to characterization. The developed colonies were counted and expressed as CFU/g for bacteria and spore-forming units per gram (SFU/g) for fungi.
Physicochemical analysis
The following physicochemical characteristics of the contaminated soil and the treated soil samples were evaluated: pH, total nitrogen, total available phosphorus, and exchangeable potassium. The pH was measured using a pH meter (Mettler Toledo Seven Compact™ pH meter S210). The exchangeable potassium content was determined following the procedure of Udo and Ogunwale (1986). The total nitrogen content was determined using the Kjeldahl technique (Bremner, 1965). The total available phosphorus content was determined using the Bray 1 method (Bray and Kurtz, 1945).
Data analysis
The experiments were performed in triplicate, data were recorded, and statistical analyses were carried out using SPSS, version 16, at the 95% confidence level. The results are presented as means and standard error of means. One-factor ANOVA was conducted upon the assumption of homogeneity of the experimental plot.
Calculation of hazard quotient and contamination factor (CF) of the TAH content in the remediated medium
The hazards quotient (HQ) expresses the probability of a contaminant being an ecological risk factor or a contaminant of potential ecological concern, whereas the CF expresses the ratio between the eventual concentrations of a pollutant and its preindustrial concentration (Ikhajiagbe and Anoliefo, 2012a):
When HQ > 1, the contaminant can cause harm. When HQ = 1, the contaminant alone is not likely to cause ecological risk. When HQ < 1, harmful effects are not likely.
Results
Physicochemical characteristics of the remediated oil-polluted substrate
Altogether, the pH of the soil, poultry droppings, and pig dung ranged from 4.8 to 7.4, with the poultry droppings showing the highest pH of 7.4 (Table 2). The total nitrogen content ranged from 0.12 to 0.46%, with the pig dung showing the highest value of 0.46% and the poultry droppings showing a value of 0.42%. Thirty days after the polluted soils were subjected to soil amendments, increases in the total nitrogen, total available phosphorus, and exchangeable potassium contents were observed (Table 3). The pH of the treated soils ranged from 4.80 to 7.27. The pH of the soil with the 1 : 1 treatment ratio was around 7. The 1 : 2 treatment ratio resulted in a slightly acidic pH of 6.57–6.77, whereas the 2 : 1 treatment ratio resulted in a more acidic pH. The total nitrogen content in the soil ranged from 0.12 to 0.52%, and the 2 : 1 treatment ratio resulted in the highest nitrogen content (0.52%).The total available phosphorus content ranged from 27.31 to 90.19 mg/kg, and the 1 : 2 treatment ratio showed the highest value of 90.19 mg/kg.
Table 2
Physicochemical characteristics of the contaminated and control soil, poultry droppings, and pig dung
Table 3
Physicochemical characteristics of the contaminated and treated soil, after 30 days of treatment
[i] pH – hydrogen ions, TN – total nitrogen (%), P – available phosphorus (mg/kg), K – exchangeable potassium (cmol/kg); mean values in the same column with the same superscripts are not significantly different from each other (P < 0.05); LSD – least significant differences; ANOVA – analysis of variance
Bacterial and fungal composition of the polluted medium
A total of nine bacterial isolates were isolated from the soil, poultry droppings, and pig dung altogether (Table 4). Pseudomonas aeruginosa and Brucella sp. were isolated from the soil, poultry droppings, and pig dung. Staphylococcus sp., Salmonella sp., P. vulgaris, and Klebsiella sp. were isolated from poultry droppings and pig dung. Among the five fungal isolates, Mucor sp. and Geotrichium sp. were not observed in the soil, but present in poultry droppings and pig dung (Table 4).
Table 4
The presence or absence of bacterial isolates in the soil, poultry droppings, and pig dung
Table 5
Fungal isolates of the soil, poultry droppings, and pig dung
| Fungi isolates | Soil | Poultry droppings | Pig dung |
|---|---|---|---|
| Aspergillus niger | + | + | + |
| Geotrichium sp. | − | + | + |
| Mucor sp. | − | + | + |
| Penicillium sp. | + | + | + |
| Saccharomyces sp. | + | + | + |
The bacterial counts were higher than the fungal counts (Table 6). The total bacterial count (TBC) ranged from 1.2 to 2.2 × 104CFU/g. In the diesel-contaminated soil, the TBC decreased from 1.5 × 104 to 1.2 × 104CFU/g (Table 6). The pig-dung-based soil amendment showed a TBC of 2.0 × 104CFU/g and an HUB content of 0.9 × × 104CFU/g. Pig manure showed a total hydrocarbonutilizing fungi (HUF) content of 0.12 × 104CFU/g. At 30 days after the treatment, significant increases in the TBC were observed in all diesel-contaminated soil samples (P < 0.05) (Table 7). When the soil was amended with the pig dung–poultry droppings mixture (pg/pd), the TBC increased from 0.4 × 104CFU/g to 1.2–2.7 × × 104CFU/g. In soils treated with 500 g of poultry dropping and 500 g of pig dung (abbreviated as Pd 500 g/Pg 500 g), the TBC and the BUB content increased by 66.66% and 171.14%, respectively. The highest HUF content was observed in the Pd 300 g/Pg 150 g treatment, which showed a 240% increase compared with the unamended diesel-contaminated soil. Moreover, increases in the HUF content were affected by soil amendments as well.
Table 6
Bacterial and fungal counts of the contaminated soil, poultry droppings, and pig dung
Table 7
Bacterial and fungal counts of the contaminated and treated soil after 30 days of exposure to experimental treatments
[i] Pd – poultry droppings, Pg – pig dung, Δ%—percentage change to background level, ΔTBCbackground – 1.5, TBC – total bacterialcount, −Δ indicates reduction compared with the background, ΔHUBbackground – 0.7, HUB – hydrocarbon-utilizing bacteria, +Δ indicates an increase compared with the background, ΔTFCbackground – 0.4, TFC – total fungi count, ΔHUFbackground – 0.5, HUF – hydrocarbon-utilizing fungi
Table 8
Bacterial isolates in the contaminated and treated soil after 30 daysof exposure to the experimental treatments
| Sample | Proteus vulgaris * | Klebsiella sp.* | Staphylococcus sp. | Pseudomonas aeruginosa * | Shigella sp. | Micrococcus sp.* | Bacillus subtilis * | Brucella sp. | Salmonella sp. |
|---|---|---|---|---|---|---|---|---|---|
| Soil | − | − | + | + | − | + | + | − | − |
| Soil (with diesel) | + | + | − | + | − | + | + | + | − |
| Pd 100 g/Pg 100 g | + | + | − | + | + | + | + | + | − |
| Pd 300 g/Pg 300 g | + | + | − | + | + | + | + | − | + |
| Pd 500 g/Pg 500 g | + | + | + | + | + | + | + | − | + |
| Pd 50 g/Pg 100 g | + | − | − | + | − | + | + | + | − |
| Pd 150 g/Pg 300 g | + | − | − | + | − | + | + | − | − |
| Pd 250 g/Pg 500 g | + | − | + | + | + | + | + | − | + |
| Pd 100 g/Pg 50 g | + | + | − | + | − | + | + | + | − |
| Pd 300 g/Pg 150 g | + | + | + | + | + | + | + | − | + |
| Pd 500 g/Pg 250 g | + | + | + | + | + | + | + | − | + |
| Percentage of occurrence % | 90.9 | 63.6 | 45.5 | 100 | 54.54 | 100 | 100 | 36.4 | 45.5 |
Five of the nine bacterial isolates showed the capacity to utilize hydrocarbons present in the soil, namely P. aeruginosa, P. vulgaris, Klebsiella sp., Micrococcus sp., and B. subtilis. P. aeruginosa, Micrococcus sp., and B. subtilis were present in all three samples – the soil, pig dung, and poultry droppings, implying that they were present in all of soil, pig dung and poultry dropping samples. Klebsiella sp. and P. vulgaris showed a percentage of occurrence higher than 60%. In addition, A. niger was detected in all experimental treatments. Saccharomyces sp. and Geotrichium sp. showed the lowest occurrence rates among the fungal species (Table 9).
Table 9
Fungal isolates in the contaminated and treated soil after 30 days of exposure to the experimental treatments
The list of aliphatic hydrocarbons present in both control and amended soils is provided in Table 10. Some of the hydrocarbons present were n-pentacosane, n-tetracosane, n-hexacosane, n-octacosane, and n-nonacosane. The control showed a TAH content of 763.40 mg/kg. However, the 2 : 1 treatment ratio of the poultry droppings and pig dung showed a TAH content as low as 38.44 mg/kg, which implied significant remediation. Bioremediation indices were computed to evaluate the bioremediation efficiency of the pig-dung-amended and poultry-dropping-amended diesel-polluted soil (Table 11). Chromatographic representations of the aliphatic hydrocarbons present in the diesel-contaminated soil and treated with poultry droppings and pig dung are shown in Figures 1–11 in Supplementary materials.
Table 10
Total aliphatic hydrocarbon content of amended and unamended diesel-contaminated soil 30 days after exposure to the experimental treatments
Table 11
Evaluation of the progress of remediation after exposure to the experimental treatments
[i] BRE – bioremediation efficiency (%); ΔCF – contamination factor; the higher the CF, the more likely it is still contaminated compared with the background value; ΔHQ – hazard quotient; when HQ > 1, harmful effects are likely due to the contaminant in question; when HQ = 1, the contaminant alone is not likely to cause ecological risk; when HQ < 1, harmful effects are not likely; TAH – total aliphatic hydrocarbon (limits for TAH 2000 mg/kg); NA – not applicable
At day 0, the TAH content of the clean soil prior to contamination was 11.2 mg/kg. This was considered the background concentration in this study. Upon contamination with diesel (also at day 0), the TAH content increased to 4028 mg/kg, thus indicating a CF of 182.86 and an HQ of 1.02. According to Ikhajiagbe and Anoliefo (2012a), a contaminant with an HQ > 1 is said to be of environmental concern. The TAH was 763.03 mg/kg after 30 days in the contaminated soil without any modification, implying a CF of 68.13 and an HQ of 0.38. With an HQ value of less than one, the contaminant (diesel) was below the limit, i.e., it was not harmful to the environment. The remediation rate was 62.74%. However, when the contaminated soils were treated with the pig dung – poultry droppings mixture, the remediation rate increased to 98.13%. The CF and the HQ were drastically decreased to 3.43 and 0.02, respectively, at that point. These results emphasize the importance of soil amendments as hydrocarbon remediation facilitators.
Discussion
Petroleum hydrocarbons enter the environment through a variety of channels, such as storage tank leaks, faulty transfer lines, and product transportation from one location to another, andtheir environmental impact can be disastrous; thus, deliberate attempts to eradicate them from the ecosystem are essential (Chikere and Ekwuabu, 2014). Reports have suggested that remediation of hydrocarbon-polluted soils can be enhanced by introducing nutrients and bacteria into them (Ikhajiagbe and Anoliefo, 2012a, 2012b; Agarry et al., 2013). The use of nutritional supplements such as animal manure (Agarry et al., 2013) and other materials like inorganic fertilizers (Margesin et al., 2001) in thebiostimulatation of degraders in contaminated environments has been extensively investigated as well.
The significant reduction in hydrocarbons in the polluted soil observedin this study wasmost likely due to the increased microbial activity in the soil due to the poultry droppings and pig dung amendment. The following microorganisms capable of metabolizing petroleum hydrocarbons were isolated from pig dung and chicken manure: Shigella sp., B. subtilis, Micrococcus sp., P. vulgaris, Klebsiella sp., Salmonella sp., Brucella sp., and P. aeruginosa, all of which had previously been documented (Williams et al., 1999; Ojumu et al., 2004; Chikere and Azubike, 2014). Pseudomonas are well-known biosurfactant-producing bacteria that can use hydrocarbons as a carbon and energy source (Rahman et al., 2003). They have been extensively investigated in the production of glycolipid biosurfactants. Biosurfactants help bacteria consume more hydrocarbons by increasing the surface area of the oil (Nikolopoulou and Kalogerakis, 2009).
Das and Chandran (2010) reported that establishing adequate nutrient concentrations is one of the most crucial factors in guaranteeing optimal microorganism growth rates for petroleum hydrocarbon biodegradation. In the present study, the amended soils showed considerable increases in soil nutrients. This was most likely due to the improvement in remediation capacity. Azubuike et al. (2016) emphasized the relevance of nutrients, especially nitrogen and phosphorus, in the biodegradation of hydrocarbons. Barrett (2008) reported that chicken droppings have considerable levels of nitrogen, phosphate, and potassium. Okolo et al. (2005) used poultry manure as an organic fertilizer in contaminated soil and found that biodegradation was improved by more than 90%in the presence of poultry manure.
Das and Chandran (2010) reported that the pH of a remediating compound should be between 6 and 9, which is similar to that of the present study. Although remediation of the TAH content of the soil was observed, the remediation rate and efficiency were enhanced when the soil was amended with either pig dung or poultry droppings.
Conclusions
The relevance of using organic manure in bioremediation to assist ecosystem processes in polluted areas cannot be overstated. Bioremediation efficiency is critical when the number and types of microbes in polluted soils or other media are increased. Organic amendments such as poultry droppings and pig dung provide a lot of benefits, especially enhancing soil parameters and microbial efficiency. Supplementing contaminated soils with the poultry droppings – pig dung mixture considerably accelerated the remediation of the diesel-contaminated soil.
