Microbiology Project Topics

Effects of Crude Oil on Some Bacteria and Mineral Constituents of Rhizosphere of Delonix Regia Hook

Effects of Crude Oil on Some Bacteria and Mineral Constituents of Rhizosphere of Delonix Regia Hook

Effects of Crude Oil on Some Bacteria and Mineral Constituents of Rhizosphere of Delonix Regia Hook

Chapter One


The objectives of this research were:

  1. To ascertain the effect of crude oil contamination on the bacterial load of rhizosphere of regia.
  2. To ascertain the effect of crude oil contamination on mineral constituents of rhizosphere of regia..
  3. To ascertain the impact of of Delonix regiaon the bacterial load and mineral constituents of crude oil contaminated soil.
  4. To identify some of the bacteria associated with the crude oil contaminated rhizosphere of regia.
  5. To determine the effects of crude oil contamination on some vegetative parameters of regia.

This work was aimed at providing information for the improvement of bacterial activity and mineral constituents of polluted soils which will be useful in managing in-situ bioremediation in order to prevent ecological damage and promote soil fertility and plant growth in oil producing areas.




The adverse effects of crude oil on crops could be due to the disruption of nutrient uptake by petroleum products and the increase in the acidity of the polluted soil. These nutrients include nitrogen, phosphorus, potassium, and calcium, which are essential for the growth and development of plants. Therefore, a decrease in their bioavailability leads to a decrease in plant growth. Aside from its phytotoxicity, excess oil can limit the availability of nitrogen (an important element of plant growth) in the soil (Njoku et al., 2009). According to Nwadinigwe and Onyeidu (2012), the soil rhizosphere of soybeans polluted with crude oil showed a decrease in nitrogen, phosphorus and soil pH. Nkwocha and Duru (2010) reported in their work on the microanalytical study on the effects of oil pollution on local plant species and food crops that potassium and magnesium had a higher mean value at the location affected by crude oil than in the control. The average pH values ​​at the areas affected by the crude oil were also in the acidic range. The effects of crude oil residues on soil chemical properties in oil sites, Momonge Wetland, China, showed higher carbon, increased pH, and lower nitrogen levels in contaminated sites compared to uncontaminated sites (Wang et al., 2010). John et al. (2011) reported that the concentration of lead, nickel, zinc, iron and manganese increased with a corresponding increase in pollution. Benka-Cooker and Ekundayo (1995) observed a significant accumulation of lead, iron and zinc in the crude oil-contaminated soil of the Niger Delta. Parameters such as electrical conductivity, available phosphorus and total nitrogen in soils exposed to crude oil were also comparatively low, while the total organic carbon was high compared to the reference location. However, crude oil may improve the mineral requirements of certain plants. Agbogidi et al. (2007) showed that organic carbon, phosphorus, calcium, magnesium and pH were higher in soil rhizosphere of Zea mays polluted with crude oil while nitrogen was reduced compared to the bare soil. This study demonstrated that crude oil contamination improved the nutrient elements including Mg2+, K+, P, Na+ that were suitable for the grown Zea mays.


When oil is spilled on a farmland, it quickly seeps into the ground and volatile fractions escape, leaving the less volatile fractions for microbial attack (Atlas, 1978). Contamination of the soil by crude oil could change the microbial density and activity even with relatively low levels of contamination (Amadi et al., 1996). Microorganisms that break down crude oil, however, are more common in oil-producing locations than in non-polluted locations (Kerry, 1990). This is because some soil microorganisms, such as bacteria, are able to aerobically degrade crude oil and use the energy gained for their growth and reproduction, and to fix nitrogen for improved plant growth (Odu, 1978). The microbial populations quantitatively reflect the degree or extent of exposure of this ecosystem to hydrocarbon contamination. The bacterial population in petroleum-contaminated soils in the Jordan desert in Iraq was analyzed in terms of soil properties. A noticeable decrease in the number and diversity of bacteria in freshly contaminated soils compared to old ones was observed (Ismail et al., 2008). Although the microbial count is not a direct measure of activity in soils, the data obtained could be used to infer the toxicity of the hydrocarbons to soil microbiota. Fresh spills and / or high concentrations of pollutants often kill or inhibit large parts of the microbial soil population, while soils with lesser or old pollution have a greater number and diversity of microorganisms (Walter et al., 1991).

Many microorganisms which play role in biodegradation have been identified. It is known that greater degradation of oil pollutants is carried out in situ by a consortium of microorganisms than a single microorganism (Nwadinigwe and Onyeidu, 2012). More than 200 species of bacteria, fungi and even algae can biodegrade hydrocarbons. The various genera that have been reported to contain hydrocarbon-degrading species include Pseudomonas, Vibrio, Corynebacterium, Arthrobacter, Brevibacterium, Flavobacterium, Sporobolomyces, Achromobacter, Bacillus, Aeromonas, Thiobacillus, Lactobacter, Staphylococcus, Penicillium and Articulosporium (Okoh, 2003). Anupama and Padamas (2009) also reported Acinetobacter sp. and Pseudomonas sp. (the most reported genus) to be active in hydrocarbon degradation. Assessing microbial response to pollution stress may provide basic information for the improvement of microbial activities in order to promote soil fertility and plant growth.





Top soil was collected from the Botanic garden, University of Nigeria, Nsukka. The seeds of D. regia were collected from the Faculty of Agriculture, University of Nigeria, Nsukka. The Bonny-light crude oil was obtained from Portharcourt.


Water was boiled at 100ºC and removed from the source of heat. The water was poured into a beaker and 100 matured seeds of D. regia were immediately introduced into the beaker. This was kept and allowed to cool for 24 hours before sowing as recommended by Delwaulle (1979).


Ninety six black, perforated polythene bags were filled, each with 18 kg of top soil and 48 soil bags were sowed with a seed of D. regia each.  After one month, 8 soil bags (4 with seedling of D. regia and 4 without seedling) were polluted with 30 ml (0.2%v/w) of crude oil. This was repeated using 150 ml (0.8%v/w) and 750 ml (4.2%v/w) of crude oil. The control was not polluted. The experiment was set up in a completely randomized design in three replicates. One month after pollution, the soil mineral constituents and total viable count of bacteria were analyzed in Soil Science and Microbiology Laboratories, respectively. Bacterial analysis was repeated after the second and third month of pollution. Some vegetative parameters were taken just before pollution and 6 months after pollution.




Six days after sowing, 85% of the seeds germinated in bagged soil. The rest germinated on the 7th day.

Bacterial Enumeration

In the 1st enumeration, the result of the unplanted soil (Np) showed that 750 ml treatment had the highest bacterial count (14.83 ± 0.18 X 106 CFU/g) while 30 ml treatment had the lowest bacterial count (10.25 ± 85  X 106 CFU/g) (Fig.1). Bacterial count decreased in 30 ml and 150 ml treatments but significantly (P<0.05) increased in 750 ml treatment compared with the control. Duncan’s multiple range test indicated that there was no significant difference in the bacterial load of soil treated with 0 ml, 30 ml and 150 ml of crude oil but they were significantly lower than that of 750 ml treatment (Fig.1). In the planted soil (P), the data on bacterial count was also reduced in 30 ml (9.50 ± 0.35  X 106 CFU/g) and 150 ml (10.50 ± 0.50  X 106 CFU/g) treatments but increased significantly (P<0.05) in 750 ml treatment (14.33 X 106 CFU/g) compared to the control (11.53 X 106 CFU/g) (Fig.2). Multiple comparison showed that bacterial load of 750 ml treatment was significantly (P<0.05) higher than the control. There was no significant difference in the bacterial load between 0 ml and 150 ml and between 30 ml and 150 ml treatments.




The consistent reduction in bacterial count in 30 ml and 150 ml treatments could be attributed to the death of microorganisms due to poor nutrient availability. The 30 ml and 150 ml treatments might not have provided adequate food for the oil eating bacteria to the level that may favor their abundance. This is similar to the findings of Ismail et al. (2008) who observed a noticeable greater decline of bacterial counts and diversity but a prevalence of the genus Pseudomonas over the other identified genera in freshly contaminated soils. The increment in bacterial load at 750 ml treatment level compared with the control could be that it was at 750 ml treatment level that the demand for crude oil caught up best with the supply of it for the oil degraders in the soil. This is in line with the statement made by American society for microbiology (ASM, 2013) that when there is a spill of crude or refined oil, the bacteria capable of degrading hydrocarbons proliferate quickly and that microbial cleanup can be considered in terms of “supply and demand”. It is also consistent with the result of Jensen (1975) who reported that the introduction of oily wastes into soil caused appreciable increases in the number of microorganisms. Certain bacteria have evolved the ability to use oil as their food and three of such bacteria were isolated in this work; Pseudomonas, Bacillus and Micrococcus species. Nwadinigwe and Onyeidu (2012) used the consortium of Pseudomonas and Bacillus to effectively remediate crude oil. Okoh (2003) included Pseudomonas and Bacillus in his list of bioremediating soil microbes.  Anupama and Padamas (2009) aslo mentioned  Pseudomonas sp as the most reported genus among oil-degraders. Ekpo and Udofia (2008) and Chickere et al. (2009) reported Micrococcus to be involved in crude oil degradation. It could be suggested that, it is the balance between the proliferation of crude-oil degraders and the death of other bacteria that are vulnerable to its toxicity that determined the number of bacterial colonies obtained in this experiment.



Crude oil caused some major changes in the soil. The predominant factor influencing bacterial availability after contamination according to this work includes contaminant concentration, some mineral elements availability and time. The underlying mechaism for the pattern of bacterial response as shown in this work is suggested to be of significant importance for predicting and managing in-situ bioremediation. Even though D. regia did not improve microbial count, it did not markedly inhibit it. The plant also had less impact on most mineral elements composition. However, it significantly reduced the build- up of carbon caused by crude oil. Also, D. regia plants were found to have a high tolerance to crude oil contamination and surprisingly, the crude oil contamination seemed to have increased the growth of the plant species.  More work is needed to test the total petroleum hydrocarbon accumulated by the plant roots and stems to test whether D. regia can actually be used for phytoremediation of crude oil.  More work is also needed to explore better plants with rhizosphere effect that favors the abundance of microbes to facilitate crude oil remediation in oil producing areas.


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