Isolation and Characterization of Hydrocarbon-degrading Bacteria in Soils of Mechanical Workshops in Maiduguri, Borno State

The study was conducted to determine the distribution of hydrocarbon utilizing bacteria in spent engine oil (SEO) contaminated soil. Five mechanical workshops within Maiduguri Metropolis. Five bulk soil sample comprising of one each of the five sites; the sites are Leventis Area on Kashim Ibrahim Way, Damboa Raod, Boiler (commonly called Bola) near Maiduguri Monday Market, Ngomari, on Kano Road, and 1000 Housing Estate, also on Kano Road. Nutrient agar was used, or isolation and enumeration total heterotrophic bacteria and Mineral salt agar was used for the isolation and enumeration of hydrocarbon utilizing bacteria. The result shows THB (92.0x104) at Damboa road (DR) to as higher and (78.8x104) at Bola area (BL) while HUB (2.0x104) at Damboa road (DR) and as high as (9.3x104) at Bola area (BL). The bacterial species isolated were species of Bacillus licheniformis, Bacillus subtilis, Bacillus coagulans, Bacillus alvei, Bacillus cereus, Bacillus lentus other are Pseudomonas aeruginosa, Klebsiella pneumonia. Bacillus licheniformis, and Bacillus subtilis are the most occurring bacterial isolates identified. The ability of those bacterial isolate to degrade hydrocarbon buoyantly will help in remediation of oil polluted environments.

Microbial studies of biosurfactant producing bacteria from crude oil contaminated soil

Soils contaminated with crude oil collected from three sites in Delta State were analyzed using serial dilution and pour plate methods. The physicochemical characteristics of the soil were determined using standard methods. The mean heterotrophic bacteria count and the mean hydrocarbon utilizing bacteria count were also determined. Blood haemolysis, foaming activity, oil spreading techniques and emulsification assay was used to screen the microorganisms for biosurfactant production. The biosurfactant producing bacteria were selected and their effect on metal removal and growth kinetics was also determined. The pH of the contaminated soil samples ranged 4.82 to 5.62. The mineral elements such as potassium (k+) and sodium (Na+) ranged from 0.20 to 0.80 Meq/100g and 0.07 to 0.81 Meq/100g respectively. The heavy metal content such as zinc (Zn2+) and lead (Pb2+) ranged from 10.13 to 19.24 mg/kg and 19.24 to 49.63 mg/kg respectively. Organic carbon and THC ranged from 5.44 to 6.87 % and 2720.00 to 3110.00 mg/kg respectively. The mean heterotrophic bacteria and hydrocarbon utilizing bacteria ranges from 1.4 ± 0.4 x 106 to 1.6 ± 0.7 x 106 and 1.0 ± 0.2 x 106 to 1.2 ± 0.4 × 106 respectively in the soil samples. Bacteria isolated and identified from the three sites include Corynebacterium spp., Bacillus subtilis., Pseudomonas aeruginosa, Micrococcus varians, Streptococcus spp., Klebsiella spp. and Enterobacter spp. with Bacillus subtilis and Pseudomonas aeruginosa having the highest prevalence and among these isolates only Corynebacterium spp., Bacillus subtilis., Pseudomonas aeruginosa and Micrococcus varians showed positive result for the production of biosurfactant. Bacillus subtilis had the highest metal removal capacity of 29.44% of 100 ppm of lead. Corynebacterium spp. and Bacillus subtilis had an optimum growth at pH 8 and 7 respectively while Pseudomonas aeruginosa and Micrococcus varians has an optimum growth at pH.

Degradation of Crude Oil by Bacteria Isolated from Various Soil Plantation at Idanre, Nigeria

Degradation of crude oil by bacteria isolated from three plantations soil at Idanre, Nigeria were comparatively investigated. Soil samples collected by hand trowel at 5cm rhizosphere of Cocoa, Orange, and Teak plantations from Idanre, Ondo State. The crude oil – forcados blend was obtained from Warri, Delta State, Nigeria. Gram negative bacteria were isolated from rhizosphere soil sample using standard microbiological methods. Preparation of Biomass was done by centrifuging nutrient broth repeatedly to wash cells. Harvested cells were obtained for the degradation of crude oil. Harvested cells were inoculated with crude oil and then incubated in a shaker. Degradation of crude oil was monitored by using spectrophotometer to read the OD at 540 nm. The bacteria isolated from rhizosphere of cocoa include: Citrobacter freundii, Yersinia pestis, Edwardsiella tarda,. Serratia marcescens. Rhizosphere of orange; Providencia stuatii while Enterobacter agglumerans, Moellerella wisconsis were isolated from rhizosphere of teak. The most effective hydrocarbon utilizing bacteria was Enterobacter agglumerans, which resulted in increase in population densities and reduction in hydrocarbon contents in the crude oil. Findings from this study shows the effectiveness of degrading hydrocarbon in liquid medium and further confirmed the potency of bacterial cells to degrade crude oil.

Evaluation of the physico-chemical state of the soil contaminant isolate indigenous bacterial species

The aim of the study is to isolate indigenous bacterial species which have the ability to degrade crude oil. Samples were collected from an oil polluted site in Ejama-Ebubu Eleme Rivers state. The samples were taken from five spot (A-E), of depth 0-15 (A1– E1) and 15-30 (A2– E2) in sterile polyethylene bags, using appropriate equipment, then taken to the laboratory for analysis, Physicochemical parameters such as pH, nitrate, sulphate, phosphate, total petroleum hydrocarbon (TPH), poly aromatic hydrocarbon (PAH), salinity, temperature, conductivity and heavy metals (iron, zinc, nikel, lead, chromium) were determined, The THB count was determined using the spread plate method on nutrient agar. Soil, sediment and water physicochemical parameters determined indicated that the samples had been exposed to hydrocarbon contamination. The Gram negative bacteria belonging to the genus pseudomonas is the most frequent. Other genera isolated were Nocardia, Micrococcus,Chromobacterium, Burkholdia, Corynebacterium. The study revealed the presence of petroleum hydrocarbons in the Ejama_Ebubu site as well as known genera of hydrocarbon utilizing bacteria. The biases associated with culture-dependent microbial enumeration techniques may limit the full description of the bacterial diversity in Ejama-Ebubu site. From the study, it is concluded that microorganisms that can degrade hydrocarbons are found in oil contaminated soil and can easily be isolated from these contaminated sites, although it is very difficult to work with aromatic hydrocarbons due to their volatility and toxic effects.

Molecular Characterisation and Plasmid Profiling of Hydrocarbon Utilizing Bacteria Isolates from Wetlands in Rivers State, Southern Nigeria

Wetlands can intercept runoff from surfaces prior to reaching open water and remove pollutants through physical, chemical, and biological processes thereby protecting and preserving the environment. Because of unsustainable oil exploration activities, most wetlands in Rivers State, Southern Nigeria have suffered severe petroleum-damaged ecosystems. This research was carried out to characterize and identify the hydrocarbon utilizing bacteria associated with crude oil polluted wetlands and to screen for the presence of plasmids that could confer resistance to antibiotics using both cultural and molecular methods. Soil samples were collected from three different wetlands across the state with hand auger at two depths of 0-15cm and 15-30cm twice monthly for three months. The presence of microbial activity was determined by the enumeration and isolation of total heterotrophic and hydrocarbon utilizing bacteria. Eight (8) most occuring hydrocarbon utilizing bacterial isolates were isolated and identified culturally and phenotypically from the 54 wetland soil samples. These bacteria isolates were confirmed to be Bacillus flexus, Bacillus subtilis, Lysinibacillus macroides, Staphylococcus aureus, Chryseobacterium aquifrigidense, Pseudomonas aeruginosa and Salmonella enterica molecularly via sequencing of the 16S rRNA gene. The most common bacteria isolated were Bacillus species, followed by Pseudomonas at a dilution of 106. Seven (7) out of the eight (8) isolates (except Salmoella enterica) showed the presence of the 25kb plasmids at various intensities.

Effect of Aerating Duration on Hydrocarbon Biodegradation in a Simulated Crude-Oil Polluted Aquatic Environment Undergoing Bioremediation

The aim of this research work was to determine the aerating duration that would be effective in enhancing hydrocarbon biodegradation rate during bioremediation of crude-oil polluted river. Sediment and river-water were placed in four glass troughs labeled CT (control), A, B, and C. The setups were polluted with crude-oil, and allowed undisturbed for 2 weeks. Subsequently, accessible crude-oil on the surface was removed; bacteria and nutrients were then added. Air was bubbled for 3 hours into setups A, B, and C, at daily, 3 days, and 7 days interval respectively. Aeration was not applied to setup CT. On day 1, 7, 14, and 21, hydrocarbon concentration was determined; populations of total heterotrophic bacteria (THB) and hydrocarbon-utilizing bacteria (HUB) were also determined. The time it will take for hydrocarbons in the setups to biodegraded “completely” was calculated using first-order reaction equation. The results obtained showed that 71.43, 86.39, 83.17, and 15.42 % hydrocarbon degradation were obtained in setup A, B, C, and CT respectively. The time it will take for hydrocarbons in the setups to biodegrade “completely” were 129, 89, 101, and 1079 days for A, B, C, and CT respectively. There was slight reduction in population of HUB in setup CT, fairly stable population in setup A, and increase in population of HUB in setups B and C. It is concluded that aerating crude-oil polluted aquatic environment for 3 hours at 3 days interval will be more effective in enhancing hydrocarbon biodegradation rate during bioremediation.

Effects of seasonal variation and locations on microbial load in sediment and water of a polluted ecosystem

Seasonal effects on microbial load of sediment and water at different locations along Bonny Estuary of Niger Delta was investigated for a period of 12 months. All analyses followed standard procedure. Results revealed that total fungi counts in sediment and water at different locations were not significantly different (p > 0.05) at both wet and dry seasons while hydrocarbon utilizing fungi showed significant differences (p < 0.05) at both seasons in both sediment and water samples. During the wet season, total faecal counts ranged from 5.0 to 10.0 x 105 CFU/g for sediment and 4.0 to 7.0x 105 CFU/g in water. In dry season, the concentration of hydrocarbon utilizing bacteria in the sediment ranged between 0.1 x 105 CFU/ml/g and 8.0 x 105 CFU/ml/g in wet season while in dry season, the concentration in water ranged between 0.1 x 105 CFU/ml/g and 6.0 x 105 CFU/ml/g at Abuloma. At Okwujagu, total heterotrophic bacteria counts in sediment ranged from 0.1 to 8.0 x 105 CFU/g in dry season. This was higher than the range 0.1 to 6.8.0 x 105 CFU/ recorded in Abuloma, Okwujagu and Slaughter at dry season. The highest vibrio counts in water (11.0 x 105 CFU/ml) for wet and (10.0 x 105 CFU/ml) for dry seasons were recorded at Slaughter. In Oginiba, the feacal count recorded 3.0 x 105 CFU/ml in water during the wet season and 2.0 x 105 CFU/ml for dry season. Generally, there were significant differences (p < 0.05) in the bacterial concentrations in both sediment and water. This showed that different seasons favour the growth of certain microbial types.

A Comparative Study on the use of Soil - Organic and Inorganic Biostimulants in the Remediation of Oily Waste

Remediation of oily waste using soil-organic (goat dung, poultry dropping) and inorganic (NPK fertilizer) nutrients was assessed for twelve weeks using culture-dependent microbiological technique and chemical procedures. The results indicate increased counts of Hydrocarbon-utilizing bacteria, fungi and actinomycetes with remediation time for both nutrient types. Bacteria in the remediated waste were members of the genera Bacillus, Pseudomonas, Acinetobacter, Alcaligenes and Serratia, fungi: Penicillium, Aspergillus and Cladosporium, and actinomycetes: Rhodococcus, Nocardia and Streptomyces for all soil-nutrient amendment techniques. pH of the NPK fertilizer ranged between 6.7 ± 0.03 and 7.3±0.06 whereas the goat dung and poultry dropping amendments was 6.5± 0.02 and 7.1 ±0.05. Dehydrogenase activity increased for the biostimulant treatment cells with remediation time. Total Petroleum Hydrocarbon reduction was 99.3 and 99.6% in organic and 99.8% for inorganic amendments. Polycyclic Aromatic Hydrocarbons of the remediated waste for both techniques revealed values below detectable limits (< 0.01) at the end of remediation period. Remediation with soil-goat dung and soil-poultry dropping amendments compared favorably with soil-NPK fertilizer technique because microbial activities were enhanced to produce eco-friendly waste. The use of soil-organic amendments is therefore a low-cost alternative biostimulant for the management of oily waste in the petroleum industry.

Myco-enhanced Bioremediation in Open Field Crude Oil Contaminated Soil Using Mucor racemosus and Aspergillus niger

Aim: To assess the Mycoremediation potential of Mucor racemosus and Aspergillus niger in open field crude oil contaminated soils in Rivers State, Nigeria. Study Design: The study employs experimental design, statistical analysis of the data and interpretation. Place and Duration of Study: Rivers State University demonstration farmland in Nkpolu-Oroworukwo, Mile 3 Diobu area of Port Harcourt, was used for this study. The piece of land is situated at Longitude 4°48’18.50” N and Latitude 6ᵒ58’39.12” E measuring 5.4864 m x 5.1816 m with a total area of 28.4283 square meter. Mycoremediation process monitoring lasted for 56 days, analyses were carried out weekly at 7 days’ interval. Methodology: Five (5) experimental plots were employed using a Randomized Block Design each having dimensions of 100 x 50 x 30 cm (Length x Breadth x Height) and were formed and mapped out on agricultural soil, each plot was contaminated with 22122.25g of Crude Oil except Control 1 and left fallow for 6 days after contamination for proper contamination and exposure to natural environmental factors to mimic crude oil spill site. On the seventh day bio-augmentation process commenced using two (2) fungal isolates namely Aspergillus niger [Asp] and Mucor rasemosus [Muc]). Two (2) control plots (P1: Uncontaminated and unamended soil - CTRL 1 US) and P2: Crude Oil contaminated but unamended soil - CTRL 2 CS); P3 = P5 were contaminated and amended/bioaugmented (P3: CS+Asp, P4: CS+Muc, P5: CS+Asp+Muc respectively. Soil profile before and after contamination was assayed while parameters like Temperature, pH, Nitrogen, Phosphorus, Potassium and Total Petroleum Hydrocarbon (TPH) contents were monitored throughout the experimental period. Microbial analyses such as Total Heterotrophic Bacteria (THB), Total Heterotrophic Fungi (THF), Hydrocarbon Utilizing Bacteria (HUB) and Hydrocarbon Utilizing Fungi (HUF) were recorded. Bioremediation efficiency was estimated from percentage (%) reduction of Total Petroleum Hydrocarbon (TPH) from day 1 to the residual hydrocarbon at day 56 of bio- augmentation/ biostimulation plots with the control. Results: Results revealed actual amount of remediated hydrocarbon and % Bioremediation Efficiency at 56 days in the different treatment plots (initial TPH contamination value of 8729.00mg/kg) in a decreasing order as follows: CS+Muc (8599.19mg/kg; 33.66%) > CS+Asp+Muc (8357.31mg/kg; 33.04%) > CS+Asp (8341.58mg/kg; 32.98%) > CTRL 2 -CS (Polluted soil without amendment) (81.06mg/kg; 0.32%). Microbiological results After fifty-six (56) days of bioremediation monitoring; %HUB were as follows; CS+Asp+Muc (45.30%) > CS+Asp (40.32%) > CS+Muc (35.01%) > CTRL 2 –CS (30.43%) > CTRL 1 – US (0%). These results indicate that the presence of the contaminated crude oil stimulated and sustained the growth of Hydrocarbon Utilizing Bacteria (HUB) in the contaminated plots (P2 - P3); more so, the higher growth in the enhanced bio-augmented plots (P3 – P5) shows the positive impact of fungal bio-augmentation in bioremediation of crude oil polluted soil. It was further observed that treatment plots with higher HUB or HUF had higher percentage (%) bioremediation efficiency; that is, the higher the sustained HUB and HUF population, the higher the %Bioremediation process. Hydrocarbon Utilizing Bacteria (Log10 CFU/g): CS+Asp (4.20) (Day 35) > CS+Muc+Asp (4.18) (Day 35) > CS+Muc (4.08) (Day 28) > CTRL 2 – CS (3.95) (Day 21) > CTRL 1 – US (3.78) (Day 35). (Fig. 3). Hydrocarbon Utilizing Fungi (Log10 CFU/g): CS+Asp (4.68) (Day 35) > CS+Muc+Asp (4.58) (Day 35) > CS+Muc (4.48) (Day 35) > CTRL 2 – CS (4.23) (Day 21) > CTRL 1 – US (2.85) (Day 42). Conclusion: Study showed that bioremediation of crude oil-contaminated soils with Bioaugmenting fungus singly may be more effective than combination with others depending on the type of substrate used, nature of the hydrocarbon utilizing organism and environmental conditions prevalent as seen in Mucor racemosus having higher bioremediation potential than when combined with Aspergillus niger. Notably, Hydrocarbon Utlilizing Bacteria (HUB) and Hydrocarbon Utilizing Fungi (HUF) which are the key players in Bioremediation has its peak count value on Day 35, this confers that nutrient renewal on bioremediation site should be at interval of 35 days for continuous effective bioremediation of hydrocarbon pollutants. It is therefore recommended that single microbes of high bioremediation potential could be used since its more effective than consortium of many hydrocarbon utilizing microbes. Also, nutrient or bio-augmenting microbes’ renewal on bioremediation site should be at an interval of 35 days for continuous effective bioremediation of hydrocarbon pollutants.

Percentage Bioremediation Assessment of Spent Mushroom Substrate (SMS) and Mucor racemosus in Hydrocarbon Contaminated Soil

Aim: The aim of the study was to assess Percentage Bioremediation of Spent Mushroom Substrate (SMS) and Mucor racemosus in hydrocarbon contaminated soil Place and Duration of Study: A portion of Rivers State University demonstration farmland in Nkpolu-Oroworukwo, Mile 3 Diobu area of Port Harcourt, Rivers State was used for this study. The piece of land is situated at Longitude 4°48’18.50’’N and Latitude 6o58’39.12’’E measuring 5.4864 m x 5.1816 m with a total area of 28.4283 m2. Bioremediation monitoring lasted for 56 days, analysis carried out weekly (per 7 days’ interval). Methodology: Five (5) experimental plots employing the Randomized Block Design were used each having dimensions of 100 x 50 x 30 cm (Length x Breadth x Height) = 150,000cm3. Baseline study of the uncontaminated and the deliberately contaminated agricultural soil was investigated for its microbiota and physico-chemical properties. Two of these plots were designated as pristine (Unpolluted soil) (CTRL 1) and crude oil contaminated soil without nutrient organics and bioaugmenting microbes (CTRL 2); these two serve as controls. Each of the experimental plots, except the control (CTRL 1), was contaminated with 2500 cm3 (2122.25 g) of crude oil giving initial Total Petroleum Hydrocarbon (TPH) value of 8729.00 mg/kg. The crude oil polluted soil in Plot 3 was further treated with 750 ml of Mucor racemosus broth (CS+Muc), Plot 4 was treated with 3000 g of Spent Mushroom Substrate (CS+SMS) while plot 5 was treated with the combination of both (CS+Muc+SMS). The plots were left for 7 days to ensure even distribution and soil-oil bonding. Sampling was done at seven-day interval (Day 1, 7, 14, 21, 28, 35, 42, 49, 56). Physicochemical parameters monitored were pH, Temperature, Nitrogen, Phosphorus, Potassium, and Total Petroleum Hydrocarbon (TPH) throughout the experimental period. Microbial parameters monitored were Total Heterotrophic Bacteria (THB), Total Heterotrophic Fungi (THF), Hydrocarbon Utilizing Bacteria (HUB) and Hydrocarbon Utilizing Fungi (HUF). Percentage (%) Bioremediation was estimated from percentage (%) reduction of Total Petroleum Hydrocarbon (TPH) from day 1 to day 56 in relation to control plots. Net % Bioremediation were also assessed to ascertain the actual potential of treatment agents singly or combined. Results: Total Heterotrophic Bacteria (THB) (CFU/g) recorded on day 7 and day 56 of the bioremediation were; day 7; CTRL 1 – US (1.07 x109), CTRL- CS (5.4 x108), CS+Muc (3.0 x108), CS+SMS (4.6 x108) and CS+Muc+SMS (5.0 x108). On day 56, data obtained were CTRL 1 –US (9.4 x108), CTRL 2 –CS (7.2 x109), CS+Muc (3.7 x108), CS+SMS (8.1x108) and CS+Muc+SMS (6.8 x108). The increase in number in the treated plots is a depiction of an increase in activity of the organism and the stimulating effect of bio-organics SMS while the untreated plot CTRL 1-US showed decrease in population at day 56. Similar trend showed for Total Heterotrophic Fungi. Generally, it was observed that the highest growth/ count was recorded at the 7th and 8th week (day 42 or day 49), at the 9th week there was an observable decrease; probably due to depletion of nutrients and other factors such as rainfall and seepage. The Net Percentage Hydrocarbon Utilizing Bacteria and Fungi (Net %HUB and Net %HUF) were highest in Crude Oil contaminated plot treated with Spent Mushroom Substrate (SMS) singly; that is (CS+SMS) (11.02% and 12.07%) and lowest in the uncontaminated soil – Control (CTRL 1 –US) (5.41% and 9.26%) respectively. The trend in decreasing order of Net % Hydrocarbon Utilizing Bacteria were as follows: CS+SMS (11.02%) > CS+Muc+SMS (10.14%) > CS+Muc (9.43%) > CTRL 2 –CS (8.1%) > CTRL 1 –US (5.41%) while Net % Hydrocarbon Utilizing Fungi followed similar trend and were: CS+SMS (12.07%) > CS+Muc+SMS (11.76%) = CS+Muc (11.76%) > CTRL 2 –CS (11.05%) > CTRL 1 –US (9.26%). Evaluation of Amount of Crude Oil or Hydrocarbon remediated and Net %Bioremediation revealed Crude Oil contaminated plot augmented with Mucor racemosus broth singly (CS+Muc) as having the highest bioremediation potential while the least is the untreated soil. The trend is as follows: CS+Muc (8599.19 mg/kg; 33.93%) > CS+Muc+SMS (8298.95 mg/kg; 32.74%) > CS+SMS (8197.03 mg/kg; 32.34%) > CTRL 2 –CS (166.54 mg/kg; 0.66%) > CTRL 1 –US (85.48 mg/kg; 0.34%) Conclusion: This shows that a single nutrient substrate or augmenting microorganism applied appropriately may have a more positive result, that is; higher bioremediation potential than combined or multiple mixed treatments. It was further observed that microbial counts decreased with time in treatments with augmenting organisms alone but increased considerably in treatments supplement with organics having its peak on the 49th day. It is therefore recommended that bioremediation of crude oil-polluted soil using bio-augmenting microorganism should be applied appropriately noting the volume: area ratio and be supplemented with efficient nutrient organics after every 49-day interval.