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.
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.
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