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

Abstract

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.

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“…Gently adding 10ml of 45% NaOH allowed the sample to be distilled into 10ml of 4% boric acid. About 50 ml distillate was collected and titrated with 0.02N H 2 SO 4 to get back a pinkish-red end point [32,33].…”

“…Ferron indicator 3 drops was made and titrated with 4Nferrons sulphate solution. Changes in color from green cast to maroon color marked the end point and tire value was taken [32].…”

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