Materials
Source of Carbon/Sample Collection
Yam and potato peels were obtained from a yam chip shop opposite Federal University Wukari, Taraba State Nigeria. Cassava root tubers for this research were harvested at a nearby farm site located along Federal University Wukari Road, in Wukari, Taraba State, Nigeria and the peeled portion properly washed with clean water for soil and dirt removal. The fresh peels were transported to the laboratory within 2 hours of procurement and washed with sterile distilled water. All the agro-waste peels were air dried for 7 days, separately grounded using mortar and pestle to obtain stock samples and thereafter stored in an airtight polythene bag.
Microorganism
The fungi used were obtained by isolation from decomposing agro-wastes and culturing in a prepared Potato Dextrose Agar (PDA) at the culture selection unit of the Department of Microbiology, Federal University Wukari, Nigeria. It was further characterized and sub cultured to yield pure strain of the organism.
Research Methods
Culture Media Preparation
The Potato Dextrose Agar (PDA) preparation was exactly according to the manufacturer recipe (39g in 1L of water). The autoclaved sterilization of media was at 121°C and 15 psi for 20 min; complete dissolution and homogeneity was achieved. Thereafter, it was cooled to 43°C followed by inclusion of a chloramphenicol capsule to 500 ml of sterile cooled PDA (Green et al., 1994). Solidified PDA was obtained by dispensing 15 ml of the media into each sterile petri dish of 8.6 cm (86 mm). Sterility was ascertained through incubation of the solidified media for 24h at 28°C before use (Cheesebrough, 1984).
Fungal Isolation
Onyike and Maduewesi (1985) described the isolation protocol employed in this study. Briefly, a small section of the cassava, yam and sweet potato peels showing advancing margin of rottenness were placed on the solidified agar. A section from each of the three samples were placed per plate. The incubation duration was 7 days at 27±2°C and fungi associated with the agro-wastes observed. Isolation frequency was as previously described by Okigbo and Ikediugwu (2000). Pure cultures were obtained by series of sub-culturing of individual isolates.
Fungal Isolates Identification
Fungal colonies were macroscopically and microscopically identified by direct low powered light microscope and identification atlas at the Department of Microbiology, University of Jos, Nigeria.
Biochemical Tests for Fungal Isolate Identification
Catalase Test: A small amount of the fungi was transfer to a clean, dry glass slide surface with the aid of a sterile teasing pin. Hydrogen peroxide (2 drops) were added, mixed and observed for bubble formation.
Indole Test: This test is based on the principle that Kovac’s reagent can combine with indole and the solution turns from yellow to cherry red. The fungi were inoculated aseptically in a sterile test tube containing 4 ml of tryptophan broth and incubated at 370C for 24-28 hours after which 0.5ml of Kovac’s reagent was added and observation for a ring formation after 20 mins. A red-violet colour ring at the top surface of the tube indicates a positive test while a yellow colour indicates a negative test. An un-incubated test tube served as control.
TSI Test: Triple sugar iron agar test is used to determine whether an organism utilize lactose and glucose or sucrose in fermentation or produce hydrogen sulphide. The isolated colony of fungi were introduced into the TSI with a sterile straight teasing pin. The medium was first agitated through the centre to the bottom and streaked with the fungi to the surface and kept in slanted position. The incubation time was 18-24hrs at 370C.
Citrate Utilization Test: This test detects the ability of micro-organism to utilize citrate as a sole source of carbon and energy. From 2% citrate, 5 ml was dispensed into various test tubes and covered with cotton wool and then incubated after inoculating with fungi. Colour variation from green to blue indicated a positive test.
Production of Bio-surfactant
Modified experiment by Michele et al. (2018) was conducted in a conical flask (250 mL) containing 25 mL of media culture (40 g/L of each agro-waste, 4% spent vegetable oil and 10 g/L yeast extract). The medium was autoclaved at 121°C for 15 minutes and inoculated after cooling. The conical flasks were incubated at 300C and manually vortexed (agitated) for 15mins 4 times daily for 21 days. An Erlenmeyer flask with 25 mL of culture medium and 4% vegetable spent oil (v/v), with no inoculation, was used as control. Afterward, the culture media was centrifuged at 3000 rpm for 10 minutes to obtain a cell free supernatant.
Bio-surfactant Quality Assessment
Emulsification Index (E24)
The emulsifying index test (E24) technique described by Cooper and Goldenberg and adapted by Kiran, et al. (2009) was used. The centrifuged supernatant (bio-surfactant) (2mL) was mixed with 2mL of diesel and kerosene separately (hydrocarbon compounds) in test tubes, and the mixture was agitated for up to 2 minutes in a constant tube agitator type vortex.
After 24 hours, the proportion of the emulsion formed was compared to the total volume of added hydrocarbon. The emulsification index was calculated by the following formula:
E24 correlates to the biosurfactant concentration ie Emulsification Index after 24 hours (%).
Oil Drop-Collapse Qualitative Test
The test was conducted in 80 x 80mm Petri dishes containing 2mL of produced biosurfactant to which 3 drops of oil was added and observed for 0, 1, 5, 30 min, 1 and 72hrs. The result was regarded positive when the oil drop dispersed. Distilled water (2ml) and 2mL 1M sodium dodecyl sulfate (SDS) in oil surfactant solution were used as negative and positive control respectively.
Redox 2, 6-dichlorophenol indophenol (DCPIP) indicator Biodegradability test
Hanson et al. (1997) described the methodology used. Briefly, in test tubes, DCPIP concentrations were adjusted to 0.010g/mL from which DCPIP solution (2 ml), spent vegetable oil (2ml) and hyphae of fungi grown in wastes corresponding to 3 mm diameter, were added to each test tube and kept at (30°C). Medium discoloring-time measurements were taken after 48 hours. DCPIP in oil and without fungal strain was used as a positive control and DCPIP without oil and without strain was used as negative control.
Iodine Test: Iodine solution (4 - 5 drops) was added into a little amount of the formulated biosurfactant and gently agitated (Mahesh et al., 2006). The bluish/dark colour formation was observed.
Saponification Test
NaOH 2% solution was added (2ml) to the small amount of biosurfactant and well agitated to observe for soap formation (Mahesh et al., 2006).
Oil Displacement Assay
To the water (20ml) measure into a petri dish was added condemned engine oil (1 ml) and 2ml of synthesized biosurfactant. The diameters of the displaced engine oils were carefully measured and recorded. These were conducted in triplicate.
Statistical Analysis
All experiments were performed in triplicate and the mean and standard deviation were calculated. Also, an analysis of variance (ANOVA) test was performed to determine any significant difference (p>0.05). The analyses were all carried out using the Statistical Package for Social Sciences (SPSS) version 20.