Sample was crushed to expose all the microorganisms present in the cassava peels.
Serial dilution of sample was carried out according to the method of Collins and Lyne (1976). Precisely 10g of the crushed cassava peels sample was introduced into conical flask containing 90ml of sterile water. This was shaken for homogenous mixture and thereafter 1ml of the a liquot was aseptically transferred into sterile test tube containing 9ml and sterile dilution blank (diluents) to give a dilution of 10-0. This was repeated until a tenfold serial dilution level was reached.
Estimation of Microbial Load of Cassava of Peels
After serial dilution process, 1ml of the dilution factor 10-5 used to seed the petri dish. Appropriately culture media were poured into the seeded plates or petri dishes. The total hetertrophic bacteria and fungi count was determined by pour plate method of Harrigan & Sabour and Dextrose Agar and Certrimide Agar.
For selective fungal growth, streptomycin 0.5mg/ml supplemented medium was used for the selective enumeration and isolation of fungi. The bacteria plate were incubated at 290C and fungi plates at room temperature (280C20C) for 7 days. Discrete colonies that emerged on the culture plates were noted and recorded as colony forming unit of cassava peels sample.
Maintenance of Pure Microbial Cultures
To maintain pure microbial culture or isolates discrete colonies from the primary plates were picked with the aid of sterile inoculating loop and sub-cultured onto a fresh agar plates nutrient agar, cetrimide agar and sabourand dextrose agar for bacteria and fungi respectively and incubated. The process was repeated to obtain a monocultures of the microbial isolates. The cultural characteristics of the colonies were observed and pure culture were stocked in an agar slants on McCartney bottles; after 24hrs the slants were preserved in the refrigerator at 40C for further use.
Characterization and Identification of Bacterial Isolates
Apart from cultural characteristics and microscopy, bacterial isolates were subjected to certain biochemical tests, the test include Gram reaction, catalase, motility coagnlase, methyl red voges proskaver (MRVP) starch hydrolysis sugar feementation citrate and urease oxidase test.
Characterization and Identification of Fungi Isolates
Morphological characteristics of fungi were noted and wet mount of each isolates were carried out using lacto-phenol in cotton blue. The microscopic characteristics were compared with the standard manual for identification of fungi Barnet & Hunter 1996.
- Gram’s Reaction
Gram stain is used differentiate between gram positive and gram negative bacteria (Fawole and Oso, 1987). A loopful of distilled water was placed on a sterile grease-free slide, the isolates was picked and smeared on the slide and allowed to air dry on bench, and then heat fixed. Each prepared slide was stained with crystal violet for 60sec. then rinsed with slow running tap water, flooded with iodine for another 60sec; rinsed with tap water. The smear was decolonized with 95% alcohol for 10…….., this was rinsed under running tap water and finally counter stain with safranin for 60sec. slides were air dried and examined under oil immersion objective (Holt et.al; 1994). A blue to purple colouration indicates gram positive bacteria while pink to red indicates a gram negative organism.
- Catalase Test:
The essence of this test is to determine the presence of catalase enzymes which catalyze the release of oxygen peroxide 3% of Hydrogen Peroxide (Hg02) solution was dropped on a sterile slide and each inoculation was placed on it and was observed for rapid appearance of gas bubbles.
- Coagulase Test:
The test is to demonstrate the ability of the organism to coagulate plasma. Using a sterile wire loop, a loopful of normal saline was dropped on a sterile slide and the test organism was picked and placed on the side, the solution was gently mixed and a drop of blood plasma was also placed and mixed, the solution was observed for one minute, positive test organism formed clumping or coagulates while the negative reaction showed no coagulation.
- Citrate Utilization
Some organism have the ability to utilize citrates as their source of carbon and energy for growth and ammonium salts as a source of nitrogen. The medium used as simmon citrate medium. The medium was dispensed into a conical flask and sterilized in an autoclave. A 24hr old culture of the test organisms were inoculated on sterile citrate agar plate and were incubated at 280C for 24hrs and colour change observed. A change in the indicator from green to blue indicates utilization of the citrate.
- Motility Test:
Some bacteria posses flagella and can therefore move. Such motility can be demonstrated by the hanging-drop technique. Test hanging method was used for this test using a hallow cavity slide, the procedure of Holt et.al; (1994) was employed to a certain the presence or absence at flagella, the organ of motility in bacteria. The preparation was observed low power objective.
- Starch hydrolysis Test:
The test organisms were streaked on starch agar and incubated at 370C for 24hrs. Thereafter, the surface of the plates were flooded with gram iodine. Some area around the line of growth showed clearing zone i.e starch were hydrocysed which were positive while some did not which show the entire medium turn blue, meaning starch has not been hydrolysed.
- Unease Test:
Heavy inoculums of the test organisms were inoculated by streaking on the surface of the medium i.e urease agar and incubated at 370C for 3-4days. Urease positive cultures produce a purple pink colour due to a change in colour of the indicator. Negative result showed no colour change.
- Oxidase Test:
This test depends on the presence of certain oxidases in bacteria to catalyze the transport of electron between electron donor in the bacteria and a redox dye. Tetramethyl – P- Phenylene diamine dihydrochlaloride. The dye will be reduced to deep purple colour for the isolates.
The whatmans filter paper was soaked in freshly prepared % solution of oxidase reagent. The colony of the test organisms were streaked on the soaked filter paper. A deep purple colouration after 5-10 seconds negative result had yellow colouration or the socked part of the paper remained unchanged.
- Sugar Fermentation
Some microorganisms are capable of metabolizing a large variety of sugars as carbon sources. The products of sugar or carbolydrates metabolism depend largely upon the type of enzymes possessed or produced by the organism. The growth medium consisted of the basal medium 0.0% of fine different types of sugars (glucose lactose, sucrose, galactose, manitol) were used. The medium was dispensed into test tube in 3ml quantity. Durham tubes were inserted into each medium and autoclaved at 1210C for 15min. using a sterile inoculating wire loop, each bacteria was suspended or inoculated into each test tube and was examined for colour charge from red to yellow and gas in Durham tubes.
- Biochemical Pretreatment of Sample
After all the biochemical test were carried out and the test organism obtained from the rotton cassava peels confirmed, the fungi Aspergillus terreus was maintained on the malt extract broth and kept at 40C while the bacteria pseudomonas floresscens was stocked on nutrient agar slant and kept at 40C for further use.
A basal medium 0.1g/100ml Cacl2.7H20, 0.1g/100ml Mgs04, 1g/100ml (NH4)2So4 and 0.05KHgPO4was prepared and 1.5g of pretreated cassava peels was dispensed into a conical flask that contain 95.5ml of the basal medium at different concentration of Alkali and Acid respectively and all the flasks were autoclaved at 1210C for 15min. After inoculation and the medium allowed to cool, 3.OX108cfu/ml of pseudomonas floresscens and Aspergillus terreus were inoculated into different concentration of Alkali and Acid. The flasks that were not inoculated served as control. The mixtures were then incubated at 500C for 24hrs for bacteria and 72hrs for fungi. After the appropriate incubation, the reducing sugar content of the hydrolysates was measured quantitatively using DNS method. (Miller 1959).
After the hydrolysis process, the concentrations that had a higher reducing sugar content were picked both for Alkali and Acid. The supernatants from the hydrolysates were centrifuged with the use of Gallenkanp centrifuge and the supernatants were transferred into another sets of conical flasks correctly labeled, autoclaved again at 1210C for 15mins and allowed to cool. Then freshly harvested cell of Aspergillus Niger was aseptically inoculated into a set of flask containing the hydrolysed supernatants of (20ml 30ml 40ml). The same procedure of sterilization was done for set and saccharonyces cerevisiae was inoculated into the same volume of (20ml 30ml and 40ml). the two organisms were combined into the third sets of hydrolysed supernatant at the same volume while the uninoculated still served as control. The flasks were corked incubated on the orbital shaker produce homogenous solution and even distribution of organisms at 280C for 5 days.