**3.3.1 Fermentation of hydrolysed rice husk**

*Zymomonas mobilis* "Local strain" was isolated from palm wine using standard solid medium. Media constituents include 5.0g of yeast extract, 20g of agar and 1000cm3 of distilled water with pH 6.8. Medium was treated with actidione (cycloheximide) to inhibit *Zymomonas mobilis* growth before autoclaving at 121oC for 15 minutes. *Zymomonas mobilis* was then inoculated into the medium and incubated an aerobically at 3oC for 24 hours. Working close to the flame (creating aseptic environment), *Zymomonas mobilis* was introduced into the conical flask containing the substrate, the flask were then shaken (agitation process) and the mouths of the conical flasks were flamed before corking back and incubating at room temperature, they were shaken at various intervals in order to produce a homogenous paste and even distribution of the organisms in the substrates. After fermentation process, the substrates were then filtered using filter cloth and collected in a conical flask, in order to separate the desired product (the filtrate) from the residue. The filtrates were then distilled at 78.3oC using alcohol distillation apparatus, round bottom flask containing the filtrate was placed in the heating mantle and the mouth fixed to the condenser, a beaker for distillate collection was placed at the end of the set up, rubber pipes

Extraction and Optimization of Oil from

wt of oil extracted (g)

S/N

solvent

respectively) yielded 24.75% and 25.03% for the replicate

% wt of oil extracted

Moringa Oleifera Seed as an Alternative Feedstock for the Production of Biodiesel 255

presented also shows that, for the extraction combination where the temperature was high (65oC), low particle size (500μm) and high extraction time (7hrs) n-hexane yielded 42.81% and 42.25% for the replicate. Ethanol at a high temperature of 75oC, low particle size (500μm) and high extraction time of 7hrs yielded 26.67% and 26.14% for the replicate. It could be observed from the Tables of result that, for treatment combination where the temperature was low (55oC) while particle size and extraction time were high (710μm and 7hrs respectively) n-hexane yielded 41.38% and 41.35% for the replicate. Ethanol at a low temperature of 65oC, high particle size and extraction time (710μm and 7hrs respectively) yielded 28.84% and 28.24% for the replicate. Finally, for extraction condition combination where all the parameters are high temperature (65oC), particle size and extraction time were (710μm and 7hr respectively) n-hexane yielded 42.03% and 42.52% for the replicate. Ethanol at a high temperature of 75oC, particle size and extraction time (710μm and 7hrs

> Temp (oC)

**Solvent = Hexane**  1 4.22 42.03 65 710 7 2 4.15 41.38 55 710 7 3 4.23 42.22 55 500 7 4 4.29 42.81 65 500 7 5 3.87 38.58 65 500 6 6 3.79 37.78 55 500 6 7 4.31 43.01 65 710 6 8 4.33 43.17 55 710 6

**Solvent = Ethanol**  1 2.48 24.75 75 710 7 2 3.76 28.84 65 710 7 3 2.22 22.16 65 500 7 4 2.67 26.67 75 500 7 5 2.09 20.82 75 500 6 6 2.00 19.90 65 500 6 7 3.55 35.32 75 710 6 8 3.89 38.71 65 710 6 Table 4.1. Oil yield at various conditions from the first run with hexane and ethanol as the

Particle Size (µm)

Resident Time (hr)

or hose were connected to the condenser to supply water from the tap for cooling the condenser to supply water from the tap for cooling the condenser and letting out water out of the condenser simultaneously. Temperature on the heating mantle was set to the standard temperature for the production of ethanol which is 78.3oC, as the filtrate was heated, the vapour rose and entered into the condenser, tap water was passed into and out of the condenser using the rubber pipes and this condenses the vapor from the heated filtrate, condensed vapour was collected into the beaker at the other end of the distillation set up as the distillate (bio-ethanol), this process was repeated for other samples. The distillate was further purified by the use of calcium oxide (lime), a basic oxide, when added to the ethanol, absorbed the water to form calcium hydroxide, an alkaline solution; calcium hydroxide formed was separated from ethanol by further distillation which leaves absolute ethanol. One cm3 of alcohol was treated with iodine and sodium hydroxide, the colour change was observed and recorded, yellow precipitate was formed, which confirm that ethanol is present. The produced bio-ethanol was characterized to determine the density, flash point, pour point.
