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Erlenmeyer flasks containing algal samples were incubated under artificial illumination with

16:8 light/dark photoperiod with 1000lux light intensity. Once the culture reached stationary
growth phase, cells were harvested by centrifugation, frozen immediately, and then

lyophilized prior to lipid analysis and subsequent in situ trans esterification. As for biodiesel
production directly from wet microalgae biomass, the microalgae biomass was harvested

and stored immediately in a refrigerator at −80°C.The algal oil extraction procedure was
adapted from the protocol described by Bligh and Dyer in 1959. The lipids extracted from

dry algal biomass was concentrated by removing water and solvent if any using rotary

evaporator at 100°C for 1hour and a dark brown layer the oil rich fraction (ORF) was
obtained. According to theory, the trans esterification requires 3 mol of alcohol per one mol

of triglyceride to produce 3 mol of fatty acid methyl esters and 1 mol of glycerol. In this

study, we investigated the percentage of moisture in biomass. Then at the optimum moisture
content, the trans esterification reaction was conducted in different methanol to oil ratio at

75°C.

RESULTS AND DISCUSSION

Optimizing Conditions of Biodiesel Production from Wet Microalgae Biomass

Biomass with distilled water were studied. The results were shown in Figure 1. From the
results obtained (Figure 1), it was observed that biodiesel yield increased on decreasing the

water content increased from 90 to 10% at the temperature of 90°C. The biodiesel yield was

92.56% when water content was 10%, and when water content was increased to 90%, the
biodiesel yield was only 12.5% (Figure. 1). This was in accordance with the previous study

that water could dramatically impede biodiesel production (Wahlen et al., 2011). The
highest yield is obtained from dry feedstock because water in biomass impedes the

transesterification process due to the reversibility of the transesterification reaction: water in
the reaction medium slows the rate of the direct reaction and promotes a reversible reaction;

at the same time, the hydrolysis process takes place of the resulting biodiesel to form fatty

acids and alcohol (Cao et al., 2013). In addition, water, an ineffective oil solvent, acts as a
barrier to oil-alcohol contact, thereby lowering the rate of the transesterification process (Liu

et al., 2007). These results suggested that water requires more energy to cause the reaction to

effectively occur only the higher temperature could partially compensate the negative effect
of water on transesterification. This is probably because high temperature could speed up the

reaction (microalgae biomass, methanol, n-hexane, and catalyst) and make trans
esterification reaction take place more effectively (Wahlen et al., 2011). Nguyen et al.,

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