Crude glycerol was obtained fromboth the Virginia Biodiesel Refinery (West
Point, VA, USA) and Seattle Biodiesel (Seattle,WA, USA). Both refineries used
alkali-catalyzed transesterification to produce the biodiesel from soybean oil. It
was found that when crude glycerol was directly mixed with artificial seawater,
soaps precipitated from the liquid, which later proved detrimental to cell growth
(data not shown). To avoid this phenomenon, the crude glycerol was pretreated
prior to use in the algal culture. The pretreatment protocol was as follows: (1) the
glycerin was mixed with distilled water at a ratio of 1:4 (v/v) to reduce the
viscosity of the fluid; (2) the pH of the fluid was adjusted to around 6.5 with
hydrochloric acid to convert the soluble soap into insoluble free fatty acids which
precipitated from the liquid; (3) precipitated solid was separated from the crude
glycerol solution by centrifugation at 5000 rpm; and (4) after separation, other
nutrients (mineral salts, nitrogen source, etc.), with additionalwaterwere added to
adjust the nutrient level (including glycerol) to a desired level