Free lipase-catalyzed biodiesel has drawn more and more attentions in recent years because of its advantages of lower cost and faster reaction rate. Utilizing free lipase to convert low quality oils such as crude vegetable oils and microbial oils is beneficial to further reduce the cost of biodiesel production. However, these oils typically contain some amount of phospholipids. Phospholipids were found to affect the lipase-catalyzed process and further influence the enzyme's thermal stability in biodiesel production process. In this work, free lipase NS81006-mediated biodiesel production from oils containing phospholipids at varied temperature was investigated systematically. It was found that the presence of phospholipids at high temperature led to a decreased fatty acid methyl esters ( FAME) yield and poor reuse stability of the lipase during NS81006-catalyzed biodiesel production process. The higher the temperature was, the greater negative effect was observed. This inhibitory effect was found to be mainly caused by the coexistence of phospholipids and methanol in the system. Based on this finding, a novel two-step enzyme-mediated process was further developed, with which the above-mentioned inhibitory effect was eliminated, and a FAME yield of 95.1% could be obtained with oils containing 10% phospholipids even at high temperature of 55 degrees C. (C) 2014 Elsevier Ltd. All rights reserved.