A series of lipase-catalysed transesterification experiments were carried out to study the effect of the presence of free fatty acids on synthesis reaction rate and the stability of the biocatalyst, and also to elucidate the underlying mechanism, which remains a subject of debate. Based on the results, the reaction rate and biocatalyst stability increased with increasing content in free fatty acids of the reaction mixture. Also, tests carried out with a mixture of triolein and linoleic acid revealed that the transesterification mechanism is a combination of direct alcoholysis of triacylglycerols and a twostep reaction involving hydrolysis of acylglycerols and further esterification of previously released free fatty acids. The time course of triacylglycerols and diacylglycerols revealed that the enzyme is similarly selective for both types of substrate. Response to Reviewers: Reviewers' comments: Reviewer #1: This work describes through a clear and convincing approach the effect of free fatty acids on reactions of transesterification. A few changes would help in improving the manuscript: 1)Methods: page 6. It is not clear how the final volume of the reaction is reached. This is relevant to understand the experimental setting. Did authors just mix fatty acids, oil and methanol in the required ratios? In this case, the final volume would be different in different reactions with some implication for the interpretation of results. The other possibility is that they keep the volume constant. But this would require adding water what is very relevant in their experiments that compare hydrolysis with alcoholysis. Please clarify in Materials and Methods and, if necessary, also in the description of the results The densities of triolein (or olive oil) and oleic acid are quite similar (0.91 g/ml and 0.89, respectively), so keeping the same final weight (8 gr) then the final volume reached for different combinations of triolein and oleic acid will be always the same, supposing that these two compounds behave as ideal liquids so the final volume is the sum of the volume of each liquid. A sentence has been included in "Materials and Methods-Transesterification" reactions for better understanding
BACKGROUND Using renewable feedstock sources for biodiesel production seems to be a promising strategy and even more so when enzymatic catalysis with lipases are used. However, it is well known that these enzymes could be inactivated by reaction conditions such as temperature or alcohol concentration. In this work, the effect of temperature and initial water activity (aw) value on immobilised recombinant Rhizopus oryzae lipase (rROL) were studied. Methanolysis and ethanolysis reactions using alperujo oil with three different stepwise addition strategies were employed. RESULTS Recombinant 1,3‐positional selective rROL covalently immobilised on polymethacrylate amino‐epoxy activated support showed maximum initial reaction rate at low aw value (0.093). It was found that 30 °C was the optimal temperature in terms of biocatalyst stability during transesterification reactions. Adding alcohol at once, ethanol was clearly a better acyl‐acceptor in terms of stability than methanol. Productivity was found to be 2‐fold higher when five pulses of ethanol were used instead of methanol. CONCLUSIONS Alperujo oil has great potential as a low cost feedstock for biodiesel production through enzymatic catalysis using a nearly semi‐continuous alcohol addition strategy. © 2017 Society of Chemical Industry
Biodiesel represents an interesting alternative to fossil fuels. Traditionally the standard method for biodiesel production from oils is alkaline-catalyzed transesterification. Chemical catalysis can be replaced by enzymatic catalysis using lipases (EC 3.1.1.3, triacylglycerol acyl hydrolases), obtained from plants, animals or microorganisms. These biocatalysts act at milder temperature and normal pressure conditions, resulting in lower energy consumption. Also, undesirable side-reactions do not occur, originating pure products. Refined vegetable oils are the most common feedstocks for biodiesel production, accounting for 70-80% of the overall biodiesel production costs. The search for low-cost feedstocks, i.e. non-edible oils and high acidic waste oils/greases, is an alternative to make biodiesel competitive. Non-regioselective and sn-1,3regioselective lipases can catalyze esterification of free fatty acids and transesterification of triacylglycerols with good yields. The lipases used as catalysts for biodiesel production must present alcohol resistance, thermo-tolerance, high stability and activity. Recently, enzymatic processes for biodiesel production have been implemented at industrial scale. Despite this trend, the conventional chemical process still remains the most popular, mainly due to the high cost of commercial lipases. This review consists of an update of the state of the art of enzymatic biodiesel production, including legislation, feedstocks, lipases used for biodiesel synthesis, the role of acyl acceptors and strategies to avoid lipase inactivation, the mechanisms proposed for biocatalysis and the enzymatic bioreactors used. In addition, the economics of the bioprocess is also presented.
The potential application of rice husk ash (RH26) as support for the immobilization of a recombinant Rhizopus oryzae (rROL) lipase as biocatalyst in the enzymatic biodiesel production using alperujo oil and the comparison with commercial hydrophobic support OD403 (RelOD) has been made. Although the specific activity (UA mg support À1 ) was around one-half lower in RH26 than in RelOD when they were used as biocatalyst in biodiesel reaction, the normalized initial rate was similar, between 1.6 and 2.4 lmol FAME mL À1 mL À1 UA À1 . Thus in terms of biocatalysis performance, rice husk as is an alternative to commercial supports. However, the main problem is the more complex recovery of RH26 for the reutilization compared with commercial ones. ARTICLE HISTORY
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