Biological catalysts for biodiesel production, including both enzymes and living organisms, are considered to be one of the most promising alternatives for future use in biodiesel production [38, 79]. They can be implemented either in solution or supported (e. g. in biological films or in packed beds). An advantage of enzymes is that they do not require the utilization of nutrients. An advantage of living organisms is that they can be genetically engineered to improve their performance, resilience, and capacity to operate in harsher conditions.

Lipases obtained from different biological sources are examples of enzymes that can be used to perform the transesterification reaction and that have shown a good tolerance to the oil FFA content [95]. Kaieda et al. [52] show that different enzymes have different capacities and report that certain lipases can be used for biodiesel production even if the oil has high water and methanol contents.

Although extensive research has been devoted to this area, the use of bio-catalysts for biodiesel production is still at the laboratory stage [1, 79, 86, 87]. They can be more efficient, selective, require a lower reaction temperature, and produce less side products or wastes, when compared with other types of catalyzed processes, but the reaction rates are much lower than for the conventional process, normally taking several hours (8-12 h) for similar conversions.

Some of the main problems include the difficulty in determining:

• What are the best enzymes or microorganisms to perform the reaction, depend­ing on the feedstocks characteristics and on the impurities that may exist?

• What are the optimum reaction conditions, in particular what are the optimal molar ratio of reactants, solvents to be used, temperature, and water content?

• How the enzymes will be used, if supported or in solution?

• How to recover and reuse the enzymes?

• How to avoid the enzymes’ deactivation, or the living organisms’ death?

Some studies can be found in literature addressing some of the problems listed above. For example, Shimada et al. [87] concluded that the best way to avoid the inhibition or deactivation of enzymes and maintain the enzyme activity for longer periods of time is their stepwise addition to the reaction mixture, in order to main­tain the oil/methanol ratio at certain optimal levels. Although the addition of co-solvents appears in some cases to have a positive effect on the enzyme stability [ 1], there is still some work to be done in order to identify the most adequate solvents and how they influence the ongoing reaction.