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. 2014 Feb 26;7(1):29.
doi: 10.1186/1754-6834-7-29.

Combining phospholipases and a liquid lipase for one-step biodiesel production using crude oils

Silvia Cesarini, Rune Falkenberg Haller, Pilar Diaz  1 , Per Munk Nielsen
Affiliations

Combining phospholipases and a liquid lipase for one-step biodiesel production using crude oils

Silvia Cesarini et al. Biotechnol Biofuels. .

Abstract

Background: Enzymatic biodiesel is becoming an increasingly popular topic in bioenergy literature because of its potential to overcome the problems posed by chemical processes. However, the high cost of the enzymatic process still remains the main drawback for its industrial application, mostly because of the high price of refined oils. Unfortunately, low cost substrates, such as crude soybean oil, often release a product that hardly accomplishes the final required biodiesel specifications and need an additional pretreatment for gums removal. In order to reduce costs and to make the enzymatic process more efficient, we developed an innovative system for enzymatic biodiesel production involving a combination of a lipase and two phospholipases. This allows performing the enzymatic degumming and transesterification in a single step, using crude soybean oil as feedstock, and converting part of the phospholipids into biodiesel. Since the two processes have never been studied together, an accurate analysis of the different reaction components and conditions was carried out.

Results: Crude soybean oil, used as low cost feedstock, is characterized by a high content of phospholipids (900 ppm of phosphorus). However, after the combined activity of different phospholipases and liquid lipase Callera Trans L, a complete transformation into fatty acid methyl esters (FAMEs >95%) and a good reduction of phosphorus (P <5 ppm) was achieved. The combination of enzymes allowed avoidance of the acid treatment required for gums removal, the consequent caustic neutralization, and the high temperature commonly used in degumming systems, making the overall process more eco-friendly and with higher yield. Once the conditions were established, the process was also tested with different vegetable oils with variable phosphorus contents.

Conclusions: Use of liquid lipase Callera Trans L in biodiesel production can provide numerous and sustainable benefits. Besides reducing the costs derived from enzyme immobilization, the lipase can be used in combination with other enzymes such as phospholipases for gums removal, thus allowing the use of much cheaper, non-refined oils. The possibility to perform degumming and transesterification in a single tank involves a great efficiency increase in the new era of enzymatic biodiesel production at industrial scale.

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Figures

Figure 1
Figure 1
Prediction profile for citric acid obtained from RSM analysis. Graphical plotting of the effect of citric/phosphoric acid, pH, and extra NaOH on FAME production, made with JMP software (SAS Institute Inc.) (Rs = 0.88). FAME, fatty acid methyl ester; RSM, response surface methodology.
Figure 2
Figure 2
Dissolving effect of methanol on oil gums. (1) Soybean oil (raw material); (2) soybean oil with 1.5 eqs MeOH; (3) soybean oil acid degummed; and (4) soybean oil acid degummed with 1.5 eqs MeOH. Image obtained after 24 h incubation at 35°C, 250 rpm agitation, and centrifugation at 2,000 rpm for 5 min.
Figure 3
Figure 3
Enzymatic activities in the combined process. Schematic representation of all possible enzymatic activities involved in the combined degumming/transesterification process. Polar glycerol-phosphatide, resulting from PLA1 + LLPL-2 activities, and the phosphodiester group resulting from PLC cleavage, migrate to the aqueous phase together with the glycerine produced during transesterification (polar compounds highlighted by rectangles). LLPL-2, lyso-phospholipase; PLA1, phospholipase A1; PLC, phospholipase C.
Figure 4
Figure 4
UPLC/MS/MS analysis of the oil phase. Comparison of the mass spectrum resulting from analysis of the oil phase of reactions TE (upper plot) and PLA1 + TE (bottom plot). Samples were run with a RP column. For both chromatograms, ion 184 m/z in positive MS/MS mode were extracted. MS/MS, tandem mass spectrometry; PLA1, phospholipase A1; RP, reverse phase; TE, transesterification; UPLC, ultra-performance liquid chromatography.
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