An industrial scale process for the enzymatic removal of steryl glucosides from biodiesel

Abstract

Background: Biodiesels produced from transesterification of vegetable oils have a major quality problem due to the presence of precipitates, which need to be removed to avoid clogging of filters and engine failures. These precipitates have been reported to be mostly composed of steryl glucosides (SGs), but so far industrial cost-effective methods to remove these compounds are not available. Here we describe a novel method for the efficient removal of SGs from biodiesel, based on the hydrolytic activity of a thermostable β-glycosidase obtained from Thermococcus litoralis.

Results: A steryl glucosidase (SGase) enzyme from T. litoralis was produced and purified from Escherichia coli cultures expressing a synthetic gene, and used to treat soybean-derived biodiesel. Several optimization steps allowed for the selection of optimal reaction conditions to finally provide a simple and efficient process for the removal of SGs from crude biodiesel. The resulting biodiesel displayed filterability properties similar to distilled biodiesel according to the total contamination (TC), the cold soak filtration test (CSFT), filter blocking tendency (FBT), and cold soak filter blocking tendency (CSFBT) tests. The process was successfully scaled up to a 20 ton reactor, confirming its adaptability to industrial settings.

Conclusions: The results presented in this work provide a novel path for the removal of steryl glucosides from biodiesel using a cost-effective, environmentally friendly and scalable enzymatic process, contributing to the adoption of this renewable fuel.

Keywords: Biofuels; Green chemistry; Synthetic biology.

Fig. 1

Schematic representation of the SGase reaction, showing the hydrolysis of β-sitosteryl glucoside, the most abundant SG in soybean oil

Fig. 2

A time course of SGase activity (expressed as conversion %) of BGTl and LacS in aqueous buffer. Reactions were performed at 70 °C in phosphate buffer at pH 6.5 for BGTl and citrate buffer at pH 5.5 for LacS. 14 µg of enzyme/mL of buffer spiked with 100 ppm SGs was used. SGase hydrolysis was measured with a coupled enzymatic fluorescence assay. Error bars show the standard deviation of three independent assays

Fig. 3

A time course of SGase activity (expressed as conversion %) of BGTl and LacS in distilled biodiesel. Reactions were performed in biodiesel/water mixtures (100:15) at 70 °C in 50 mM phosphate buffer (pH 6.5) for BGTl and 50 mM citrate buffer pH 5.5 for LacS. 14 µg of enzyme/g of biodiesel spiked with 100 ppm SGs was used. Error bars show the standard deviation of three independent assays

Fig. 4

Optimization of the enzymatic process. Influence of reaction factors on the efficiency of BGTl-mediated hydrolysis of SGs (expressed as conversion %) in commercial soybean oil biodiesel/water mixtures. Experimental conditions: a 100:15 biodiesel/water mixtures, 5 µg of enzyme/g biodiesel, 70 °C, and 50 mM phosphate or citrate buffers of different pHs, b 100:15 biodiesel/water mixtures, 5 µg of enzyme/g biodiesel, 70 °C, phosphate buffer (pH 6.75) at different concentrations, c 100:15 biodiesel/water mixtures, 5 µg of enzyme/g biodiesel, 20 mM phosphate buffer (pH 6.75), 70 °C, and different concentrations of NaCl, d 100:15 biodiesel/water mixtures, 5 µg of enzyme/g biodiesel, 20 mM phosphate buffer (pH 6.75), 20 mM NaCl, at different temperatures, e 100:15 biodiesel/water mixtures, 20 mM phosphate buffer (pH 6.75), 20 mM NaCl, 65 °C, and different concentrations of BGTl, f biodiesel/water mixtures with different water content, 5 µg of enzyme/g biodiesel, 20 mM phosphate buffer (pH 6.75), 20 mM NaCl, 65 °C. Error bars show the standard deviation of three independent assays

Fig. 5

Effect of agitation on BGTl-mediated hydrolysis of SGs in a 15 L reactor. A time course of the hydrolysis of SGs (expressed as conversion %) in commercial biodiesel containing 65 ppm of SGs/water mixtures (100:4.5) using 7 mg of enzyme/kg biodiesel at 65 °C in 20 mM phosphate buffer pH 6.75 and 20 mM NaCl and at different agitation speeds. Error bars show the standard deviation of three independent assays

Fig. 6

BGTl-mediated removal of SGs in a 20 ton industrial reactor. a A time course of the hydrolysis of SGs (expressed as conversion %) in a commercial biodiesel containing 75 ppm of SGs/water mixture (100:4.5) using 7 mg of enzyme/kg biodiesel at 65 °C in 20 mM phosphate buffer pH 6.75 and 20 mM NaCl and at 43 rpm. Error bars show the standard deviation of three independent assays. b SPE-GC-FID analysis of treated biodiesel samples. Traces are shifted on y axis for clarity. Peaks are labeled as follows: Std, cholesteryl glucoside standard; 1 campesteryl glucoside; 2 stigmasteryl glucoside and 3 β-sitosteryl glucoside