Bioconversion of crude glycerol to glycolipids in Ustilago maydis Yanbin Liu, Chong Mei John Koh, Lianghui Ji ⇑ Biomaterials and Biocatalysts Group, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore article info Article history: Received 28 July 2010 Received in revised form 22 November 2010 Accepted 23 November 2010 Available online 28 November 2010 Keywords: Biosurfactant Crude glycerol Glycolipids Fed-batch fermentation Ustilago maydis abstract Ustilago maydis is known to produce glycolipid-type biosurfactants. Here, we show that U. maydis is able to efficiently convert biodiesel-derived crude glycerol to glycolipids. We have optimized the medium composition and environmental factors for bioconversion of crude glycerol to glycolipids. The synthetic medium (MinCG) contains 50 g L 1 crude glycerol and 20.3 mg L 1 ammonium citrate as the carbon and nitrogen sources, respectively. The supplementation of trace amount of amino acids, Group-B vitamins and precursors of glycolipids, mannose and erythritol, also improved the final yield. At pH 4.0 and 30 °C, 32.1 g L 1 total glycolipids was produced in a 8.2-day fed-batch bioprocess. Methanol at 2% or above severely inhibited cell growth and production of glycolipids. Our results suggest that U. maydis is an excellent host for the bioconversion of crude glycerol to value-added products. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Crude glycerol is a by-product of the biodiesel industry, accounting for approximately 10% (v/v) of the oil transesterifica- tion reaction (Wang et al., 2009). It is sometimes regarded as a waste product because of the cost associated with its disposal (Yazdani and Gonzalez, 2007). To date, utilization of glycerol, par- ticularly crude glycerol, remains limited although there has been a number of reports on the conversion of glycerol to value-added products through chemical methods (Johnson and Taconi, 2007) or biological methods (Athalye et al., 2009; Papanikolaou and Aggelis, 2002). The end products include 1,3-propanediol, dihy- droxyacetone, ethanol, succinate, propionic acid, glyceric acid, cit- ric acid, hydroxypyruvic acids, polyhydroxyalcanoate, pigments and biosurfactants (da Silva et al., 2009). In recent years, remark- able exploration on the utilization and conversion of crude glycerol are on the way with improving yields (Ethier et al., 2010; Moon et al., 2010; Sabourin-Provost and Hallenbeck, 2009). Biosurfactants have been a subject of notable interest in recent years owing to their low toxicity, biodegradability and structural diversity. A small number of microorganisms have been reported to produce biosurfactants, which have diverse industrial applica- tions, such as enhanced oil recovery, crude oil drilling, lubrication, surfactant-aided bioremediation, health care and food (Cameotra and Makkar, 2004). Glycolipids contain a carbohydrate (monosac- charide, disaccharide or oligosaccharide) and one or more lipophilic moieties consisting of saturated, unsaturated and/or hydroxylated fatty acids or fatty alcohols. The amphiphilic nature of the gly- colipids make them very promising as a biosurfactant (Morita et al., 2009). Certain strains of Ustilago maydis were reported to secrete large amounts of glycolipid type biosurfactants, mainly mannosylerythr- itol lipids (MEL) and ustilagic acid (UA) under nitrogen-limiting conditions (Hewald et al., 2005). Both MEL and UA have excellent surface-active properties. Although glycolipids can be also pro- duced by some species in Pseudomonas, Candida, Rhodococcus and Pseudozyma genera, U. maydis is by far the best-characterized spe- cies. With the wealth of genetic resources, genomic information, and genetic and molecular tools (see review by (Brefort et al., 2009)), U. maydis is a superior host for the bioengineering of im- proved strains for bioconversion of glycerol and other renewable resources. Recently, refined glycerol has been successfully applied to the production of rhamnolipid and MEL-type biosurfactants (Morita et al., 2007; Rahman et al., 2002). It has also been reported that Candida bombicola is able to produce 60 g L 1 sophorolipid from a biodiesel co-product stream (BCS), which is composed of 40% glyc- erol and 34% hexane-solubles (Ashby et al., 2005). Here, we present a simple method for conversion of crude glycerol to UA and MEL in U. maydis. 2. Methods 2.1. Source of crude glycerol and U. maydis strains Crude glycerol was obtained from Biofuel Research Pte. Ltd. (Singapore). The sample was derived from alkali-catalyzed 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.115 ⇑ Corresponding author. Tel.: +65 6872 7483; fax: +65 6872 7007. E-mail address: jilh@tll.org.sg (L. Ji). Bioresource Technology 102 (2011) 3927–3933 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech