Short Communication Whole-cell based solvent-free system for one-pot production of biodiesel from waste grease Aitao Li, Thao P.N. Ngo, Jinyong Yan, Kaiyuan Tian, Zhi Li ⇑ Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore article info Article history: Received 21 February 2012 Received in revised form 8 March 2012 Accepted 9 March 2012 Available online 20 March 2012 Keywords: Biodiesel Grease Free fatty acid Whole-cell Biotransformation abstract A whole-cell based solvent-free system was developed for efficient conversion of waste grease to biodie- sel via one-pot esterification and transesterification. By isolation and screening of lipase-producing strains from soil, Serratia marcescens YXJ-1002 was discovered for the biotransformation of grease to bio- diesel. The lipase (SML) from this strain was cloned and expressed in Escherichia coli as an intracellular enzyme, showing 6 times higher whole-cell based hydrolysis activity than that of wild type strain. The recombinant cells were used for biodiesel production from waste grease in one-pot reactions containing no solvent with the addition of methanol in several small portions, and 97% yield of biodiesel (FAME) was achieved under optimized conditions. In addition, the whole-cell biocatalysts showed excellent reusabil- ity, retaining 74% productivity after 4 cycles. The developed system, biocatalyst, and process enable the efficient, low-cost, and green production of biodiesel from waste grease, providing with a potential indus- trial application. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Biodiesel, a mixture of fatty acid methyl esters (FAME), is receiving increasing attention as an environmentally friendly and renewable energy with better combustion performance. However, the current price of biodiesel is too high, since most biodiesel plants are using refined vegetable oils (0.70–1.28 USD/L) (Baig and Ng, 2010) as the feedstock which contributes nearly 80% of the overall biodiesel production cost (Lam et al., 2010). In addition, using vegetable oils for fuel production directly competes with food supplies, causing further difficulties for the use of these re- sources. On the other hand, waste grease which contains a high amount of free fatty acid (FFA, 15–40 wt.%) is a cheap (<0.3 USD/ L) (Canakci, 2007), non-edible, and easily available resource, repre- senting a promising alternative feedstock for biodiesel production with the potential of reducing production costs and avoiding waste disposal. However, there is a significant technical challenge in using waste grease to produce biodiesel. The traditional alkaline catalysis cannot be used due to the soap formation caused by the high percentage of FFA in grease. A two-step process consisting of acid-catalyzed esterification of FFA to reduce FFA content and subsequent transesterification of the remaining triglycerides with a base catalyst could be employed. However, the use of liquid acid generates environmental pollution and corrosion, and the conver- sion achieved with solid acid catalysts is not high enough (Wan Omar et al., 2009). Enzyme could catalyze both esterification of FFA and transeste- rification of triglyceride with methanol into biodiesel in one-pot under mild reaction conditions (Fig. 1(a)), providing green and effi- cient process. Many extracellular lipases (free or immobilized en- zymes) were examined for biodiesel production (Ranganathan et al., 2008; Li et al., 2011; Liu et al., 2011). However, the efficien- cies and conversions are unsatisfactory for high FFA-containing feedstock (>15 wt.%). For example, immobilized Pseudomonas cepa- cia Lipase (PS-30), Candida antartica Lipase (gran-C.a.), or Ther- momyces lanuginose Lipase (gran-T.l.) achieved low conversions of grease to FAME (Hsu et al., 2002). In addition, the cost of enzymes is high due to the complicated recovery, purification, and immobi- lization steps required. Compared with free and immobilized enzyme, microbial cells containing intracellular lipase are much cheaper and more conve- nient for biodiesel production, and they can be easily prepared in large amounts. A few whole-cell systems (e.g., fungi and yeast) were successfully used for biodiesel production from refined Soy- bean oil or Jatropha oil (Fukuda et al., 2008). However, transforma- tion of pre-acidified rapeseed oil (67 mol% FFA content) with methanol in tert-butanol using Rhizopus oryzae cells gave an unsat- isfactory yield of FAME (70%) (Li et al., 2007) and required tert- butanol as a solvent which adds to process costs and generates waste. Thus far, the use of whole-cell biocatalysts for biodiesel production from resources containing high FFA content is still a 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.03.034 ⇑ Corresponding author. Tel.: +65 6516 8416; fax: +65 6779 1936. E-mail address: chelz@nus.edu.sg (Z. Li). Bioresource Technology 114 (2012) 725–729 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech