Session 26 - Environmental Biotechnology and Bioenergy

Aim of this study is to explore the effect of sodium lignosulfonate (SL), a paper industry by-product, on cell growth and lipid production by the yeast Rhodosporidium toruloides, cultivated on xylose-based media, that mimic the principal waste-stream originated from paper production facilities (viz. the spent-sulfite liquor). Yeast lipids present increasing interest as alternative non-food feedstocks for biodiesel production. Strains DSM 4444 and NRRL Y-27012 were shake-flask cultured under nitrogen-limiting conditions using xylose at 50 g/L, and SL was added at varying concentrations. Finally, a fed-batch bioreactor trial of the strain NRRL Y-27012 with optimum SL addition was carried out.
In the strain DSM 4444, maximum lipid production was obtained in media supplemented with 20 g/L SL, where lipid of 4.8 g/L occurred. In NRRL Y-27012 strain, maximum lipid production was seen with the addition of 10 g/L SL (lipid =5.3 g/L). In fed-batch bioreactor experiments carried out with the strain NRRL Y-27012, lipid =17.0 g/L (corresponding dry biomass =29.7 g/L) was achieved. The yield of lipid produced per unit of xylose consumed was ≈0.19 g/g. Lipids containing increased concentrations of oleic acid, constituting thus perfect materials amenable to be converted into “2nd generation” biodiesel were synthesized.

Aim of the present study was to assess the ability of four Yarrowia lipolytica strains (ACA-DC 50109, LFMB Y-20, ATCC 20460 and LMBF Y-45) to grow on biodiesel-derived crude glycerol, the principal residue-stream deriving from biodiesel manufacture. Initial trials were carried out in shake-flasks under nitrogen limitation (initial glycerol Glol0~40 g/L, initial nitrogen ~0.35 g/L), that favor the production of cellular lipids and/or extra-cellular secondary metabolites like citric acid (CA). All strains produced appreciable dry cell weight (DCW) quantities (up to 13.0 g/L). The strain ACA-DC 50109 produced CA in concentrations up to 16.0 g/L, while lipid in DCW values of ~15% w/w were recorded. In the next stage, this strain was cultured on media with higher nitrogen limitation (Glol0~50 g/L, initial nitrogen ~0.15 g/L) in batch-bioreactor and shake-flask experiments, and comparable DCW (up to 8.0 g/L) and CA (25-28 g/L) quantities were reported for these trials. Lipid production was higher in the batch-bioreactor experiment. In fed-batch bioreactor trials performed thereafter, a maximum CA quantity of 66.1 g/L (conversion yield 0.66 g per g of glycerol) was obtained. Cellular lipids of all tested strains were mainly composed of the fatty acids Δ9C18:1, Δ9,12C18:2 and C16:0.

We have previously reported that a cell-free extract prepared from Geobacillus thermodenitrificans UZO 3 reductively cleaves diaryl ether bonds of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), a dioxin with the highest toxicity, in a sequential fashion producing 3’,4’,4,5-tetrachloro-2-hydroxydiphenyl ether (TCDE) as the intermediate, and 3,4-dichlorophenol (DCP) as the final reaction product. The detection of TCDE implicated the discovery of an unprecedented dioxin-degrading enzyme that reductively cleaves the diaryl ether bonds. In this study, we report the cloning and sequencing of the dioxin reductive etherase gene dreE which codes for the 2,3,7,8-TCDD-degrading enzyme. We showed that dreE was expressed in Escherichia coli and that the product of the expression could reductively cleave diaryl ether bonds of 2,3,7,8-TCDD to produce TCDE. Furthermore, we established that the amino acid sequence encoded by dreE was homologous to an enzyme with yet unknown function that is encoded by a gene located in the riboflavin (vitamin B2) biosynthesis operon in Bacillus subtilis. We also showed that the amino acid sequence possesses a coenzyme A (CoA) binding site that is conserved in the N-acyltransferase superfamily. For the first time, the degradation of 2,3,7,8-TCDD at the molecular level using a enzyme of bacterial origin has been demonstrated.