Improved glycerol utilization by a triacylglycerol-producing Rhodococcus opacus strain for renewable fuels

Abstract

Background: Glycerol generated during renewable fuel production processes is potentially an attractive substrate for the production of value-added materials by fermentation. An engineered strain MITXM-61 of the oleaginous bacterium Rhodococcus opacus produces large amounts of intracellular triacylglycerols (TAGs) for lipid-based biofuels on high concentrations of glucose and xylose. However, on glycerol medium, MITXM-61 does not produce TAGs and grows poorly. The aim of the present work was to construct a TAG-producing R. opacus strain capable of high-cell-density cultivation at high glycerol concentrations.

Results: An adaptive evolution strategy was applied to improve the conversion of glycerol to TAGs in R. opacus MITXM-61. An evolved strain, MITGM-173, grown on a defined medium with 16 g L(-1) glycerol, produced 2.3 g L(-1) of TAGs, corresponding to 40.4% of the cell dry weight (CDW) and 0.144 g g(-1) of TAG yield per glycerol consumed. MITGM-173 was able to grow on high concentrations (greater than 150 g L(-1)) of glycerol. Cultivated in a medium containing an initial concentration of 20 g L(-1) glycerol, 40 g L(-1) glucose, and 40 g L(-1) xylose, MITGM-173 was capable of simultaneously consuming the mixed substrates and yielding 13.6 g L(-1) of TAGs, representing 51.2% of the CDM. In addition, when 20 g L(-1) glycerol was pulse-loaded into the culture with 40 g L(-1) glucose and 40 g L(-1) xylose at the stationary growth phase, MITGM-173 produced 14.3 g L(-1) of TAGs corresponding to 51.1% of the CDW although residual glycerol in the culture was observed. The addition of 20 g L(-1) glycerol in the glucose/xylose mix resulted in a TAG yield per glycerol consumed of 0.170 g g(-1) on the initial addition and 0.279 g g(-1) on the pulse addition of glycerol.

Conclusion: We have generated a TAG-producing R. opacus MITGM-173 strain that shows significantly improved glycerol utilization in comparison to the parental strain. The present study demonstrates that the evolved R. opacus strain shows significant promise for developing a cost-effective bioprocess to generate advanced renewable fuels from mixed sugar feedstocks supplemented with glycerol.

Keywords: Adaptive evolution; Co-fermentation; Glycerol utilization; Renewable fuels; Rhodococcus opacus; Triacylglycerol.

Figure 1

Growth of R. opacus MITGM-173 on varying concentrations of glycerol. Glycerol concentrations of defined media were 16, 40, 80, 120, 160, and 200 g L⁻¹ in shake flasks. Values and error bars represent the mean and s.d. of triplicate experiments.

Figure 2

TAG production from glycerol and/or glucose by R. opacus MITGM-173. (a-c) Time course kinetics of TAG production as fatty acids. The strain was grown in defined media containing 16 g L⁻¹ glycerol (a), a mixture of 8 g L⁻¹ glycerol and 8 g L⁻¹ glucose (b), and 16 g L⁻¹ glucose (c) in shake flasks. Values and error bars represent the mean and s.d. of triplicate experiments. (d) Thin-layer chromatography analysis of the crude organic extracts obtained from the cells grown on glycerol (a), glycerol/glucose (b), and glucose (c) for 6 days. Lipids were extracted and separated on a silica gel plate as described in the “Methods” section. Lipid standards of TAG (1,2-dioleoyl-3-stearoyl-rac-glycerol), DAG (1,2-dipalmitoyl-rac-glycerol), and MAG (DL-α-palmitin) were used to identify the Rf value for TAG under the conditions used. Lanes: 1, crude lipid extract (10 μg) on glycerol; 2, crude lipid extract (10 μg) on glycerol/glucose; 3, crude lipid extract (10 μg) on glucose; S, TAG (3 μg)/DAG (3 μg)/MAG (3 μg) mixtures. (e) Fatty acid composition as percentage of total fatty acids (g g⁻¹) of lipids from the cells growing in the defined medium containing glycerol (a), glycerol/glucose (b), or glucose (c) for 6 days. Data are results of triplicate experiments, ±s.d.

Figure 3

Response surface plot of the effect of glycerol and (NH ₄ ) ₂ SO ₄ concentrations on TAG production. As fatty acids by R. opacus MITGM-173. Curves and points represent predicted values and experimental data, respectively.

Figure 4

Time course of TAG production as fatty acids from glycerol by R. opacus MITGM-173. Performed under the optimized conditions. The strain was grown in a modified defined medium containing 96 g L⁻¹ glucose and 6.55 g L⁻¹ (NH₄)₂SO₄ in bioreactors. Values and error bars represent the mean and s.d. of triplicate experiments.

Figure 5

TAG production from mixed substrates of glucose, xylose, and glycerol by R. opacus MITGM-173. (a-c) Time course kinetics of TAG production as fatty acids. The strain was grown in modified defined media supplemented with 5.56 g L⁻¹ (NH₄)₂SO₄ containing a mixture of 40 g L⁻¹ xylose and 40 g L⁻¹ glucose (a), a mixture of 40 g L⁻¹ xylose, 40 g L⁻¹ glucose and 20 g L⁻¹ glycerol (b), and a mixture of 40 g L⁻¹ xylose and 40 g L⁻¹ glucose with pulse loading of 20 g L⁻¹ glycerol after 2 days of cultivation (c) in bioreactors. Values and error bars represent the mean and s.d. of triplicate experiments. (d) Thin-layer chromatography analysis of the crude organic extracts from the cells growing on xylose/glucose (a), xylose/glucose/glycerol (b), and xylose/glucose with pulse loading of glycerol (c) for 7 days. Lanes: 1, crude lipid extract (10 μg) on xylose/glucose; 2, crude lipid extract (10 μg) on xylose/glucose/glycerol; 3, crude lipid extract (10 μg) on xylose/glucose with pulse loading of glycerol; S, TAG (6 μg)/DAG (3 μg)/MAG (3 μg) mixtures. (e) Fatty acid composition as percentage of total fatty acids (g g⁻¹) of lipids from the cells growing in the defined medium containing xylose/glucose (a), xylose/glucose/glycerol (b), or xylose/glucose with pulse loading of glycerol (c) for 7 days. Data are results of triplicate experiments, ±s.d.