Electronic Supporting Information

Extraction and purification of violacein from Yarrowia lipolytica cells with surfactants

Mariam Kholany1, Pauline Trebulle2,3, Margarida Martins1, Sónia P. M. Ventura1, Jean-Marc Nicaud2, João A. P. Coutinho1*

1CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

2Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France

3University of Lille, 42, rue Paul Duez, Lille, 59000, France

*Corresponding author

Email: jcoutinho@ua.pt

Figure S1: A. Plasmids encoding the five genes involved in the violacein pathway, constructed through Golden Gate Assembly. B. Y. lipolytica violacein producing strains JMY7019 containing the two expression cassettes VioABE and VioCD.

Figure S2: Chemical structure and abbreviation name of the compounds used in this work.

Figure S3. Photos taken to the extracts obtained after the first screening with ethanol, water (controls) and tensioactive and non-tensioactive compounds.

Figure S4. Photos taken to the extracts and biomass pellets obtained after the consecutive extractions for the best and worst solvents, respectively, aqueous solution of Tween 20 and ethanol.

Figure S5. Photos of Y. lipolytica cells growth after violacein extraction using a Tween 20 aqueous solution and ethanol (control).

Figure S6: Binodal curves of the ternary systems composed of [Ch]X + Tween 20 + water at 298 K and atmospheric pressure in molality units.

Figure S7. Binodal curves of the ternary systems in weight percentage units composed of [Ch]X + Tween 20 + water at 298 K and atmospheric pressure.

Figure S8. Extraction efficiencies obtained for violacein (purple bar) and total protein (blue bar) of each ABS composed of Tween 20 + [Ch]X + water.

Figure S9. 1H NMR of the Tween 20 (top phase) and cholinium acetate (bottom phase) in deuterated DMSO.

Figure S10. 1H NMR of violacein-rich sample after 2-methyl-THF back-extraction and solvent evaporation. Main bands associated with Tween 20 and cholinium acetate are highlighted in yellow and purple, respectively. Water peak is identified in blue.

Figure S11. 1H NMR of violacein-rich sample after ethyl acetate back-extraction and solvent evaporation. Main bands associated with Tween 20 and cholinium acetate are highlighted in yellow and purple, respectively. Water peak is identified in blue.

Table S1. Solvents tested to promote the back-extraction at an solvent:sample phase ratio of 0.5.Solvent Tested ResultToluene Formed bubbles, lost most of the purple colour of violaceinn-Hexane Jellified

3-Hexyloxypropan-1,2-diol [6.0.0] Formed two phases, inviable to separate mixture from sample1,3-dipropoxypropan-2-ol [3.0.3] Formed two phases, inviable to separate mixture from sample1:1 Menthol:thymol Milky solution, the purple colour of violacein disappearedEthyl acetate Formed two phases, possible to separate from sample2-Methyl-THF Formed two phases, possible to separate from sample

Table S2. Experimental weight fraction compositions (%w/w) of the binodal curve for the system [Ch][Ac] (1) + Tween 20 (2) + H2O (3) at (25 ± 1) °C.

Table S3.

Experimental weight fraction compositions (%w/w) of the binodal curve for the system [Ch]Cl (1) + Tween 20 (2) + H2O (3) at (25 ± 1) °C.

[Ch][Ac] (1) + Tween 20 (2) + H2O (3)100 w1 100 w2 100 w1 100 w2 100 w1 100 w277.55 5.74 34.68 21.82 15.17 30.9774.34 6.79 33.89 22.44 14.70 31.1871.35 7.70 33.29 22.58 14.18 31.4267.60 9.45 32.69 22.72 13.68 31.5763.26 10.99 31.89 23.18 13.19 32.0859.45 12.23 31.07 23.78 12.74 32.3357.89 12.89 30.21 24.08 12.35 32.5356.28 13.54 29.25 24.81 11.95 32.6154.91 14.07 28.33 25.01 11.60 32.7052.40 14.92 27.71 25.36 11.22 32.7850.90 15.29 26.79 25.77 10.88 32.9049.28 16.35 25.79 26.23 10.48 33.2448.15 16.82 24.77 26.85 10.22 33.4147.02 16.90 23.91 27.21 9.91 33.4746.02 17.32 23.08 27.68 9.64 33.5544.20 18.00 22.25 28.16 9.36 33.7443.31 18.24 21.23 28.23 9.08 33.8741.66 18.96 20.53 28.7340.77 19.22 19.81 28.8839.93 19.43 18.94 29.0538.94 20.05 18.31 29.2238.10 20.24 17.45 29.9237.10 20.82 16.92 30.4736.39 21.05 16.37 30.7035.31 21.67 15.70 30.71

Table S4.

Experimental weight fraction compositions (%w/w) of the binodal curve for the system [Ch][DHP] (1) + Tween 20 (2) + H2O (3) at (25 ± 1) °C.

[Ch]Cl (1) + Tween 20 (2) + H2O (3)100 w1 100 w2 100 w1 100 w2 100 w1 100 w276.00 7.14 34.97 26.75 20.77 35.5974.00 7.87 34.28 26.99 20.37 35.9271.13 9.11 33.23 27.72 20.15 36.0168.52 10.21 32.64 28.00 19.83 36.3365.13 12.23 32.08 28.31 19.62 36.3562.48 13.10 31.25 28.91 19.41 36.4560.25 14.18 30.48 29.43 19.09 36.6758.64 14.76 30.01 29.65 18.78 36.9756.91 15.48 29.57 30.00 18.32 37.3255.16 16.24 28.88 30.49 18.13 37.3452.88 17.64 28.23 30.9850.89 18.24 27.77 31.1349.45 18.86 27.19 31.5347.42 20.35 26.79 31.6646.16 20.94 26.18 32.1845.01 21.56 25.41 32.4544.09 22.09 24.83 32.9342.86 22.58 24.41 33.1741.90 22.99 23.86 33.5740.94 23.38 23.36 33.9840.25 23.65 23.07 34.0339.34 24.12 22.59 34.4238.15 24.94 22.12 34.7437.43 25.19 21.67 35.1136.26 26.05 21.39 35.19

Table S5.

Experimental weight fraction compositions (%w/w) of the binodal curve for the system [Ch][Bic] (1) + Tween 20 (2) + H2O (3) at (25 ± 1) °C.

[Ch][DHP] (1) + Tween 20 (2) + H2O (3)100 w1 100 w2 100 w1 100 w2 100 w1 100 w270.22 3.43 33.17 15.62 17.66 21.8066.04 4.31 32.53 15.97 17.28 21.9863.92 5.05 31.98 16.27 16.86 22.1261.78 5.83 31.39 16.46 16.53 22.3258.59 6.39 30.79 16.70 16.07 22.6356.02 7.69 30.27 16.94 15.74 22.7653.60 8.20 29.61 17.18 15.37 22.8651.82 8.80 29.11 17.44 15.05 23.0250.28 9.45 28.15 17.94 14.73 23.1448.98 9.90 27.00 18.29 14.46 23.2847.72 10.47 25.96 18.57 14.20 23.3946.44 10.97 25.43 18.71 13.89 23.6644.59 11.32 25.01 18.87 13.54 23.7643.62 11.72 24.64 19.0542.62 12.09 24.27 19.2241.64 12.51 23.57 19.5940.83 12.90 22.86 19.8939.99 13.28 22.10 20.0538.77 13.59 21.56 20.3337.97 13.98 20.99 20.6436.78 14.21 20.23 20.9936.01 14.59 19.56 20.9335.41 14.89 19.10 21.1334.72 15.21 18.55 21.5934.02 15.47 18.06 21.65

[Ch][Bic] (1) + Tween 20 (2) + H2O (3)100 w1 100 w2 100 w1 100 w278.39 5.79 28.35 30.8475.55 6.56 27.05 31.5773.42 7.20 25.63 32.4669.57 9.08 24.47 33.1667.30 9.95 23.46 33.7864.04 11.67 22.45 34.3959.86 13.58 21.60 35.0157.28 14.93 20.50 35.6054.50 16.59 19.54 36.1751.39 17.74 18.69 36.7248.74 19.52 17.93 36.9347.29 20.01 17.04 37.5045.71 21.07 16.13 38.2043.93 22.01 15.39 38.8042.37 22.87 14.88 39.1440.57 24.0339.25 24.7038.19 25.2336.80 26.1135.78 26.6034.61 27.4133.79 27.8532.94 28.2931.57 28.9429.93 29.88

Table S6. Experimental weight fraction compositions (%w/w) of the binodal curve for the system [Ch][DHC] (1) + Tween 20 (2) + H2O (3) at (25 ± 1) °C.

Table S7. Correlation parameters used to describe the experimental binodal data by

Eq. 2, and respective standard deviations (σ) and correlation coefficients.

ILs A ± σ B ± σ 105 (C ± σ) R2

[Ch][DHP] 105.35 ± 2.38 -0.23 ± 0.01 7.1 ± 0.3 0.9956

[Ch][DHC] (1) + Tween 20 (2) + H2O (3)

100 w1 100 w2 100 w1 100 w2

50.65 21.42 14.37 45.6045.94 24.20 13.32 46.3540.47 27.73 12.46 46.9336.26 30.48 11.74 47.4031.95 33.35 11.13 47.7929.02 35.32 10.57 48.2226.82 36.77 10.04 48.6124.26 38.53 9.46 49.0622.77 39.52 9.16 49.2821.14 40.63 8.36 49.9019.52 41.80 7.94 50.1917.63 43.32 7.67 50.3616.51 44.1015.38 44.94

[Ch][Ac] 121.30 ± 2.75 -0.19 ± 0.01 3.4 ± 0.1 0.9974[Ch][Bic] 110.33 ± 2.16 -0.16 ± 0.01 1.76 ± 0.01 0.9975[Ch]Cl 113.64 ± 3.22 -0.16 ± 0.01 1.89 ± 0.01 0.9938[Ch][DHC] 97.12 ± 2.15 -0.12 ± 0.01 1.23 ± 0.01 0.9991

Table S8. Average of the experimental weight fraction compositions (wt%), extraction efficiencies of violacein and total protein content (EEviolacein and EETP) and respective standard deviations (σ) for the partition systems investigated.

ABS

100 Mass fraction composition / (wt%) EEviolacein (%) EETP (%)

[Tween 20]M [IL]M

[Ch]Ac 30.06 40.01 100 42.9 ± 0.2

[Ch]Bic 29.58 49.83 100 44 ± 1

[Ch]Cl 30.03 39.97 100 52.6 ± 0.6

[Ch]DHP 30.07 39.89 100 55 ± 2

[Ch]DHC 30.00 39.96 100 56 ± 1