Technical meeting December 1 st 2014 Dr. Daniele Spinelli Laboratory Manager

WPs - Solaris Biotechnology WP1 – Preliminary process design, selection of process components, supplier and market research Task 1.1 (M1-M12) – Set up of batch fermentation process WP2 – Process component characterisation and optimisation Task 2.2 (M13-M30) – Experimental screening and optimization of downstream procedures

Task 1.1 – Set up of batch fermentation process for metabolic description and downstream processing Microorganism: Clostridium acetobutylicum DSM 792 (freeze dried by DSMZ) Re-activation and crioconservation (20% glycerol at T = -80°C) Culture medium: Yeast Extract 5 g/L and D-Lactose (2-100 g/L) Temperature: 35°C Initial pH: 6-7 Anaerobic conditions: nitrogen stream

Task 1.1 – Set up of batch fermentation process for metabolic description and downstream processing Variation of culture medium to investigate effects on butanol yield: Yeast extract (1 g/L) KH 2 PO 4 (0.5 g/L) K 2 HPO 4 (0.5 g/L) p-aminobenzoic acid (0.001 g/L) Thiamin (0.001 g/L) Biotin (1x10 -5 g/L) MgSO 4 ·7H 2 O (0.2 g/L) MnSO 4 ·7H 2 O (0.01 g/L) Fe 2 SO 4 ·7H 2 O (0.01 g/L) NaCl (0.01 g/L) Ammonium Acetate (2.2 g/L) The Scientific Wolrd Journal, Volume 2014, Article ID g/L using 50 g/L date fruit

Task 1.1 – Set up of batch fermentation process for metabolic description and downstream processing Concentration vs time profiles: Lactose Acetic acid Butyric acid Ethanol Acetone Butanol Biomass density pH monitored in order to investigate acidogenesis and solventogenesis phases Time: h Gas chromatography UV/visible spectrophotometry

Adsorption Task 2.2 – Experimental screening and optimization of downstream procedures, generation of data for mathematical model verification Butanol can be desorbed by increasing the temperature to around 200°C. Greatly decrease of energy costs as ordinary distillation would require 73.3 MJ/kg butanol, while adsorption only would need 8.2 MJ/kg.

Task 2.2 – Experimental screening and optimization of downstream procedures, generation of data for mathematical model verification Biomass and Bioenergy 60 (2014)

Task 2.2 – Experimental screening and optimization of downstream procedures, generation of data for mathematical model verification Liquid-liquid extraction Appropriate organic solvent: - compatible with the bacteria used for fermentation - high distribution coefficient for butanol (minimize the amount of solvent needed and the product recovery cost) - if the products are recovered from the solution by distillation, the solvent should be less volatile than the products. - barely soluble in water, this to minimize solvent losses. i.e. oleyl alcohol and oleyl alcohol/decane (50 wt%)

Task 2.2 – Experimental screening and optimization of downstream procedures, generation of data for mathematical model verification High distribution coefficient for butanol (primary C6-C11 alcohols) ABE Solv2 remove Solv1 from the fermentation broth (C9-C12 alkanes  no azeotropes by distillation) Novel dual extraction process for ABE fermentation Separation and Purification Technology 124 (2014) Best case: Solv1: Decanol Solv2: Decane ~ 4 MJ/kg butanol

Task 2.2 – Experimental screening and optimization of downstream procedures, generation of data for mathematical model verification Gas stripping Oxygen-free nitrogen or fermentation gases (hydrogen and carbon dioxide) are bubbled through the fermentation broth to strip away acetone, butanol and ethanol. Inert gas will be sparged through the fermentation broth during fermentation and volatile butanol will vaporize and go out with the gas stream in the top of the reactor.

Task 2.2 – Experimental screening and optimization of downstream procedures, generation of data for mathematical model verification Biomass and Bioenergy 60 (2014)

Task 2.2 – Experimental screening and optimization of downstream procedures, generation of data for mathematical model verification Environmental Engineering and Management Journal 11 (2012), 8, Eicosanol (high affinity towards the butanol and low affinity towards the water) butanol-water solution butanol-eicosanol solution

Task 2.2 – Experimental screening and optimization of downstream procedures, generation of data for mathematical model verification Pervaporation This method involves the selective transport by diffusion of some components thrugh a membrane. A vacuum applied to the side of permeate. The permeated vapours should be condensed on low pressure side. Membrane in this case ought to be a hydrophobic polymer since transportation of organic components from the fermentation broth is preferred. Polydimethylsiloxane membranes and silicon rubber sheets are generally used for the pervaporation process. The drawback of the method can be high costs to produce low pressure at low pressure side of the membrane.

Task 2.2 – Experimental screening and optimization of downstream procedures, generation of data for mathematical model verification i.e. silicalite- 1/polydimethylsiloxane (PDMS) hybrid membranes (98 mg butanol/g) Separation and Purification Technology 79 (2011) 375– 384

Task 2.2 – Experimental screening and optimization of downstream procedures, generation of data for mathematical model verification SS—steam stripping distillation; GS—gas stripping; Perv—pervaporation; Ext—liquid–liquid extraction; Ad—adsorption on to silicalite Bioprocess Biosyst Eng (2005) 27: 215– % -33% -20% -3% MethodMJ/kg butanol Steam stripping24.2 Direct distillation18.4 Extraction (oleyl alcohol)13.3 Gas stripping13.8 Adsorption-desorption8.2 Extraction (mesitylene)4.8 √ Separation and Purification Technology 124 (2014) 18-25

Task 2.2 – Experimental screening and optimization of downstream procedures, generation of data for mathematical model verification Appl Microbiol Biotechnol (2014) 98:3463–3474