*2.6. Bottle and Bioreactor Experiments*

Frozen stocks of *L. fermentum* were prepared from cells growing exponentially on Mann, Rogosa and Sharpe (MRS) broth and stored at −80 ◦C after the addition of a 20% *v*/*v* glycerol solution. Bottle experiments were performed in 100 mL screw-cap bottles with a working volume of 90 mL and incubated at 37 ◦C and 150 rpm in a rotary shaker incubator (model Minitron, Infors, Bottmingen, Switzerland) for 24 h. Modified semi-define medium SGSL [23] contained the following per liter: 10 g yeast extract; 10 g soy peptone; 2 g Na3C6H5O7; 0.5 g L-ascorbic acid; 0.5 mL Tween80; 0.25 g MgSO<sup>4</sup> · 7 H2O; 0.2 g NaCl; 0.05 g MnSO<sup>4</sup> · H2O. The concentrated and spray-dried UF retentate (UF\_Ret20Pow) was used for growth experiments. In particular, the medium consisted of 1 ± 0.1, 2 ± 0.2 and 4 ± 0.2% UF\_Ret20Pow reconstituted in SGSL medium. UF\_Ret20Pow 4 ± 0.2% reconstituted in distilled water was also tested. Control experiments on SGSL supplemented with 30 g/L glucose or lactose were also performed. Samples were withdrawn at time 0, and after 8 and 24 h of growth to analyze optical density (600 nm) carbon sources consumption, and acid and ethanol production. All bottle experiments were performed in triplicate. Viability was evaluated by serially diluting the samples and plating on MRS-agar medium. Plates were incubated at 37 ◦C for 36 h before counting viable cells. Each sample was analyzed in triplicate.

Bioreactor experiments were performed in a Biostat CT plus (Sartorius Stedim, Gottingen, Germany) bioreactor with a working volume of about 2.2 L. Temperature was controlled at 37 ◦C, pH at 6.1 and agitation was fixed at 150 rpm. Fermentation medium containing UF\_Ret20Pow reconstituted in water or in water supplemented with salts present in the SGSL medium and 2 g/L of yeast extract and soy peptone (1/5th of that present in SGLS) were used for bioreactor growth. Before each experiment, a concentrated stock solution of *L. fermentum* was inoculated in 0.25 L of SGSL medium at 37 ◦C and 150 rpm and grown for 8 h. The pre-culture was then transferred to the bioreactor with a peristaltic pump (model 313 U, Watson-Marlow, England) to reach up to 10% (*v*/*v*) of the working volume inside the fermenter. Stirring was set to 150 rpm and air was sparged at a constant flow of 0.44 vvm. A constant pH of 6.1 was maintained by addition of NaOH 10 M and 30% *v*/*v* H2SO<sup>4</sup> solutions. Experiments lasted up to 24 h. Samples were withdrawn during the experiments to analyze cell density (600 nm), cell viability, carbon source consumption, and acid and ethanol production. Viability was evaluated as previously described. Batch experiments on UF\_Ret20Pow only were repeated 4 times whereas those on UF\_Ret20Pow supplemented with salts and complex nitrogen sources were performed in duplicate.
