2.1. Microorganism and culture conditions
Zymomonas mobilis subs. mobilis type strain DSM 424 (DSMZ:
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH),
originally isolated in Mexico from a fermented Agave sap to obtain pulque, an alcoholic beverage containing 4 to 6% (v/v) ethanol (Swings and
De Ley, 1977), and Lactobacillus sanfranciscensis DSM 20663 were used
in this study. Z. mobilis was weekly maintained in DSM liquid medium
having the following composition (g/L): Bacto-peptone (Costantino
SpA, Turin, Italy) 10, yeast extract (Costantino SpA) 10, glucose 20. Biomass production was carried out in IC G20 liquid medium (Musatti et
al., 2015) having the following composition (g/L): Bacto-peptone 10, casein enzymatic hydrolysate (Costantino SpA) 10, glucose 20. For both
media the pH was 6.8 and sterilization 112 °C for 30 min.
L. sanfranciscensis was maintained and cultivated in MRSm medium
having the following composition (g/L): casein enzymatic hydrolysate
10, meat extract (Merck K GaA, Darmstadt, Germany) 5, yeast extract
5, K2HPO4 (Sigma Aldrich, Missouri, USA) 2.6, KH2PO4 (Sigma Aldrich)
4, NH4Cl (Sigma Aldrich) 3, cysteine-HCl (Sigma Aldrich) 0.5, Tween
80 (Merck K GaA) 0.5, maltose (Merck K GaA) 10, pH 6.2 and sterilization at 112 °C for 30 min. After sterilization 1 mL/L of minerals and vitamins mix (sterilized by filtration) was added. The mix had the following
composition (g/50 mL): MgSO4 (Sigma Aldrich) 10, MnSO4 (Sigma Aldrich) 1.9, thiamine (Merck K GaA) 0.01, niacin (Merck K GaA) 0.01,
folic acid (Carlo Erba, Cornaredo, Italy) 0.01, pyridoxal (Sigma Aldrich)
0.01, pantothenic acid (BDH Chemicals, London, England) 0.01, cobalamin (Carlo Erba) 0.01.
Stock cultures of both microorganisms were stored at −80 °C in the
same media (DSM for Z. mobilis and MRSm for L. sanfranciscensis) added
with 20% (v/v) glycerol (VWR International, Leuven, Belgium).
2.2. Lactobacillus and Zymomonas biomass production
Z. mobilis was grown in 1 L flasks containing 600 mL of IC G20 medium, inoculated with a 5% (v/v) of a 9 h pre-culture in the same medium.
Cultures were incubated at 30 °C for 14–16 h in stationary conditions.
L. sanfranciscensis was grown in 1 L flasks containing 600 mL of
MRSm medium, inoculated with 2% (v/v) of a 24 h pre-culture in the
same medium. Cultures were incubated at 30 °C for 16–24 h in stationary conditions.
The determination of the biomass growth was performed by
centrifuging 200 mL of culture broth at 8600 ×g for 20 min at 5 °C in a