Lactose

Complex microbiota from an anaerobic membrane bioreactor (AnMBR) fed with protein- and lipids-rich dairy wastewater was used as inoculum, the inoculum was taken at Biothane—Veolia Water Technologies Techno Center Netherlands B.V Research Facilities, in Delft, The Netherlands. The volatile suspended solids (VSS) content of the inoculum was 14.5 g VSS·kg⁻¹ wet weight, and 3 g VSS was added to each reactor (working volume of 2 L) to achieve an initial microbial concentration of 1.5 g VSS·L⁻¹.
Three substrates were used in the experiment: A) casein (Fisher Scientific), with casein as the sole carbon source, B) lactose (Sigma Aldrich), with lactose as the sole carbon source, and C) mixture of casein and lactose as carbon sources, further referred to as MIX, with a ratio of 50%: 50% in terms of chemical oxygen demand (COD). The addition of nutrients was based on the anaerobic fermentation process growth media protocol of Hendriks et al. (2018 (link)). The substrates contained 6000 ± 100 mg COD·L⁻¹ and 800 ± 50 mg N·L⁻¹.

The two mutational reporter strains used are STL20589 (QP5) and STL20590 (QP6), which are derivatives of Escherichia coli K-12 MG1655 (Bachmann 1972 ). The strains were grown at 37° in Luria broth (LB, Lennox formulation) medium, consisting of 1% Bacto-tryptone, 0.5% yeast extract, 0.5% sodium chloride and, for plates, 1.5% Bacto-agar. Tetracycline (15 µg/mL) was used for genetic selection. Lac⁺ reversion mutants were selected on lactose minimal medium containing 56/2 salts (Willetts et al. 1969 (link)), 0.2% lactose (Sigma Aldrich, St. Louis, MO, USA), 0.001% thiamine (Sigma Aldrich), and 2% agar (ThermoFisher, Sparks, MD, USA). X-gal (40 µg/mL) and IPTG (0.1 mM) (Both from Gold Bio, St. Louis, MO, USA) were included in lactose selection medium as a visual aid for counting colonies.