Glucose

For biofilm formation, overnight cultures were diluted to an OD₆₀₀ of 0.1 in TSBy supplemented with 50 mM glucose (Carl Roth GmbH + Co. KG) and subsequently applied to either 6-well plates (for microscopic analysis; Greiner Bio-One GmbH, Frickenhausen, Germany) or 96-well plates (for analysis of metabolic activity) and incubated in the dark for 5 h and 24 h at 37 °C under aerobic conditions without agitation, respectively. Formed biofilms were washed once with 0.2 mM NaCl or binding buffer and then incubated with different concentrations of AgAu NPs in 0.2 mM NaCl or binding buffer for 18 h at 37 °C under aerobic conditions and agitation at 300 rpm.

We screened individual media components, using a Plackett–Burman design with 12 ((n₁ + n₂) + 1) runs, 9 (n₁) factors, and 2 (n₂) dummies, to estimate the occurring falsification related to cumulative interaction components, as shown in Table 1. We optimized the process with regard to a maximum specific pDNA yield (ng pDNA/ODV) (OD₆₀₀ × culture volume = ODV) and the E. coli biomass (determined as OD₆₀₀). The following components were used for this purpose: LB powder (consists of 10 g/L trypton, 5 g/L yeast extract, 10 g/L NaCl) (Carl Roth), meat peptone (Fluka Analytical), casein peptone (Carl Roth), yeast extract (Carl Roth), glucose (Carl Roth), glycerol (Carl Roth), phosphate (Carl Roth), NaCl (Carl Roth), and MgSO₄ (Carl Roth). Experiments were performed in 500 mL shake flasks and harvested and tested after 18 h.

All laboratory manipulations with Mycoplasma were carried out under BSL2 conditions (Mwirigi et al., 2016 (link)). Mycoplasma mycoides subsp. capri, Mycoplasma mycoides subsp. mycoides and Mycoplasma capricolum subsp capricolum (Table 2, Table 3) were cultured in Pleuropneumonia Like-Organism (PPLO) broth (Difco™ PPLO Broth) media prepared as follows: 21 g of PPLO was dissolved in 700 ml of distilled water and autoclaved for 15 min at 121 °C. The mixture was cooled in a water bath to 55 °C and supplemented with phenol red (Carl Roth GmbH) to a final concentration of 3%, 200 ml horse serum (Sigma), 0.25% of glucose (Carl Roth GmbH), 0.15% of penicillin G (Carl Roth GmbH) and 0.25% of thallium acetate (Carl Roth GmbH). A forty-eight well plate was used to grow Mycoplasma strains where they were incubated at 37 °C for a minimum period of seven days. Growth of Mycoplasma cells was determined by color change from red to yellow (Stemke and Robertson, 1982 (link)), as a result of pH change due to the growth of Mycoplasma strains. Stock cultures of both Mmc, Mmm and Mcc liquid cultures were grown to a density of approximately 1–3×10⁶ cells per ml as measured by the colony forming units method (Stemke and Robertson, 1982 (link)) and cryopreserved at −80 °C.

Basic medium (B medium) for Staphylococci consisted of Soy Peptone (10 g; Plato, Koblenz, Germany), Yeast Extract (5 g; Deutsche Hefewerke, Nuernberg, Germany), NaCl (5 g; Carl-Roth, Karlsruhe, Germany), Glucose (1 g; Carl Roth) and K₂HPO₄ (1 g; Applichem, Darmstadt, Germany). Deionized water was added to a final volume of 1 liter and pH was adjusted to 7.2.
LB medium for E. coli consisted of Peptone (10 g; Plato), Yeast Extract (5 g; Deutsche Hefewerke) and NaCl (5 g; Carl Roth). Deionized water was added to a final volume of 1 liter and pH was adjusted to 7.2. MacConkey agar was prepared according to the manufactures’ instructions (Carl Roth). Minimal media M9 for staphylococci consisted of Na₂HPO₄ (6 g; Carl Roth), KH₂PO₄ (3 g; Fisher Chemicals), NH₄Cl (1 g; Merck Darmstadt, Germany) and NaCl (0.5 g; Carl Roth). Deionized water was added to a final volume of 1 L, the media was autoclaved and supplemented with thiamine (2 µg/mL; Sigma Aldrich, Munich, Germany), MgSO₄ (1 mM; Carl Roth) and casamino acids (0.2%; BD Bioscience, Heidelberg, Germany). Cells were routinely grown at 37 °C either in liquid culture in baffled Erlenmeyer flasks (1:5 ratio) with shaking or on 1.5% agar plates.

Corn cobs are agro-wastes grown in Zagorje county, Croatia and harvested in October 2016. Dried lignocellulosic biomass was shredded to smaller pieces using a garden shredder (Hurricane HMH 200, Germany), cut with a cutting mill (Retsch SM 2000, Germany) and sieved through a 1 mm screen. Biomass was stored in the plastic container at room temperature (18–21 °C) in the dark. It contained approximately 5% (g g⁻¹) of moisture.
Commercially available enzyme mixes Celluclast 1.5 L (Sigma, St. Louis, MO, USA), Viscozyme L (Sigma, St. Louis, MO, USA), and Cellic CTec2 (Novozymes, Copenhagen, Denmark) were used for hydrolysis of lignocellulosic biomass. The cellulase activity was determined according to the International Union of Pure and Applied Chemistry [27 (link)]. The filter paper activity of Celluclast 1.5 L and Viscozyme L was 65.2 and 22.2 filter paper units mL⁻¹ (FPU mL⁻¹), respectively. Glucose was purchased from Carl Roth GmbH (Karlsruhe, Germany), yeast extract and peptone were purchased from Merck Millipore (Darmstadt, Germany), mineral salts were obtained from Kemika (Zagreb, Croatia), and chloroform and methanol were purchased from Fisher Scientific (Cleveland, OH, USA).