Z. mobilis Zm6 and S. cerevisiae strain CEN.PK113-7D strain were used for adaptive evolution by co-culture. Cells were grown in growth medium containing glucose (20 g/L or 100g/L), yeast extract (5 g/L), NH4SO4 (1 g/L), KH2PO4 (1 g/L) and MgSO₄ (0.5 g/L). The growth medium was flushed with nitrogen gas prior to culture. The co-culture was grown at 30 °C in a tightly capped test tube with shaking at 200 rpm throughout the study.
For serial co-culture, 10 μL of Zm6 overnight anaerobic culture and S. cerevisiae anaerobic culture were used as starters. The mixed cultures typically spent all nutrients within a day. The fully grown cultures (10 μL) were transferred to fresh identical medium the following day and the co-culture continued. After the transfer was repeated 5 times, we observed that Z. mobilis competes out in the co-culture under the condition, as shown by viable counts of the co-culture. To maintain balanced co-culture, we inoculated additional S. cerevisiae overnight culture 40 μL, in addition to 10 μL of previous co-culture, upon each transfer from 6th round.
For Escherichia. coli co-culture with Z. mobilis, E. coli strain K12 grown in LB medium under aerobic condition was used as a starter. We started co-culture with mixing 10 μL of fully grown monocultures of Z. mobilis and E. coli. During the experiments, we learned that inoculating 10 μL of fully grown co-culture of previous round with 20 μL of fully grown E. coli monoculture upon a transfer gave a good balance for continuation of co-cultures, which we performed for all transfer. All lines were replicated for whole passages.
After the serial co-culture of 50 transfers, cells were streaked out on the solid identical medium. Isolated pure strains are designated as follows, Zs100: Z. mobilis Zm6 derived strain obtained from last round of Z. mobilis vs S. cerevisiae serial co-culture supplemented with glucose 100 g/L. Zs100R is obtained from a parallel replicate of Z. mobilis vs S. cerevisiae glucose 100 g/L. Zs20: Zm6 derived strain obtained from the last round of serial co-culture with S. cerevisiae supplemented with 20 g/L glucose, and Zs20R was obtained from a last culture of parallel run of Zs20R. Similarly, Ze20 designates Z. mobilis strain obtained from last round of Z. mobilis vs E. coli co-culture with 20 g/L glucose, and Ze20R as a parallel replicate. As a control, the 50 times serial transfer of Z. mobilis monocultures grown in the same complex medium with 20 g/L glucose or 100 g/L glucose were performed. Z20 designates Z. mobilis strain obtained from a last round of Z. mobilis monoculture with 20 g/L glucose, and Z100 designates Z. mobilis strain obtained from a last round of monoculture with 100 g/L glucose.
Characterization of growth and ethanol production
Growth profiles of all strains were analyzed using flat bottom 96-well microplate in plate-reader spark 20M (Tecan) by measuring its absorbance at 600 nm. Overnight anaerobic monoculture was used as an inoculum. Temperature control was set at 30 °C. Three technical replicates were repeated for each time point. The used medium in plate reader was same as for co-culture, with supplement of glucose 20 g/L. We also measured growth profiles in test tubes under anaerobic condition using spectrophotometer (VWR), showing similar trends from microplate experiments.
Acetate, lactate, ethanol and glucose in the overnight monoculture of generated Z. mobilis strains were measured using Waters 2695e Alliance HPLC (Waters) with Hi-plex column (300 x 7.7 mm, Agilent). The spent medium of anaerobic overnight culture in the test tube was analyzed for HPLC analysis. The collected supernatant was filtered through 0.2 μm Supor® polyether sulfone membrane (PALL) before the analysis. HPLC analysis was run under the condition; 0.05 M sulfuric acid as mobile phase at a flow rate of 0.8 mL/minute. External standard curve was used for converting obtained peaks to concentration of analytes.
Growing sample was directly mounted on Phosphate-buffered saline (PBS)-agarose pad before imaging. Zeiss Axio Imager Z2 microscope (ZEISS) equipped with camera Axiocam MR R3 (ZEISS) was used for capturing phase contrast images. Software ZEN 3.1 (ZEISS) was used for image analysis.
Whole genome sequencing
Total DNA was extracted by combining a lysozyme treatment  and D-neasy blood tissue kit (Qiagen). Whole genome of the co-cultured Z. mobilis strains was sequenced by GATC re-sequencing service (INVIEW Genome sequencing). The reference sequence was our lab stock Zm6 strain which was previously sequenced [18, 19].