The microbiome of both the sputum samples and the media from the WinCF columns after incubation in the pH and oxygen experiments were analyzed using 16S rRNA gene amplicon sequencing. For the sputum samples, an aliquot of the sample was frozen at −80°C before sequencing in batch with all other samples. DNA extraction, 16S rRNA gene variable region 4 (V4) polymerase chain reaction, and amplicon preparation for sequencing were performed according to protocols benchmarked for the Earth Microbiome Project found here (see Supplementary Methods).

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