Fecal samples from three healthy individuals were collected into sterile containers, sealed, and transferred to an anaerobic chamber within 1 h of defecation. Samples were manually homogenized and subdivided into sterile vials, which were stored at −80 °C until use. Prior to MBRA inoculation, fecal samples were resuspended at 25 % w/v in anaerobic phosphate buffered saline in the anaerobic chamber, vortexed for 5 min, and centrifuged at 201×g. For the pooled sample, equal amounts of each fecal sample (by mass) were combined prior to vortexing.
In order to analyze the impact of freezing upon MBRA cultivation, one fecal donor (donor A) provided a second sample ~3 months post initial donation (donor A2). This sample was collected and transferred to the anaerobic chamber within 1 h of defecation. Following manual homogenization and subdivision into sterile vials, a portion of the sample was flash frozen in liquid nitrogen for 45 min (frozen), whereas the other sample was maintained in the anaerobic chamber until inoculation. Both fresh and frozen samples were then inoculated into triplicate reactors and analyzed as described below. Analysis of this data revealed that there was little impact on communities cultivated from frozen samples compared to freshly voided samples (see Additional file11 for an NMDS ordination of the Bray-Curtis dissimilarities between these different samples as well tests of community similarity and dispersion).
MBRA were prepared for use as previously described [29 (link)] and inoculated with 4 ml of fecal slurry. Bioreactor medium was prepared as described [29 (link)], except that 1 g/L of taurocholic acid was replaced with 0.5 g/L of bovine bile, which was added prior to autoclaving. There were multiple reasons for substituting bovine bile for taurocholate. (1) Bovine bile is a complex mixture of bile salts as well as other constituents of bile (e.g., fatty acids, cholesterol, inorganic salts) and is more commonly used in medium for cultivation of human fecal communities than taurocholate alone. (2) Taurocholate was originally included in our medium to promote germination of C. difficile spores; subsequent studies have shown that bovine bile is sufficient to support germination under our reactor conditions (Auchtung and Britton, unpublished results). (3) Bovine bile is significantly less expensive than taurocholate (>10-fold lower cost).
After inoculation, fecal bacteria were allowed to equilibrate for 16–18 h prior to the initiation of flow. After equilibration, a 1-ml sample was removed (day 1 sample) and flow commenced at 1.875 ml/h (8-h retention time). Reactors were then sampled daily for 20 additional days (days 2–21). Cells were pelleted from samples by centrifugation at 21,000×g. Supernatants were discarded, and pellets were stored at −80 °C until further processed.
In order to analyze the impact of freezing upon MBRA cultivation, one fecal donor (donor A) provided a second sample ~3 months post initial donation (donor A2). This sample was collected and transferred to the anaerobic chamber within 1 h of defecation. Following manual homogenization and subdivision into sterile vials, a portion of the sample was flash frozen in liquid nitrogen for 45 min (frozen), whereas the other sample was maintained in the anaerobic chamber until inoculation. Both fresh and frozen samples were then inoculated into triplicate reactors and analyzed as described below. Analysis of this data revealed that there was little impact on communities cultivated from frozen samples compared to freshly voided samples (see Additional file
MBRA were prepared for use as previously described [29 (link)] and inoculated with 4 ml of fecal slurry. Bioreactor medium was prepared as described [29 (link)], except that 1 g/L of taurocholic acid was replaced with 0.5 g/L of bovine bile, which was added prior to autoclaving. There were multiple reasons for substituting bovine bile for taurocholate. (1) Bovine bile is a complex mixture of bile salts as well as other constituents of bile (e.g., fatty acids, cholesterol, inorganic salts) and is more commonly used in medium for cultivation of human fecal communities than taurocholate alone. (2) Taurocholate was originally included in our medium to promote germination of C. difficile spores; subsequent studies have shown that bovine bile is sufficient to support germination under our reactor conditions (Auchtung and Britton, unpublished results). (3) Bovine bile is significantly less expensive than taurocholate (>10-fold lower cost).
After inoculation, fecal bacteria were allowed to equilibrate for 16–18 h prior to the initiation of flow. After equilibration, a 1-ml sample was removed (day 1 sample) and flow commenced at 1.875 ml/h (8-h retention time). Reactors were then sampled daily for 20 additional days (days 2–21). Cells were pelleted from samples by centrifugation at 21,000×g. Supernatants were discarded, and pellets were stored at −80 °C until further processed.
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