Figure 6.
(A) Workflow for enrichment culturing to obtain Hyp-degrading environmental microbes. Hyp-containing supplements were added to certain samples before inoculation into the Hyp enrichment medium. Turbidity and Hyp degradation were observed in all initial cultures before passaging into fresh medium. All cultures were incubated anaerobically at 30ºC in sealed vessels. (B) High Performance Liquid Chromatography-Refractive Index Detector (HPLC-RID) traces for the four enrichments further passaged for sequencing to show Hyp degradation. Samples were derivatized with nitrosonium, generated from potassium nitrite and hydrogen chloride, to obtain N-nitroso derivatives of Pro and Hyp for separation on an Aminex HPX-87P column.69 The previously reported method was modified to include an isocratic elution at 60ºC for 40 minutes.