Fig. 2

Graphic summary outlining the bioassays and fed-batches. Bioassay 1 was used to determine the growth parameters and yield for anaerobic growth in the designed medium, which was then used in an initial attempt at high cell density cultivation in a fed-batch (Fed-batch 1; Strain: Pd1222). There were indications of toxicity when mixing glucose into the acid reservoir. To address this, we designed a bioassay to test the different feed compositions and combinations (Bioassay 2). Based on the results, we concluded that the acid, feed, and trace elements should be provided from three separate reservoirs. Cells and liquid samples from Bioassay 1 and Fed-batch 1 were analyzed by ICP-MS to determine the trace element content, which was used to re-design the trace element solution (TRES-2, Table 1) for Fed-batch 2. Fed-batch 2 (Strain: Pd1222_mC-nirS) had the expected biomass yield, but the growth rate was lower than expected based on Bioassay 1. During Fed-batch 2, cell-free liquid samples were analyzed by ICP-MS, HPLC, and HS-GC to determine the concentrations of trace metals and selected metabolites. Cells were analyzed by fluorescence microscopy to determine the expression of mCherry-nirS. At the end of Fed-batch 2, cells and reactor liquid were separated and used in Bioassay 3 and 4, respectively. Bioassay 3 aimed to test the presence of inhibitory or toxic compounds in the liquid by incubating with fresh cells. In addition, the toxicity of Fe(II) and Fe(III) under anoxic conditions was assessed. Bioassay 4 tested the fitness of the reactor cells by inoculation in a fresh medium. Bioassays 3 and 4, and the results from the liquid analysis, prompted us to re-design the trace element solution prior to Fed-batch 3 by reducing the Fe content. In Fed-batch 3 (Strain: Pd1222) we sustained a robust anaerobic HCDC, however, glucose excess resulted in PHA accumulation and the growth rate remained low