Reactors were maintained by feeding 125 ml of new substrate
daily and removing digestate to keep the operating volume at 4 L.
The test CSTR began with 1 g of glycerol (with 124 g of primary)
and was increased to 10 g, with one additional gram being added
every 3–4 weeks. The feeding schedule shown in Table 2 was followed.
The stepwise increase in glycerol addition was done to
allow sufficient time to buffer changes in primary, and to allow
the microorganisms to adjust to new feed stock. Each new batch
of primary required TS/VS analysis to determine the OLR. OLR
was determined using Eq. (2), where M is total daily mass input
flow in kilograms, TS and VS are percentages based on substrate
and V is reactor volume in cubic meters.