BACKGROUNDThe hybrid fed-batch bioreactor (HFBB) operation is a novel design method for high-cell-density fermentations. It employs fed-batch and batch operations, starting with fed-batch that in turn shifts to the next, and proceeds in successive cycles to enhance production and productivity by controlling the substrate uptake rate. RESULTSThe method was tested for the production of recombinant human growth hormone (rhGH) in Pichia pastoris under GAP promoter with carbon sources of (i) glucose and (ii) pre-treated hydrolyzed sugar-beet molasses containing equimolar glucose and fructose. The superior strategies were: (i) GH2, a HFBB with glucose feeding composed of t(FB)=1.5h fed-batch periods with continuous-feed stream designed with the pre-determined growth rate mu=0.10h(-1), which shift in turn to t(B)=0.5h batch periods; and (ii) MH2, a HFBB with molasses feeding designed in a similar way to GH2. The highest rhGH production attained in GH2 was 611mgdm(-3) at 8h, whereas it was 625mgdm(-3) at 13.5h in MH2. In GH2, rhGH production increased 1.37-fold in half the cultivation time of GH2. However, in MH2 with dual carbon sources enriched with other substrates, higher rhGH production was obtained at the cost of lower productivity. CONCLUSIONThe HFBB is a powerful design method with a single carbon source which improves the productivity of high-cell-density fermentations. However, with molasses upon shifting to batch operations, the uptake of accumulated fructose is continued. Thus, the glamour of the HFBB is hindered for complex media fermentations to an extent, because multi-carbon sources contribute to the growth during the batch periods by lengthening the cultivation time. (c) 2018 Society of Chemical Industry