Semi-continuous cultivation and experimental set-up in PBR
For semi-continuous cultivation, culture dilution with Zarrouk´s medium was carried out on alternate days maintaining OD750 = 2.0 (~1 gDW L-1) as the starting point for the next production cycle. The experiment was continued over a time course of 43 days in this semi-continuous operation mode. The experimental program was divided into three phases with eight PBRs designated in alphabetical order from A to H. These eight PBRs were divided in four sets of duplicate PBRs indicated as ‘AB’ for A and B, ‘CD’ for C and D, etc. (Figure 1). OD750nm was monitored as a surrogate for biomass concentration, with the OD-concentration relationship periodically monitored. That relationship (typically DW in g L-1 = 0.5*OD750nm) varied only slightly during the course of these experiments as discussed above.
In Phase I, four reactors each were cultivated at constant 20 °C (AB, CD) and 35 °C (EF, GH) for 15 days. In Phase II, the four reactors at 20°C were shifted to 30 °C (CD) and 35 °C (AB) whereas the other set of four reactors at 35°C were shifted to 20 °C (EF) and 30°C (GH). The Phase II shift was started on the 15th day and kept in place until the 33rd day. To acclimate the cultures to the newer temperature conditions, cultures were grown in batch mode for four days (15 – 19) without performing any dilution. In the final Phase III, the culture conditions were shifted from constant temperature to dynamic summer temperature profiles (derived from historical climate data for Fort Myers, Florida) with hourly changes in temperature over the course of 24 hours. This phase lasted from day 33 to day 43. In this phase, the cultures grown at constant 20 °C (EF) and 30 °C (CD) in Phase II were shifted to average summer profile with 35/21 °C as maximum/minimum temperatures, the culture at constant 35 °C (AB) was moved to extreme summer profile with 39/26 °C as maximum/minimum temperatures, and final set of two reactors cultured at constant 30°C (GH) were shifted to constant day (31 °C) and night (22 °C) temperature based on summer average of day and night temperatures. Summer is defined as June 1 through August 31 for creating these profiles from historical data.