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.