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 to opposing temperatures was started on the
15th day and kept in place until the
33rd day. To acclimate the cultures in the newer
temperature conditions and examine the acclimation effect, 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.