Chlorophyll and Phycocyanin Production
During the cultivations, chlorophyll (Chl-a) content and phycocyanin
(PC) content for Arthrospira were monitored. The two main
components of the PC content were determined separately: allophycocyanin
(aPC) and C-phycocyanin (cPC) via spectroscopic determination (Yoshikawa
and Belay, 2008). In addition, whole cell (WC) absorption spectra were
measured (Figure 4) for qualitative pigment analysis. Results for
pigment content are summarized in Table 1 and displayed in detail in
Figure 5. All quoted contents are expressed as a percentage of whole
cell dry weight.
First, from the Phase I results in Table 1, good reproducibility between
the biological duplicates (AB-CD and EF-GH) is found for all the
measurements. It can also be seen from Figure 5 that the AB and CD
experiments at 20 °C are very stable in their pigment content, both PC
and Chl-a. That is not the case for the 35 °C results where both PC and
Chl-a are decreasing steadily throughout the Phase I residence time in
Figure 5c. The decline in PC is about 20% and the decline in Chl-a is
about 40% over the course of the Phase I experiments at 35 °C. It is
unlikely that the degradation in pigment content in either case is due
to thermal damage to the pigments, as these pigments are known to be
stable to much high temperatures. It is more likely due to a slow
alteration of the photosynthetic apparatus, which we regard as
biologically irreversible (as distinguished from recovery from culture
turnover in the semi-continuous mode). The decrease in PC content could
in fact be a consequence of the decrease in Chl-a content as the light
harvesting machinery re-balances the optimal ratio for these pigments.
In Phase II, the AB culture goes from 20 °C to 35 °C with an initial
sharp increase in PC content followed by a slow decline (Figure 5a). The
Chl-a content stays constant initially and then declines slowly over the
Phase II residence time, the overall decrease being similar to that for
EF in Phase I (Figure 5c). The CD culture, which transitioned from 20 °C
to 30 °C (Figure 5b), shows much more stable behavior, with a slower
increase in PC content before reaching an apparent steady concentration
of about 12%. The Chl-a concentration is stable at about 1.7%, similar
to Phase I at 20 °C. There is no indication of instability at 30 °C. The
CD culture, transitioned from 35 °C to 20 °C (Figure 5c), shows a steady
decline in PC to the expected level for 20 °C production
(~8 %). The Chl-a content increases slowly, eventually
reaching the level expected for 20 °C production
(~1.5%). The time scale for these changes are
consistent with the expected time scale for culture turnover (roughly
20-30% per 2 day cycle). At the end of Phase II, the EF culture is
almost exactly at the expected pigment contents found in the AB and CD
Phase I experiments. The GH culture, transitioned from 35 °C to 30 °C
(Figure 5d), achieves an overall increase in PC content and Chl-a
content, with a slightly enhanced time scale for reaching stable levels
for both pigments.
Phase III observations from Figure 5 are consistent with the above
observations. AB (35 °C to ExSP) shows an initial decline in pigment
contents and then some recovery over time. CD shows essentially no
change in going from 30 °C to AvSP. EF shows expected changes in going
from 20 °C to AvSP. GH (30 °C to CtSP) shows little or no change in
pigment content.
According to the literature for shorter duration experiments, there is a
narrow temperature range between 35 °C and 37 °C for optimal growth with
40 °C being definitely detrimental for Arthrospira (Kumar et al.,
2011; Torzillo et al., 1991b). Our results suggest extended periods at
35 °C are also not favorable for sustained growth, though the effects
are largely reversible on a culture basis and most of the variation is
in pigment production. A similar trend was seen in whole cell spectrum
in Figure 4, where relatively higher peak at ~680nm
(Chl-a) and lower peak at 620 nm (cPC) was found at lower temperatures,
and thus indicates a higher Chl-a to PC ratio for low temperature
cultures compared to those after prolonged high temperature exposure.
The culture at 35 °C turned bluish green with Chl-a reduction (by
>50 %) after prolonged exposure to this modestly elevated
temperature. The spectra in Figure 4 are consistent with this visual
observation. These results are generally consistent with Watras et al.,
(2017) where a progressive decrease in chlorophyll and phycocyanin
fluorescence with increasing temperature was reported in most of the
cultures of green and blue-green algae (e.g., Scenedesmus
dimorphus, Selenastrum minutum, and Synechococcus leopoliensis ).