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 ).