Nitrogen availability and cell density each affects growth and cellular astaxanthin content of
The green microalga,
In a previous study, we demonstrated that a record high astaxanthin productivity of 17.1 mg L-1 d-1 can be obtained from
In this study, the combined effect of nitrogen concentration and algal biomass density (which affects light intensity at the single cell level) on astaxanthin production potential was investigated in
For the IBD experiment, the culture medium was free of nitrogen, and the IBDs were set as 0.1, 0.5, 0.8, 1.5, 2.7, 3.5, and 5.0 g L-1 DW. For the nitrogen concentration experiment, the BG-11 medium containing 0, 0.375, 0.75, and 1.5 g L-1 sodium nitrate were prepared, which corresponded to the 0, 4.4, 8.8, 17.6 mM nitrate, respectively. More than 90% of the cells for inoculation were in a palmella stage (non-flagellated green vegetative cells) and less than 10% were green swimming cells with two flagella. The average chlorophyll content of inoculum cells was 3.0 ± 0.8% DW. The maximum photochemical efficiency of photosystem II (
Algal biomass concentration was measured by a gravimetric method. Briefly, an aliquot of culture sample (
, where W1 and W0 represent the weights of the filter paper with and without the algae (g), respectively. Specific growth rate (μ, d-1) was calculated as:
, where t represents the cultivation time at the red stage. The biomass productivity (g L-1 d-1) was calculated as:
For red stage biomass productivity, DWt and DW0 represent biomass densities at day t and day 0 of red stage, respectively, and t represents the cultivation time of the red stage. For overall biomass productivity, DWt and DW0 represent biomass densities at day t of the red stage and biomass at day 0 of the green stage, respectively and t represents the entire cultivation time (green stage plus red stage). An average biomass productivity of the green stage culture of
The cellular astaxanthin content was measured by reverse-phase high performance liquid chromatography according to Wang et al. (2013). Chlorophyll concentration of algal samples was calculated according to Lichtenthaler and Wellburn (1983). Optical densities at 647 and 663 nm were measured using a spectrophotometer (DU- 650; Beckman and Coulter, Fullerton, CA, USA).
The nitrogen concentration of the culture (in the form of sodium nitrate) was measured by Quikchem8500 (Lachat, Milwaukee, WI, USA) according to the manufacturer’s instructions. Aliquots of 10 mL culture were centrifuged at 10,000 ×g for 10 min, and the supernatants saved for nitrate analysis.
In our previous study we demonstrated that under nitrogen-replete conditions the
In this study, we took an additional step to determine whether the relationship between IBD and growth and astaxanthin content was influenced by nitrogen depletion. We utilized the same experimental setting used in the previous study except that at the red stage no nitrate was provided to the cultures. The results showed that the maximum specific growth rate of
The biomass densities of the different IBD cultures increased gradually during the red stage with more biomass accumulation in the cultures of higher IBDs (Fig. 1B). However, when the biomass increases in the individual cultures were calculated on a per IBD basis, a reverse relationship between IBD and biomass productivity was evident. The higher IBDs resulted in lower net biomass yields (Fig. 1C). When the IBD was 0.1 g L-1, the cells died and the cultures bleached in one or two days due to high light-induced photooxidative cell death (Han et al. 2012).
When the IBD was 0.5 g L-1 or higher, the cellular astaxanthin content increased gradually and reached the highest level on day 10, and thereafter it remained the same or declined. A clear trend was that the lower the IBD the higher the rate of astaxanthin accumulation and thus the higher cellular astaxanthin content. As a result, the highest astaxanthin content of 3.3% dry weight was obtained in the 0.5 g L-1 IBD cultures (Fig. 1D).
Based on the above and our previous experiments (Wang et al. 2013), a general trend was observed that for a given IBD, a nitrogen-deplete culture reduces the final biomass density but increases the maximum cellular astaxanthin content. Regardless of the nitrogen status, the optimal IBD of 0.5-0.8 g L-1 DW was higher than the 0.29 g L-1 applied to an outdoor horizontal tubular photobioreactor (4.85 cm inner diameter, 50 L culture volume) (Torzillo et al. 2003) and 0.4 g L-1 in an outdoor horizontal airlift tubular (30 mm inner diameter, 55 L culture volume) and an outdoor vertical bubble column photobioreactor (20 cm inner diameter, 55 L culture volume)
[Fig. 1.] Effects of initial biomass densities (IBDs) on the growth and astaxanthin content of Haematococcus pluvialis in nitrogen-deplete culture medium under outdoor conditions. (A) The specific growth rate (μ d-1) as a function of IBD and time at the red stage. (B) Net volumetric biomass increase (g L-1) as a function of IBD and time at the red stage. (C) Net biomass increase (g g-1) as a function of time on a per IBD basis. (D) Cellular astaxanthin content (% DW) as a function of IBD and time at the red stage. Data were means ± standard deviations of six measurements from three individual columns (two measurements per column). The values in the legend represent different IBD treatments (g L-1).
(Garcia-Malea Lopez et al. 2006), but lower than that (1.3 g L-1) started in an outdoor horizontal tubular photobioreactor (30 mm inner diameter, 2,000 L culture volume) (Aflalo et al. 2007).
The maximum volumetric biomass productivity in the red stage occurred on day 1 of cultivation. As the IBD increased from 0.5 to 3.5 g L-1, the maximum volumetric biomass productivity increased from 0.37 ± 0.01 to 1.39 ± 0.02 g L-1 d-1. Further increasing in IBD resulted in a slightly reduced productivity. As the cultivation proceeded, the volumetric biomass productivities of all the treatments decreased with distinct patterns, where more dramatic decreases in productivity occurred in the cultures of higher IBDs (Fig. 2A).
Because the volumetric astaxanthin productivity is a function of biomass density and the cellular astaxanthin content, the trend for astaxanthin productivity was somewhat different from that of biomass productivity. As shown in Fig. 2B, the maximum volumetric astaxanthin productivity at the red stage increased from 6.14 ± 0.19 mg L-1 d-1 in the IBD 0.5 g L-1 cultures, to 10.51 ± 1.49 mg L-1 d-1 in the IBD 0.8 g L-1 cultures, to 13.31 ± 1.41 mg L-1 d-1 in the IBD 1.5 g L-1 cultures, and to 18.71 ± 2.69 mg L-1 d-1 in the IBD 5.0 g L-1 cultures. The volumetric astaxanthin productivity of 18.71 ± 2.69 mg L-1 d-1 was the highest ever achieved at the red stage of outdoor
Since the red stage was part of the
During the entire cultivation period (
[Fig. 2.] Effects of different initial biomass densities (IBDs) on the biomass productivity and astaxanthin productivity of Haematococcus pluvialis in nitrogen-deplete culture medium under outdoor conditions. (A & B) Results on the red stage. (C & D) Results on the green + red stages. The average biomass productivity of green stage culture of H. pluvialis was 0.1 g L-1 d-1. Accordingly, the time (n) for preparing the inoculum densities of 0.1, 0.5, 0.8, 1.5, 2.7, 3.5, and 5.0 g L-1 DW were calculated to be n = 1, 5, 8, 15, 27, 35, and 50 days, respectively. Data were means ± standard deviations of six measurements from three individual columns (two measurements per column). The values in the legend represent different IBD treatments (g L-1).
plus red stage) the volumetric biomass productivity of the different IBD cultures decreased dramatically. The time required for achieving maximum biomass productivity was also changed. When the IBD was low and moderate (0.5, 0.8, and 1.5 g L-1 DW), the maximum biomass productivity occurred within the first 2 to 4 days of the red stage. In contrast, the maximum biomass productivity was obtained at the end of the red stage when the IBDs were high (2.7, 3.5, and 5.0 g L-1 DW) (Fig. 2C).
Likewise, when the green stage was taken into account, the overall volumetric astaxanthin productivities of the different IBD cultures were reduced considerably as compared to that obtained from the red stage only. However, the trend of volumetric astaxanthin productivity as a function of IBD was drastically different from that obtained from the red stage. The maximum astaxanthin productivity of 4.47 ± 0.03 mg L-1 d-1 was obtained in the IBD 0.5 g L-1 cultures, and it increased to 5.61 ± 0.03 mg L-1 d-1 when the IBD was raised to 0.8 g L-1, above which the higher the IBD the lower the maximum astaxanthin productivity (Fig. 2D).
Because the nitrogen availability for achieving maximum growth was mutually exclusive from achieving maximum cellular astaxanthin content, we selected 0.8 g L-1 as the optimal IBD to further optimize the initial nitrogen concentration for maximum astaxanthin production. To this end, the initial nitrogen concentrations of 0, 4.4, 8.8, and 17.6 mM nitrate were chosen and the results are shown in Fig. 3A. At the red stage the nitrate concentrations in the 4.4, 8.8, and 17.6 mM nitrate cultures decreased at more or less the same rate, indicating that
[Fig. 3.] Effects of nitrate concentrations on nitrate uptake kinetics (A), growth (B), and astaxanthin content (C) of Haematococcus pluvialis under outdoor conditions. Data were means ± standard deviations of six measurements from three individual columns (two measurements per column). The values in the legend represent different initial nitrogen concentration treatments. The initial biomass density was 0.8 g L-1 for this experiment.
A reverse relationship between the initial nitrogen concentration and the maximum cellular astaxanthin content was observed, i.e., the lower the initial nitrogen concentration, the more rapid increase in cellular astaxanthin content and the higher the maximum cellular astaxanthin content. The highest cellular astaxanthin content of 3.84 ± 0.05% DW was obtained in the nitrogen-depleted cultures, whereas the lowest astaxanthin content of 2.20 ± 0.14% DW was measured in the 17.6 mM nitrate cultures (Fig. 3C).
The volumetric biomass productivity as a function of initial nitrogen concentration is shown in Fig. 5A. The fact that the biomass productivity of the nitrate-deplete cultures were identical to that obtained in the nitrate-limited or nitrogen-replete cultures for the first two days suggested that the amount of intracellular nitrogen was sufficient to sustain initial vigorous growth. The highest biomass productivity of 0.59 ± 0.01 g L-1 d-1 was obtained in the 4.4 mM nitrate cultures. The culture with initial nitrate concentration that was lower or higher than this level resulted in reduced biomass productivity. A possible interpretation is that during the transformation of
[Fig. 4.] Photomicrographs of Haematococcus pluvialis cells grown in outdoor glass columns containing 0 or 17.6 mM nitrate medium. Cell images A, B, C, D, and E were taken on days 1, 3, 4, 7, and 10 of the nitrogen-deplete culture, respectively. Cell images F, G, H, I, and J were taken on days 1, 3, 4, 7, and 10 of the 17.6 mM nitrate culture, respectively. Scale bars represent: A-J, 20 μm.
[Fig. 5.] Effects of nitrate concentrations on the biomass productivity and astaxanthin productivity of Haematococcus pluvialis under outdoor conditions. (A & B) Results on the red stage. (C & D) Results on green + red stages. The green stage duration was n = 8 days. Data were means ± standard deviations of six measurements from three individual columns (two measurements per column). The values in the legend represent different initial nitrogen concentration treatments. The initial biomass density was 0.8 g L-1 for this experiment.
occurred on day 10 or longer. At the red stage, the highest astaxanthin productivity of 16.0 ± 0.15 mg L-1 d-1 was obtained in the 4.4 mM nitrate cultures (Fig. 5B).
When the green stage was taken into account, the maximum volumetric biomass productivity of the nitrate-deplete cultures was 0.20 ± 0.002 g L-1 d-1. When the initial nitrate concentrations were 4.4, 8.8, and 17.6 mM, the maximum biomass productivities increased to 0.27 ± 0.004, 0.29 ± 0.002, and 0.30 ± 0.01 g L-1 d-1, respectively (Fig. 5C). The maximum astaxanthin productivity of 8.9 ± 0.08 mg L-1 d-1 was obtained in the 4.4 mM nitrate cultures, and reduced astaxanthin productivities occurred in cultures with lower or higher nitrogen concentrations (Fig. 5D).
Developing an understanding of the combined effect of algal biomass density and nitrogen availability on growth and astaxanthin synthesis of
The maximum biomass production and maximum cellular astaxanthin content of