Cellular Growth and Carbon Utilization
To advance our knowledge of inorganic carbon utilization during TAG accumulation in C. reinhardtii, batch cultures were grown under 5% CO2 sparge until near ammonium depletion. At which time, experiments were initiated that analyzed C. reinhardtii while being sparged with atmospheric air (0.04% CO2), with and without 50 mM bicarbonate added, and cultures that were maintained at 5% CO2. This allowed for comparison of cells while utilizing CO2, at high and low concentrations, or low CO2 with supplementary bicarbonate as an inorganic carbon source. Figure 1 shows cell growth (A), medium ammonium concentrations (B), and medium nitrate concentrations (B) for C. reinhardtii grown under 14:10 h light/dark cycle in Sager's minimal medium. Sager's minimal medium was chosen, in contrast to Tris–acetate–phosphate medium (commonly known as TAP), to minimize the heterotrophic activity and thus maximize the autotrophic properties of C. reinhardtii. Prior to ammonium depletion, the cultures maintained exponential growth and exhibited a 1.6 day−1 maximum specific growth rate (10.4 h doubling time). Medium ammonium became depleted near 2.8 days and further cell cycling was arrested in the cultures to which bicarbonate was added. However, cultures sparged with 5% CO2 or atmospheric air, without added bicarbonate, continued to divide an average of 1.7 more times. Previous studies on the Chlorophyte Scenedesmus sp. WC-1 showed a similar cessation of cell cycling upon a 50 mM bicarbonate addition and is comparable with the cell cycle arrest observed in C. reinhardtii (Gardner et al., 2012). Medium nitrate was not utilized by any of the cultures and the slight concentration decrease in the bicarbonate added cultures, visible in Figure 1B, is attributed to culture media dilution from the sodium bicarbonate addition.
Cellular properties such as cell concentration, degree of aggregation, and cell size can be monitored over the course of a batch growth experiment using an optical hemocytometer. It was observed that air aeration, without bicarbonate addition, resulted in cells that were smaller than the cells maintained at 5% CO2. This can be observed in the micrographs and fluorescent images of Figure 2 (comparison of A and B). However, as time progressed these smaller cells gradually grew into larger cells and this difference became less evident (note—the images taken in Fig. 2 were captured within 1 day of culture harvest). Additionally, both cultures maintained in air or on 5% CO2, without added bicarbonate, retained their flagella and motility. However, cultures to which bicarbonate was added shed their flagella and formed membrane bound, incompletely divided cells (Fig. 2C). This caused a cessation of cellular motility and increased cell size. Furthermore, the cells maintained an incomplete division state until the end of the experiment. This would not be evident if only cell number were reported.
Figure 2. Transmitted micrographs, Bodipy 505/515 and Nile Red epifluorescent images (left to right) of C. reinhardtii CC124, lipid vacuoles stain blue and yellow with Bodipy 505/515 and Nile Red, respectively. Images were taken prior to culture harvest for cultures grown on 5% CO2 switched to air (A), maintained on 5% CO2 (B), and grown on 5% CO2 which was switched to air and the addition of 50 mM sodium bicarbonate (C). Cells imaged are representative cells for each respective culture and all micrographs are at the same magnification.
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Final cell concentration and DCW are given in Table I. Cultures maintained on 5% CO2 and those sparged with air without added bicarbonate had the highest number of cells. Cultures to which bicarbonate was added had less than half as many cells. In contrast, cultures to which bicarbonate was added had twice the biomass yield compared to “no-bicarbonate” added cultures. This argues that the added bicarbonate caused a change in metabolism to shift the cells from a growth state to a product formation state as evident by the cessation of cellular division and higher biomass yield. Additional evidence of increased TAG and starch storage supporting this observation is further discussed below.
Table I. Comparison of final average and standard deviation of culture cell number, biomass yield, fluorescence TAG accumulation, and starch properties of C. reinhardtii cultured in Sager's minimal medium during 14:10 h light–dark cycling (n = 3).
|Gas-sparge during NH depletion||Time of HCO3 addition (day)||Cell concentration (×107 cells mL−1)||Dry weight (g L−1; DCW)a||Total Nile Red fluorescence (×103 units)||Nile Red specific fluorescence (units cell−1)b||Total starch (g L−1)||Specific starch (µg cell−1)c|
|5% CO2||N/A||2.31 ± 0.09||0.48 ± 0.05||4.5 ± 1.4||2.0 ± 0.6||0.06 ± 0.007||0.3 ± 0.04|
|Air||N/A||2.01 ± 0.30||0.55 ± 0.06||1.8 ± 0.4||0.9 ± 0.3||0.04 ± 0.005||0.2 ± 0.01|
|Air||2.8||0.66 ± 0.04||1.14 ± 0.11||8.8 ± 1.1||13.5 ± 2.2||0.79 ± 0.21||11.7 ± 3.3|
Since carbonate speciation and carbon species concentrations are a function of pH and total DIC, these parameters were monitored throughout the experiments. Figure 3 shows total DIC (A) and medium pH (B) for the C. reinhardtii cultures. Again, all cultures were initially grown on 5% CO2 and began with 1.4 mM C and a pH of 6.8. Through 2.8 days, there was a decrease in pH to 5.0 along with a DIC decrease to 0.2 mM C. At the time of medium ammonium depletion (2.8 days), the cultures to which bicarbonate was added increased in DIC to 52.1 mM C (50 mM C targeted) and showed an initial increase in pH to 7.9. By the end of the light cycle (3.0 days), the pH had risen to pH 9.3 and DIC had decreased to 50.2 mM C. Over the next 14:10 h light/dark cycle, beginning with 10 h dark, the DIC decreased at a rate of 0.87 mM C h−1 to 29.9 mM C and the pH increased to 10.0. This decrease in DIC concentration during the dark cycle is presumably due to CO2 off gassing as the medium was not in carbon equilibrium due to the high bicarbonate addition and algal photosynthesis was not active. In contrast, the DIC decrease in the light could be a combination of CO2 off gassing and algal photosynthesis consuming the DIC. At 4 days, the DIC increased due to in-gassing during the dark and decreased due to photosynthetic utilization during the light hours. The remainder of DIC data points were taken at the end of the light cycle, thus increased DIC from dark cycle in-gassing is not shown. By the end of the experiment, there were 26.2 mM C remaining with a final medium pH of 10.5. Abiotic DIC equilibrium was calculated for Sager's minimal medium with a 50 mM sodium bicarbonate addition, using the chemical equilibrium model Visual Minteq (ver 3.0, KTH Department of Land and Water Research Engineering) and is shown by the dashed line in Figure 3A. Comparison of the abiotic carbon equilibrium model with the DIC data, from 4.4 d through the remainder of the experiment, suggests active bicarbonate utilization by C. reinhardtii due to the carbon concentration being below equilibrium and the pH remaining high, indicating bicarbonate was the predominate DIC species.
Figure 3. C. reinhardtii CC124 average and standard deviation of medium DIC concentration (A) and medium pH (B). Arrow indicates time of medium NH depletion and inorganic carbon adjustment, dashed line represents geochemical modeling prediction (Visual Minteq ver 3.0) of carbon equilibrium with 50 mM sodium bicarbonate addition, and the bar represents the light and dark times of the light cycle. Note—a split scale was used on the y-axis of the DIC plot to better visualize the data. Growth was maintained in Sager's minimal medium illuminated with a 14:10 h L:D cycle (n = 3).
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The medium pH in the cultures to which bicarbonate was not added and the cultures sparged with 5% CO2 remained low, but there was an increase in pH from 5.0 to 5.7 after ammonium in the medium became depleted. The DIC concentration in the 5% CO2 sparged cultures increased after ammonium depletion and remained at 0.55 mM C throughout the remainder of the experiment. The DIC concentration in the cultures without added bicarbonate was below the detection limit of 0.01 mM C for the remainder of the experiment after the gas-sparging was switched from 5% CO2 to air. This suggests that the air sparged cultures, without added bicarbonate, were carbon limited after the aeration shift and could be the reason that cellular growth, after 2.8 days, produced smaller cells that gradually grew into larger cells, as shown in Figure 2 and previously discussed. Furthermore, the pH difference between the bicarbonate amended cultures and the “no-bicarbonate” cultures could be the reason the bicarbonate cultures shed their flagella. Historically, medium pH has been used to detach C. reinhardtii's flagella, however, a low pH treatment is traditionally used (Harris, 1989). Additional experimentation is needed to elucidate if pH or high DIC was the reason for C. reinhardtii's flagella detachment.
Total and Specific Lipid Accumulation
To gain an accurate assessment of the accumulated lipids of C. reinhardtii, culture TAG properties were tracked throughout the experiments by using the Nile Red fluorescent staining method, GC analyses were performed at the end of the experiments on both extracted lipids and in situ transesterified FAMEs, and both Bodipy 505/515 and Nile Red fluorescent images were taken to visually confirm TAG accumulation. This approach allowed for monitoring neutral TAG accumulation during ammonium depletion and quantification of the final concentration of FFAs, MAGs, DAGs, TAGs, and biofuel potential in each experiment. Figure 4 shows total Nile Red fluorescence (A) and Nile Red specific fluorescence (B) for the C. reinhardtii experiments. Nile Red fluorescence has previously been shown to correlate with neutral TAG and has become a generally accepted screening method for analyzing TAG in algal cultures (Chen et al., 2009; Cooksey et al., 1987; da Silva et al., 2009; Elsey et al., 2007; Gardner et al., 2011, 2012; Lee et al., 1998; Liu et al., 2008; Yu et al., 2009). Prior to ammonium depletion, the cultures show low Nile Red signals. After becoming ammonium depleted (2.8 days), the Nile Red fluorescence increased in both the 5% CO2 sparged cultures and in the bicarbonate amended cultures but remained low in the air sparged cultures where no bicarbonate was added. The low Nile Red signal observed in the air sparged cultures without added bicarbonate is presumably due to carbon limitation, as previously discussed (shown in Fig. 3).
Figure 4. C. reinhardtii CC124 average and standard deviation of total Nile Red fluorescence (A), insert is the Nile Red fluorescence of the three cultures constituting the average for the cultures maintained on 5% CO2, and Nile Red specific fluorescence at and post-NH depletion (B). Arrow indicates time of medium NH depletion and inorganic carbon adjustment. Growth was maintained in Sager's minimal medium illuminated with a 14:10 h L:D cycle (n = 3).
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The rate of fluorescence increase was highest in the 5% CO2 sparged cultures, but only increased for 1.2 days. The rate of fluorescence increase in the bicarbonate added cultures was slower, but increased over the next 3.2 days. One of the 5% CO2 sparged cultures seemed to exhibit a lower Nile Red fluorescence during TAG accumulation (Fig. 4A insert), and explains the large variation shown in the triplicate standard deviation for that system. It is unclear why this culture did not exhibit as high of a Nile Red signal, given that it did not show significant variation in the cell density, ammonium utilization, DIC, or pH.
Nile Red specific fluorescence is calculated by normalizing the total Nile Red fluorescence with 10,000 cells. It offers insight into the amount of TAG per cell, and/or the metabolic state of the cultures (Gardner et al., 2011, 2012). The Nile Red specific fluorescence increased in both the 5% CO2 sparged cultures and the bicarbonate amended cultures over 2.2 days after ammonium depletion. However, the Nile Red specific fluorescence decreased in the 5% CO2 sparged cultures after this time. The bicarbonate amended cultures continued to increase after ammonium depletion throughout the end of the experiment. By the end of the 7th day of culturing, the bicarbonate added cultures exhibited a significantly higher TAG per cell as compared to the other systems monitored, although for a short time the 5% CO2 sparged cultures displayed higher TAG accumulation per cell (discussed below).
Final Nile Red fluorescence properties for the experiments are given in Table I. At the end of the experiments, Nile Red fluorescence and Nile Red specific fluorescence in the bicarbonate added, the 5% CO2 sparged, and the air sparged cultures without bicarbonate added was highest to lowest, respectively. This trend can be compared to the fluorescence images of Figure 2, where both fluorescent stains Bodipy 505/515 and Nile Red, that are commonly used to assess TAG vacuoles in algal cells, were used to visually confirm the trend in Nile Red fluorescence values (Bertozzini et al., 2011; Chen et al., 2009; Cooksey et al., 1987; Cooper et al., 2010; Work et al., 2010). Furthermore, the C. reinhardtii biomass was extracted for quantitative analysis of biofuel precursors and results are given in Table II. The Nile Red fluorescence correlates directly with % TAG, r2 = 0.998, but not with % FFA, MAG, DAG, or biofuel potential. In addition, comparing the Nile Red fluorescence trend over time (Fig. 4) with the % FFA, MAG, DAG, and TAG (Table II), it can be seen that for the 5% CO2 cultures TAGs accumulated and then likely degraded into FFAs and DAGs. This de-convolutes the discrepancy between the Nile Red fluorescence being less in the 5% CO2 sparged cultures, as compared to the bicarbonate amended cultures, at the end of the experiments but having a higher sum of biofuel precursors, and explains the higher Nile Red signal observed at 5 days in the 5% CO2 sparged cultures (Fig. 4). These observations would suggest that TAG was degraded for cellular energy requirements. This is supported by the cultures maintaining motility and observations of starch/chlorophyll data discussed below.
Table II. Comparisons of percent (w/w) extractable neutral constituents of biofuel and total biofuel potential of C. reinhardtii cultured in 5% CO2, air, or air with 50 mM HCO during ammonia depletion (combined extraction or in situ transesterification of triplicate cultures).
|Aeration||Free fatty acid (%)a||Mono-glyceride (%)a||Di-glyceride (%)a||Tri-glyceride (%)a||Sum of extracted (%)||Total biofuel potential (%)b|
|5% CO2 → air||2.61||0.25||3.39||2.07||8.32||15.59|
|5% CO2 → air + HCO||1.94||0.33||3.21||7.59||13.07||19.61|
The difference between the total biofuel potential and the sum of extracted biofuel precursors represents the polar lipid contribution to biofuel capability. Fatty acids contained in polar molecules (e.g., phospholipids) have been shown to contribute to biofuel potential (Wahlen et al., 2011). The difference between the biofuel potential and the sum of extractable precursors is 6.8 ± 0.4% for all of the C. reinhardtii experiments. Thus, the polar lipid influence on biofuel potential is similar for each inorganic carbon substrate tested. Furthermore, Table III details the composition of the FAMEs produced during in situ transesterification. Table III also shows similar profiles between the C. reinhardtii experiments, albeit, subtle differences where observed in the degree of saturation of the C16 palmitic acid methyl ester.
Table III. Comparisons of percent composition of in situ transesterified FAMEs from C. reinhardtii cultured with 5% CO2, air, or air with 50 mM HCO during ammonia depletion (combined transesterification of triplicate cultures).
|FAME||5% CO2 → air + HCO (%)||5% CO2 → air (%)||5% CO2 (%)|
Total and Specific Starch Accumulation
The C. reinhardtii wild type has been shown to accumulate starch during N-depleted conditions (Ball et al., 1990), and because starch is a direct competitor to TAG as a carbon storage molecule, it was monitored throughout ammonium depletion. Figure 5 shows total starch (A) and specific starch (B) for the C. reinhardtii experiments beginning near ammonium depletion. All cultures had low levels of starch upon ammonium depletion (2.8 days); however, the 5% CO2 sparged cultures rapidly increased in starch concentration within 0.2 day. After which the starch concentration decreased rapidly to ∼200 µg mL−1 and slowly decreased throughout the remainder of the experiment. The cultures to which bicarbonate was added accumulated starch, reaching ∼1050 µg mL−1 at 5 days, which was higher than the 5% CO2 sparged cultures; however, maximum starch concentration may have been missed in the 5% CO2 sparged cultures because of the fast rate of accumulation/degradation and limited sampling during that time. After peak starch was realized, the bicarbonate added cultures slowly decreased in starch concentration to ∼750 µg mL−1. The cultures to which no bicarbonate was added and was sparged with air did not accumulate starch throughout the experiment which is further evidence of the carbon limitation previously discussed.
Figure 5. C. reinhardtii CC124 average and standard deviation of total starch (µg mL−1) (A) and specific starch (B), both at and post-NH depletion. Arrow indicates time of medium NH depletion and inorganic carbon adjustment. Growth was maintained in Sager's minimal medium illuminated with a 14:10 h L:D cycle (n = 3).
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Specific starch concentration is calculated by normalizing the total starch with 100,000 cells, and the value offers insight into the amount of starch per cell. As shown in Figure 5B, 0.2 days after ammonium depletion, the 5% CO2 sparged cultures had the highest specific starch. However, the starch appeared to rapidly degrade over the next day and the starch content per cell continued to decrease throughout the remainder of the experiment. The bicarbonate amended cultures accumulated starch for 2.2 days after ammonium depletion and then starch remained high throughout the remainder of the experiment, with a possible decrease after 2.2 days (albeit no statistical difference between 2.2 and 4.1 days after ammonium depletion). Final measured starch concentrations are given in Table I. Starch is clearly remaining in the bicarbonate added cultures compared to the “no-bicarbonate” cultures.
Algal carbon reallocation from starch into lipid or a switch in metabolic pathways to form lipid in preference to starch during nutrient limitation has been hypothesized in the past (Roessler, 1990), but specific mechanisms were not studied at the time. Additional evidence has been gathered using advanced spectroscopic analysis (Giordano et al., 2001; Murdock and Wetzel, 2009), and it was recently pointed out that additional experimentation in support of this hypothesis is needed (Merchant et al., 2012). Comparison of the total, and specific, starch versus Nile Red fluorescence (Figs. 5 and 4, respectively) provides additional evidence for this hypothesis. In the cultures, to which 5% CO2 was sparged and possibly in the cultures to which bicarbonate was amended, starch accumulated to maximum values and then decreased as TAG accumulated (Nile Red signal) to a maximum value. However, the bicarbonate amended cultures maintained high starch accumulation as maximum TAG was realized. Essentially, bicarbonate caused the cultures to maximize carbon storage metabolites in the form of starch and biofuel precursors.
Chlorophyll State During Lipid Accumulation
To ascertain the underlying health of the C. reinhardtii cultures during ammonium depletion and metabolite accumulation, chlorophyll concentrations were monitored. Figure 6 shows chlorophyll a (A), chlorophyll b (B), and total chlorophyll (C) for the experiments reported. During exponential growth and up to 4 days there are no discernible differences in chlorophyll concentrations between the different cultures. After which there was a decrease through 7 days for all the cultures. However, the rate of decrease was highest to lowest in the cultures to which 5% CO2 was sparged, bicarbonate was added, and air sparged without added bicarbonate, respectively.
Figure 6. C. reinhardtii CC124 average and standard deviation of chlorophyll a (A), chlorophyll b (B), and total chlorophyll (C). Arrow indicates time of medium NH depletion and inorganic carbon adjustment. Growth was maintained in Sager's minimal medium illuminated with a 14:10 h L:D cycle (n = 3).
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The decreased chlorophyll concentrations toward the end of the experiment can be compared with the decrease in DIC utilization rate (Fig. 3) observed in the cultures to which bicarbonate was added, suggesting a slowing of DIC utilization. Further, the cultures to which 5% CO2 was sparged had the lowest amount of chlorophyll at the end of the experiment, and was degraded in both TAG and starch (Table I and Figs. 4 and 5, respectively), suggesting non-optimal health. Further, at the end of the experiment the cultures to which air was sparged without added bicarbonate had the highest amount of remaining chlorophyll, but never accumulated TAG or starch, an indication of carbon limitation (Fig. 3).