Gene expression of carbonic anhydrase and rubisco under high CO2 stress
Chlorella PY-ZU1 had a higher biomass yield (2.85 g L−1) and shorter growth cycle (7 days) when cultivated in SE medium under continuous aeration with 15% (v/v) CO2 gas. This CO2 concentration is equivalent to that of flue gas from most coal-fired power plants. By contrast, the biomass concentration of Chlorella PY-ZU1 was only 1.30 g L−1 after 10 days of cultivation under air. It was previously reported that high CO2 induced algae growth [23]. Carbon fixation and nitrogen metabolism are the two most important aspects of primary cell metabolism. Therefore, we expected to observe significant differences in the expression of genes encoding enzymes of carbon fixation and nitrogen metabolism.
When aerated into the microalgal suspension, CO2 first dissolves in the medium, and then transfers from the extracellular culture medium through the cell membrane to the intracellular chloroplast. Then, CO2 is converted by ribulose-1,5-bisphosphate (RuBP) upon the catalysis of RuBP carboxylase (rubisco) to 3-phosphoglycerate (PGA), the precursor of structural materials in microalgal cells [24]. Rubisco (rbcS, EC4.1.1.39), the first enzyme of the Calvin cycle, fixes CO2 into the three carbon atoms of RuBP (\({\rm CO}_{2} + {\rm C}5\mathop{\longrightarrow}\limits^{{\rm Rubisco}}2{\rm C}3\)). The transcript abundance of RuBP increased slightly from 11,043.90 to 11,478.37 under high CO2 (15% CO2), whereas it was highly expressed under both high (15% CO2) and low (air) CO2 (Fig. 2). A 2-fold increase in transcript abundance was observed for PGK (EC2.7.2.3), which catalyzes the phosphorylation of 3-PGA to 1,3-bisphosphoglycerate. Moreover, triose phosphate isomerase (tpiA, EC5.3.1.1), which reversibly converts GAP into dihydroxyacetone phosphate (DHAP), increased by approximately 10.4-fold. The transcript abundance of a series of enzymes that converts GAP to sedoheptulose-7-phosphate (S7P), such as fructose-1,6-bisphosphatase aldolase (fbaB, EC4.1.2.13), increased more than 2-fold, whereas those of transketolase (tkt, EC2.2.1.1) and sedoheptulose-1,7-bisphosphatase (SBPase, EC3.1.3.37) improved slightly. Ribose-5-phosphate isomerase (rpiA, EC5.3.1.6), which converts R5P into ribulose-5-phosphate (Ru5P), increased by 7.8-fold. Phosphoribulokinase (prkB, EC2.7.1.19), which phosphorylates Ru5P into RuBP, increased by 4.0-fold. Therefore, almost all of the enzymes involved in the Calvin cycle had increased transcript abundances (Additional file 1) under high CO2. The results indicated that the whole carbon fixation process was driven by 15% CO2 gas. Therefore, the growth rate of Chlorella PY-ZU1 increased under 15% CO2 compared with under air (Fig. 1).
Moreover, rubisco was still highly expressed even Chlorella PY-ZU1 was cultivated under limited CO2, such as air. The high expression of rubisco was caused by CCM in microalgae cells. Rubisco is only activated when CO2 concentration is greater than its K
m (CO2), because that CO2 is the only carbon source that rubisco can utilize. However, when aerated with air, 99% of carbon in the culture is in the form of HCO3
− [1]. CO2 concentration in the medium hardly meets the requirements of rubisco because of the low solubility of CO2. Gene transcript abundance of CAs in Chlorella PY-ZU1 pyrenoids increased to 5190 from 39 under cultivation with 15% CO2. CAs expression increased to maintain high CO2 concentration in pyrenoids. Furthermore, CCM was simultaneously activated as most of the dissolved inorganic carbon, HCO3
−, was transferred by pump through the chloroplast membrane into the internal pyrenoid; HCO3 was then converted by CAs to CO2 as function (2) in the chloroplast to meet the needs of rubisco (Fig. 2) [25]. However, CCM occurred at the cost of ATP. Increased CCM expression consumed more energy for CO2 transfer, thus decreasing the energy available for carbon fixation and other pathways of photosynthetic growth. Conversely, when cultivated under continuous aeration with 15% CO2, CAs was barely expressed in Chlorella PY-ZU1 pyrenoids. CCM was inactive. The CO2 that diffused directly into pyrenoids by high CO2 osmotic pressure was sufficient for rubisco. Therefore CO2 transfer pathway was simplified. Hence, more ATP was available for photosynthesis to promote growth.
$${\rm HCO}_{3}^{ - } + {\rm H}^{ + } \mathop{\longrightarrow}\limits^{{{\text{Carbonic anhydrase}}}}{\rm CO}_{2} +{\rm H}_{2} {\rm O}$$
(2)
CAs and rubisco are the most important genes of carbon fixation pathways. To confirm the transcriptome results, the expression levels of genes encoding CA and rubisco were measured by qRT-PCR under different cultivation times (Fig. 3a). Under high CO2, the CAs transcript level was significantly reduced to 0 (The original data showed in Additional file 2), which is consistent with the transcriptome results (Fig. 2). Moreover, the study conducted by Fan et al. reported a similar, remarkably decreased CAs expression when oleaginous Chlorella cells were exposed to 5% CO2 [26]. This result is also consistent with previous reports that CO2 concentration significantly affects CAs activity in Chlorella pyrenoidosa cells, and that elevating CO2 concentration decreases CAs activity [27].
Rubisco expression levels increased under high CO2 (Fig. 3b). This result is consistent with the transcriptome results. Under 15% CO2, the enhanced rubisco expression of Chlorella PY-ZU1 cells might induce more CO2 to directly permeate intracellular pyrenoids for conversion into cellular energy storage molecules and to promote ATP conversion to glucose, thereby improving the photosynthetic efficiency of microalgae. Therefore, high CO2 concentrations reduced the ATP consumption of CO2 transfer. On the other hand, high CO2 concentrations improved CO2 conversion and photosynthetic efficiency, thus eventually reducing the growth cycle and increasing the biomass yield (2.85 g L−1) of Chlorella PY-ZU1.
In addition, the time when rubisco had highest transcriptional level (24 h of high CO2 and 48 h of air) and the time CAs had lowest transcriptional level under air (48 h) were also the time when maximum microalgae growth rate was achieved (24 h of high CO2 and 48 h of air) (Fig. 1). That fully illustrated that CAs and rubisco had the ability to regulate microalgae growth.
Response of nitrogen metabolism and chlorophyll synthesis to high CO2 stress
Similar to carbon fixation, the transcript abundance of important enzymes in nitrogen metabolism, including those of nitrate reductase, nitrite reductase and glutamate dehydrogenase (ghdA), also increased under high CO2 (Fig. 4a). Nitrogen is an essential element for chlorophyll and protein synthesis. After nitrate reductase and nitrite reductase increased, nitrate ions absorbed by microalgae were immediately catalyzed to nitrite ions and then to ammonia through a series of reduction reactions to synthesize nitrogenous compounds, such as amino acids, which in turn increased the efficiency of nitrate ion uptake from the medium by microalgae cells. On the aspect of nitrogen source consumption (in this study, the nitrogen source was sodium nitrate), Chlorella PY-ZU1 consumed almost all of the 3 mM nitrate during the first 2 days, especially on the first day (Fig. 4b) when cultivated under continuous aeration with 15% CO2. By contrast, Chlorella PY-ZU1 only consumed 1.26 mM nitrate during the first 2 days, and nitrate was not completely consumed until the sixth day when cultivated under air. On the genetic level, the transcript abundance of nitrate reductase, the first enzyme in nitrogen metabolism, increased by approximately 10-fold by the 24th h and 8-fold by the 48th h under high CO2 compared with that under air (Fig. 4c). The higher transcript expression of nitrate reductase accelerated the transformation of nitrate to nitrite and O2 catalyzed by nitrate reductase (Function 3). This higher gene expression might manifest by the rapid consumption of nitrate. Furthermore, by the catalysis of the up-regulated nitrite reductase, more nitrite was converted to amino acid synthesis precursors, such as ammonia (Function 4), thereby accelerating the synthesis of proteinaceous materials, such as chlorophyll (Fig. 4d).
$$2{\rm NO}_{3}^{ - } \mathop{\longrightarrow}\limits^{{\rm Nitrate}\,\, {\rm reductase}}2{\rm NO}_{2}^{ - } + {\rm O}_{2}$$
(3)
$${\rm NO}_{2}^{ - } + 6{\rm NADPH}\mathop{\longrightarrow}\limits^{{\rm Nitrite}\,\,{\rm reductase}}{\rm NH}_{4}^{ + } + 2{\rm OH}^{ - } + 6{\rm NADP}$$
(4)
Under 15% (v/v) CO2, 23.65 mg L−1 of chlorophyll was produced during the first day of cultivation. Chlorophyll concentration remained stable in the range of 23–26 mg L−1. All of the 3 mM nitrate in the medium was consumed during the following 3 days (Fig. 4d). Chlorophyll was vital in photosynthesis and allowed Chlorella PY-ZU1 cells to absorb energy from light. Moreover, light conversion efficiency is linearly correlated with chlorophyll content [1, 28]. Increased chlorophyll provided more energy for photosynthetic reactions, thereby improving the photosynthetic growth rate of Chlorella PY-ZU1. However, the high nitrogen consumption during the first 3 days resulted in nitrogen deficiency in the following days under 15% CO2. Microalgae consumed its chlorophyll to maintain cell growth under nitrogen deficiency from the 4th day, and the chlorophyll content of Chlorella PY-ZU1 decreased. By contrast, chlorophyll content of Chlorella PY-ZU1 still increased when cultivated under air. Given that chlorophyll synthesis is almost directly proportional to nitrate concentration in the culture medium, the chlorophyll contents of Chlorella PY-ZU1 were almost the same under different CO2 conditions by the end of the cultivation period [1, 29]. However, during cultivation, the chlorophyll content of Chlorella PY-ZU1 cultivated under 15% CO2 was always higher than that of under air, which resulted in higher microalgae growth rate (Fig. 1).
Analysis of different concentrations of CO2 transport and fixation mechanisms
Figure 5 shows the biomass productivity of Chlorella PY-ZU1 cultivated in optimized SE medium under different CO2 concentrations. Excessively low (<1%) and high (>30%) CO2 concentration could restrain microalgae growth and resulted in lower biomass yield (<2 g L−1). However, when cultivated under 1% CO2, the biomass yield drastically increased by 130.2% to 3.73 g L−1 compared with the 1.62 g L−1 obtained by cultivation under 0.5% CO2. The drastically increased biomass yield in response to higher CO2 concentrations indicated some changes in the pathway of CO2 transfer and utilization by microalgae. CCM will work when microalgae is cultivated under limited CO2 conditions, such as air. However, it will not work if the CO2 that directly diffused to pyrenoids by extra- and intracellular CO2 osmotic pressure is sufficient for rubisco, more energy was concentrated for cell growth. Thus, biomass productivity was dramatically enhanced [11]. An external concentration of 0.5% CO2 was too low for Chlorella PY-ZU1 because it could not supply enough CO2 through osmosis to rubisco. However, 1% external CO2 could overcome diffusion resistance to enable CO2 flux from the external medium to the cytoplasm. Moreover, enough CO2 diffusion by high CO2 stress to the cytoplasm resulted in non-operational CCM. Therefore, >1% external CO2 concentration maintained enough CO2 in pyrenoids for rubisco only through direct diffusion, which is dependent on extra- and intra-cellular CO2 osmotic pressure. When cultivated under 3–30% CO2, Chlorella PY-ZU1 had stable, higher biomass yields of 4.60–4.78 g L−1. Therefore, 3–15% was ideal CO2 concentration for Chlorella PY-ZU1 growth. However, when CO2 concentration exceeded 30%, excess CO2 inhibited microalgal growth and sharply decreased biomass yield (1.89 g L−1). Therefore, >30% external CO2 is too high for Chlorella PY-ZU1 given the overly acidic culture after aeration with higher CO2 concentrations [26, 27].