Acetate and Bicarbonate Assimilation and Metabolite Formation in Chlamydomonas reinhardtii: A 13C-NMR Study

Abstract

Cellular metabolite analyses by 13C-NMR showed that C. reinhardtii cells assimilate acetate at a faster rate in heterotrophy than in mixotrophy. While heterotrophic cells produced bicarbonate and CO2 aq, mixotrophy cells produced bicarbonate alone as predominant metabolite. Experiments with singly 13C-labelled acetate (13CH3-COOH or CH3-13COOH) supported that both the 13C nuclei give rise to bicarbonate and CO2 aq. The observed metabolite(s) upon further incubation led to the production of starch and triacylglycerol (TAG) in mixotrophy, whereas in heterotrophy the TAG production was minimal with substantial accumulation of glycerol and starch. Prolonged incubation up to eight days, without the addition of fresh acetate, led to an increased TAG production at the expense of bicarbonate, akin to that of nitrogen-starvation. However, such TAG production was substantially high in mixotrophy as compared to that in heterotrophy. Addition of mitochondrial un-coupler blocked the formation of bicarbonate and CO2 aq in heterotrophic cells, even though acetate uptake ensued. Addition of PSII-inhibitor to mixotrophic cells resulted in partial conversion of bicarbonate into CO2 aq, which were found to be in equilibrium. In an independent experiment, we have monitored assimilation of bicarbonate via photoautotrophy and found that the cells indeed produce starch and TAG at a much faster rate as compared to that in mixotrophy and heterotrophy. Further, we noticed that the accumulation of starch is relatively more as compared to TAG. Based on these observations, we suggest that acetate assimilation in C. reinhardtii does not directly lead to TAG formation but via bicarbonate/CO2 aq pathways. Photoautotrophic mode is found to be the best growth condition for the production of starch and TAG and starch in C. reinhardtii.

Quantitative analysis of the chemotaxis of a green alga, Chlamydomonas reinhardtii, to bicarbonate using diffusion-based microfluidic device

“…The cia5 mutant, which cannot acclimate to the low CO 2 condition due to the lack of CCM, was grown in the presence of 5% CO 2 . All cultivations were conducted under a photoautotrophic condition in 100 ml Erlenmeyer flask, with 50 …”

“…8 C. reinhardtii is able to uptake inorganic carbon (HCO 3 À and CO 2 ) and efficiently convert them into organic compounds in the light phase by the means of CCM and photosynthesis. 50 Thus, they can successfully cover their carbon requirement. In the dark phase, however, the light-dependent carbon assimilation process would stop, and the dark respiration starts to increase the internal production of CO 2 .…”

The Relationship of Triacylglycerol and Starch Accumulation to Carbon and Energy Flows During Nutrient Deprivation in Chlamydomonas.

“…The observation that TAG accumulates during nitrogen deprivation in Chlamydomonas cells cultured heterotrophically in the dark separates the induction of TAG accumulation from photosynthesis. High levels of TAG accumulation have been reported in heterotrophically grown Chlorella species (Miao and Wu, 2004;Liu et al, 2011), and TAG accumulation was also observed in heterotrophically cultured Chlamydomonas after carbon starvation (Singh et al, 2014) and during N deprivation in the presence of high levels (60 mM) of acetate (Fan et al, 2012).…”

Protein and lipid dynamics in photosynthetic thylakoid membranes investigated by in-situ solid-state NMR

“…Cells were nevertheless exposed to a low light intensity, close to the compensation point, to maintain their photosynthetic metabolism active. Also cells were cultivated in flasks, where CO2 diffusion from the atmosphere is limited in order to further stimulate acetate assimilation from the medium [33]. Three rounds of cultivation in labeled medium were performed in these conditions with a tenfold dilution at every step.…”