Metabolic survey of Botryococcus braunii: Impact of the physiological state on product formation

Abstract

The microalga Botryococcus braunii is widely regarded as a potential renewable and sustainable source for industrial applications because of its capability to produce large amounts of metabolically expensive (exo-) polysaccharides and lipids, notably hydrocarbons. A comprehensive and systematic metabolic characterization of the Botryococcus braunii race A strain CCAP 807/2 was conducted within the present study, including the detailed analysis of growth-associated and physiological parameters. In addition, the intracellular metabolome was profiled for the first time and showed growth- and product-specific fluctuations in response to the different availability of medium resources during the cultivation course. Among the identified metabolites, a constant expression of raffinose was observed for the first time under standard conditions, which has until now only been described for higher plants. Overall, the multilayered analysis during the cultivation of strain CCAP 807/2 allowed the differentiation of four distinct physiological growth phases and revealed differences in the production profiles and content of liquid hydrocarbons and carbohydrates with up to 84% of organic dry weight (oDW). In the process, an enhanced production of carbohydrates with up to 63% of oDW (1.36±0.03 g L-1) could be observed during the late linear growth phase, whereas the highest accumulation of extracellular hydrocarbons with up to 24% of oDW (0.66±0.12 g L-1) occurred mainly during the stationary growth phase. Altogether, the knowledge obtained is potentially useful for the general understanding of the overall physiology of Botryococcus braunii and provide important insights into the growth behavior and product formation of this microalga, and is thus relevant for large scale biofuel production and industrial applications.

Fig 1. Growth performance analysis of Botryococcus braunii CCAP 807/2 and differentiation into different growth stages.

a. BODIPY505/515 staining of cell colonies after 12 days of cultivation. (i) DIC image of single BODIPY505/515-stained colony, (ii) chlorophyll autofluorescence channel, (iii) BODIPY channel, false colored green, (iv) Merge of DIC, BODIPY, chlorophyll autofluorescence images. Bar 25 μm. Yellow and white arrows indicate cytoplasmic oil bodies and extracellular hydrocarbons, respectively. b. Determination of total chlorophyll and organic dry weight (oDW) of the cell biomass during cultivation period of 30 days. c.Illustration of the color change of the cultures during the course of the cultivation. Images show the cultures after 6, 12 and 21 days of culturing as well as the corresponding biomass dry weight. d. Measurement of the C/N ratio and nitrogen (NO₃-N) content in the media supernatant during cultivation period of 30 days. Numbers at the top of the graph (I to IV) represent the proposed distinct growth stages of B. braunii cells, referred as Phases I–IV. Error bars represent standard error of mean value of three biological and three technical replicates (SE; n = 9).

Fig 2. The overall intracellular metabolome profile of Botryococcus braunii CCAP 807/2, containing all identified metabolites during the proposed growth stages, referred as Phases II (linear phase), III (stationary phase) and IV (decline phase).

a. Non-targeted metabolome profile of primary metabolites showing the comparison of relative abundance level of metabolites, divided into three different categories based on the related metabolic pathways, thus (i) glycolysis intermediates, sugars and sugar alcohols, (ii) amino acids and other related metabolites and (iii) citric acid cycle intermediates, terpenoids, steroids and vitamins. b. Intracellular pigments with relative abundances of (i) chlorophylls and (ii) carotenoids. c. Gravimetrically determined total lipid content, containing polar (P lipids) and non-polar lipid (N-P lipids) and expressed as percentage of dry biomass weight. d. Polar lipid fraction with relative abundance level of fatty acids. e. Non-polar lipid fraction with relative abundance levels of (i) fatty acid and (ii) hydrocarbons. f. Comparison of hydrocarbons and fatty acids derived from the total non-polar lipid fraction on the basis of the relative abundance levels, considering Phase II as 100%. Metabolites were identified by ‘a’ comparison with the NIST 05 library and Golm Metabolome Database (Lib) and verified with purified standards; ‘b’ only via above mentioned databases with RSI value above 750. ‘c’ marks the identified hydrocarbons via mass spectra of GC-MS and available literature [10,17]; ‘#’ not detected. Error bars represent standard deviation (SD). Asterisks represent p-values as determined via Student’s t-test (* = < 0.05, ** = < 0.01).

Fig 3. Extracellular product formation of Botryococcus braunii CCAP 807/2 in form of carbohydrates and hydrocarbons during the proposed growth stages referred as Phases I (lag phase), II (linear phase), III (stationary phase) and IV (decline phase).

a. Determination of organic dry weight (oDW) and C/N ratio of the whole culture broth (containing cells and supernatant) over the period of cultivation for 30 days. b. Determination of total carbohydrate concentration in the whole culture broth and the cell-free supernatant. c. Quantification of total extractable hydrocarbons via GC-FID at each time point during cultivation (except for day 12 –lost samples (##)). Error bars represent standard error (SE; n = 9 for a, n = 12 for b) and standard deviation (SD) for c.