Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway

Abstract

Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D31-16:0 (palmitic) acid yielded D31-18:0 (stearic) acid, D29-18:1 (oleic and cis-vaccenic) acids, and D29-heptadecene. These findings showed that loss of the carboxyl group of a C18 monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C21 hexaene, without detectable C15-C19 hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C16 and C18 fatty acids into alka(e)nes by a new, light-dependent pathway.

Figure 1.

Long-chain alka(e)nes are synthesized by cells of C. reinhardtii and C. variabilis NC64A. A, Chromatogram of the unsaponifiable cell material analyzed by GC-MS. The long-chain hydrocarbon region is magnified (inset). 17:1-alk, n-heptadecenes; 17:0-alk, n-heptadecane; 15:0-alk, n-pentadecane. B, Mass spectrum of the C. reinhardtii peak at 14.3 min in A. This spectrum is identical to that of a commercial standard of 1-heptadecene. C, Quantification of alka(e)nes detected in C. reinhardtii and C. variabilis after transmethylation of cells and GC-MS/FID analysis. Values are mean of three biological replicates, and error bars represent sd; nd, not detected.

Figure 2.

The heptadecene isomers are 7-heptadecene and 8-heptadecene. Cells were saponified and a solvent extract was reacted with DMDS before analysis by GC-MS. The major diagnostic ions expected for DMDS derivatives of 7-heptadecene and 8-heptadecene and the mass spectra obtained for the two C. variabilis n-heptadecene isomers modified by DMDS are shown.

Figure 3.

C. reinhardtii and C. variabilis hydrocarbons derive from fatty acids. Cells were cultivated for 5 d in presence of deuterium-labeled (D₃₁) palmitic acid, collected, and directly transmethylated. The transmethylation extract was analyzed by GC-MS/FID. A, Part of the GC-MS chromatogram showing endogenous and labeled C. reinhardtii FAMEs. B and D, Part of the chromatograms showing C. reinhardtii (B) and C. variabilis (D) hydrocarbons. C and E, Mass spectra of the C. reinhardtii peak at RT = 9.2 min (C) and the C. variabilis peak at RT = 8.9 min (E). An interpretation of the fragments observed is shown in red and is consistent with the compound being D₂₉ n-heptadecene and D₃₁ n-heptadecane, respectively.

Figure 4.

Heptadecene content varies with growth and culture conditions in C. reinhardtii after cell transmethylation products were analyzed by GC-MS/FID. A and B, Heptadecene content (A) and growth curve (B) of cells grown for 9 d in flasks under mixotrophic (TAP medium) or photoautotrophic (minimal medium) conditions. C and D, cells were grown for 2 d in TAP medium (C) or minimal medium (D) and changed to the same culture medium devoid of nitrogen (TAP-N and MM-N). Values are means of four biological replicates, and error bars represent sd. Asterisks denote significant differences at *P < 0.05, **P < 0.01, and ***P < 0.001 in a two-sided t test.

Figure 5.

C. reinhardtii heptadecene synthesis is strictly dependent on light and increases with light intensity. A, Variation of heptadecene during day-night cycles. Cells were grown in flasks under a day-night cycle (12 h light/12 h dark) for 3 d and then diluted at the beginning of the day and samples were collected during the next 24 h. Values are means of three biological replicates, and error bars represent sd. B, Heptadecene synthesis in dark- and light-grown cultures. Cells were grown in flasks in TAP medium at 120 µmol photons m⁻² s⁻¹ and then diluted in the same medium to be grown for an extra 5 d either in the same condition or in the dark (i.e. under heterotrophic conditions). Values are means of three biological replicates, and error bars represent sd; nd, not detected. C, Quantification of heptadecene in isolated chloroplasts. Whole cells or isolated intact chloroplasts were transmethylated and heptadecene was quantified using GC-MS/FID. The proportion of cell heptadecene present in the chloroplast was calculated using the chlorophyll content of isolated chloroplasts and assuming all chlorophyll was present in chloroplasts. Values are means of three biological replicates, and error bars represent sd. D, Kinetics of heptadecene synthesis upon light exposure. Cells were grown for 5 d in the dark in TAP medium and heptadecene was quantified at various times after exposure to light (120 µmol photons m⁻² s⁻¹) in presence or absence of the photosynthesis inhibitor DCMU. Values are means of three biological replicates, and error bars represent sd; nd, not detected. Using two-sided t tests, no significant differences were found at P < 0.01 with and without DCMU. E, Influence of light intensity on heptadecene content per cell volume. Cells were grown in minimal medium at different light intensities in a photobioreactor operated at constant cell density. Cells cultures were stabilized for 3 d at a constant light intensity (starting at 125 µmol photons m⁻² s⁻¹) before switching to the next higher intensity (up to 300 µmol photons m⁻² s⁻¹). Cells aliquots were harvested from stabilized cultures at each intensity to measure heptadecene content. Values are means of three biological replicates, and error bars represent sd.

Figure 6.

Long-chain hydrocarbons are synthesized by Nannochloropsis sp. Strains were cultivated for 5 d, and hydrocarbons were analyzed by transmethylation of whole cells and GC-MS/FID. Values are means of three biological replicates, and error bars represent sd.

Figure 7.

Possible pathways for 7-heptadecene synthesis in C. reinhardtii. Two major possibilities are presented based on findings of this work and reactions known in other organisms. The specificity for cis-vaccenic acid would be brought by a phospholipase A1 or another type of lipase depending on the lipid used as substrate. Left side: cyanobacterial- or plant-type pathway with acyl reduction and formal decarbonylation of aldehyde intermediate. Chloroplast localization would involve acyl-acyl carrier protein (acyl-ACP) substrate, while other subcellular localizations would involve acyl-coenzyme A (acyl-CoA) substrate. Right side: bacterium-type pathway with decarboxylation of a free fatty acid (but without formation of terminal double bond). Decarbonylase and decarboxylase are indicated with quotes because decarbonylation and decarboxylation might be only formal and not actual reactions. Regulation by light may occur at the step of free fatty acid generation and/or at its conversion to heptadecene.