Microalgae as bioreactors for bioplastic production

Abstract

Background: Poly-3-hydroxybutyrate (PHB) is a polyester with thermoplastic properties that is naturally occurring and produced by such bacteria as Ralstonia eutropha H16 and Bacillus megaterium. In contrast to currently utilized plastics and most synthetic polymers, PHB is biodegradable, and its production is not dependent on fossil resources making this bioplastic interesting for various industrial applications.

Results: In this study, we report on introducing the bacterial PHB pathway of R. eutropha H16 into the diatom Phaeodactylum tricornutum, thereby demonstrating for the first time that PHB production is feasible in a microalgal system. Expression of the bacterial enzymes was sufficient to result in PHB levels of up to 10.6% of algal dry weight. The bioplastic accumulated in granule-like structures in the cytosol of the cells, as shown by light and electron microscopy.

Conclusions: Our studies demonstrate the great potential of microalgae like the diatom P. tricornutum to serve as solar-powered expression factories and reveal great advantages compared to plant based production systems.

Figure 1

In vivo localization studies on PhaA, PhaB and PhaC expression in P. tricornutum. Sequences for PhaA (ketothiolase), PhaB (acetoacetyl-CoA reductase) and PhaC (PHB synthase) of R. eutropha H16 were introduced as GFP fusion proteins in P. tricornutum. All three enzymes were expressed in the heterologous system and accumulate in the cytosol as no specific targeting signal was added. Plastid autofluorescence is shown in red and GFP fluorescence is depicted in green. Scale bar represents 10 μm. PAF - plastid autofluorescence, Re - R. eutropha H16

Figure 2

Fluorescence and electron microscopic analyses on PHB accumulation in P. tricornutum. Cytosolic expression of enzymes PhaA, PhaB and PhaC of R. eutropha H16 induces the formation of granule-like structures that are stained by the lipophilic dye Nile red as visualized by fluorescence microscopy (A: NO₃^-). Under non-induced conditions no such granules were observed (A: NH₄⁺). Electron microscopic analyses confirm cytosolic accumulation of electron-translucent granules (exemplarily marked by arrows) in cell lines expressing bacterial enzymes of the PHB pathway (C-F). PHB granules are about 0.1-0.3 μm in size and were not observed under non-induced conditions (B). Scale bar represents 1 μm (B-D) and 500 nm (E/F). DIC - differential interference contrast, G - golgi apparatus, Mt -mitochondrium, Nu - nucleus, PAF - plastid autofluorescence, Pl - plastid, V - vacuole

Figure 3

Quantification of PHB synthesis in the cytosol of P. tricornutum. The level of PHB synthesis for five transgenic P. tricornutum cell lines (No. 8, 11, 15, 18, 28) was analyzed by gas chromatography coupled to mass spectrometry. After 7 days of PhaA/PhaB/PhaC expression PHB levels of 8.0 to 10.6% of algal dry weight were detected. Wild type cells were negative for PHB synthesis. dwt - dry weight, wt - wild type