RNAi Knock-Down of LHCBM1, 2 and 3 Increases Photosynthetic H2 Production Efficiency of the Green Alga Chlamydomonas reinhardtii

Abstract

Single cell green algae (microalgae) are rapidly emerging as a platform for the production of sustainable fuels. Solar-driven H2 production from H2O theoretically provides the highest-efficiency route to fuel production in microalgae. This is because the H2-producing hydrogenase (HYDA) is directly coupled to the photosynthetic electron transport chain, thereby eliminating downstream energetic losses associated with the synthesis of carbohydrate and oils (feedstocks for methane, ethanol and oil-based fuels). Here we report the simultaneous knock-down of three light-harvesting complex proteins (LHCMB1, 2 and 3) in the high H2-producing Chlamydomonas reinhardtii mutant Stm6Glc4 using an RNAi triple knock-down strategy. The resultant Stm6Glc4L01 mutant exhibited a light green phenotype, reduced expression of LHCBM1 (20.6% ±0.27%), LHCBM2 (81.2% ±0.037%) and LHCBM3 (41.4% ±0.05%) compared to 100% control levels, and improved light to H2 (180%) and biomass (165%) conversion efficiencies. The improved H2 production efficiency was achieved at increased solar flux densities (450 instead of ∼100 µE m−2 s−1) and high cell densities which are best suited for microalgae production as light is ideally the limiting factor. Our data suggests that the overall improved photon-to-H2 conversion efficiency is due to: 1) reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses) near the photobioreactor surface; 2) improved light distribution in the reactor; 3) reduced photoinhibition; 4) early onset of HYDA expression and 5) reduction of O2-induced inhibition of HYDA. The Stm6Glc4L01 phenotype therefore provides important insights for the development of high-efficiency photobiological H2 production systems.

Oxygenic photosynthesis: translation to solar fuel technologies

“…The reported hydrogen production rates of stm6 were over 10-fold higher compared to wild type, with ~540 ml of H 2 produced per liter culture over 10-14 days at a maximal rate of 4 ml per hour [51]. More recently, the same group reported another mutant [53] with the truncated light harvesting antenna of PSII, the LHCII system. This mutant exhibited even higher H 2 production rates compared to stm6 due to better penetration of light into the dense culture inside the bioreactor and minimization of solar energy conversion losses due to the diminished non-photochemical quenching and photoinhibition in this LHCII-depleted mutant under increased light intensities promoting biomass production [53].…”

“…More recently, the same group reported another mutant [53] with the truncated light harvesting antenna of PSII, the LHCII system. This mutant exhibited even higher H 2 production rates compared to stm6 due to better penetration of light into the dense culture inside the bioreactor and minimization of solar energy conversion losses due to the diminished non-photochemical quenching and photoinhibition in this LHCII-depleted mutant under increased light intensities promoting biomass production [53]. Therefore, truncation Carbon concentrating mechanisms, carbon fixation pathways and carbon utilization strategies adopted by various autotrophic organisms, leading to bio-based products.…”

Antenna size reduction in microalgae mass culture

“…An alternative approach to light dilution is to genetically engineer the microalgal cells to reduce the size of their antenna complexes (Mussgnug et al 2007, Ort et al 2011, Formighieri et al 2012, Perrine et al 2012, Kwon et al 2013, Oey et al 2013. Antennae are light-harvesting systems that are evident in all known photosynthetic organisms (Grossman et al 1995).…”

“…The creation of such antenna size mutants, i.e., microalgae with a reduced pigment content, has been proposed as a potential solution (Mussgnug et al 2007, Ort et al 2011, Formighieri et al 2012, Perrine et al 2012, Kwon et al 2013, Oey et al 2013) to light saturation. The lower pigmentation of the mutants allows increased biomass concentrations with the same light gradient in the photobioreactor , Mussgnug et al 2007).…”

“…Therefore, an alternative is reducing the light uptake per cell by genetically reducing the size of the light harvesting antennae. Reducing the chlorophyll content to overcome the oversaturation bottleneck is a promising technique that may increase biomass productivity of a mass culture exposed to full sunlight (Mussgnug et al 2007, Melis 2009, Ort et al 2011, Formighieri et al 2012, Perrine et al 2012, Kwon et al 2013, Oey et al 2013. In this chapter, both the potential and the limitations of antenna size reduction are discussed.…”