It’s green light for algae! The molecular secrets of green light mediated growth

Overview

Microalgae are currently at the forefront of research being the cornerstone of aquatic food webs, negatively impacting ecosystems via eutrophication and harmful blooms and emerging as a sustainable solution for a range of biotechnological applications. One of the most important drivers of microalgae biomass is light. Recent research from our team has made significant impact by challenging the paradigm that white light is the most important for microalgae growth. Instead, we showed that dim green light at night, boosted algal biomass compared to other dim night colours. A positive effect of green light is increasingly supported by studies in land plants. Our project aims to capitalise on our recent findings by investigating the molecular mechanism involved in green light mediated growth. Employing a multifactorial experiment of different light colours and intensities and analysing transcriptional changes of genes at different circadian time points, we will disentangle whether green light stimulation is due to better resonance with the circadian clock and/or to specifically adapted photoreceptors to dim green light. Our findings will impact photoecology by i) revealing how algae use green light in the light limited deep sea and ii) optimising industrial microalgae production that is increasingly relying on artificial light.

In plant and algae photobiology, green light is a particularly understudied colour as it is less efficiently absorbed by chlorophyl compared to red and blue lights, that are major components of photosynthetically active radiation. In addition, no dedicated green light receptor has been thus far identified. However, green light can be absorbed by the blue light receptors, the cryptochromes, as well as rhodopsin-based photosensory systems regulating phototaxis1. Recent studies on economically important plant species have highlighted a possible role for green light in increasing plant productivity2. However, further investigation is necessary to uncover the molecular mechanism underlying this response. A similar situation applies also to microalgae. Comparisons between different light colours revealed that the growth and biomass of the green algae Tetraselmis increased under white and red light but not under green or blue green light3. However, recent findings from our research on the effects of artificial light at night showed that both biomass and photosynthetic activity of Tetraselmis was boosted under dim green light at night compared to continuous broad-spectrum light, rich in blue and red wavelengths (Figure 1).

In contrast to previous comparative studies using continuous light of different wavelengths, our study employed a mixed photoperiod whereby white light was supplied for 12hr during day and coloured light was supplied for 12hr during night. In addition, our study was the first to employ dim light at night. Therefore, the interesting questions stemming from our recent findings on the stimulatory effect of green light are (a) whether microalgae have developed photoreceptors that are activated by a different colour at night-time versus daytime because this resonates better with their endogenous circadian clock and (b) whether microalgae have developed new or modulate existing photoreceptors that are activated under dim green light. The later could have developed as an evolutionary mechanism of sequestering green wavelengths which reach deepest into the ocean.

Currently, no dedicated green light receptor has been identified in nature. Moreover, although the rhodopsin-based photosensory systems that are responsive to green light, have been proposed as an alternative mode of solar energy capture in marine bacteria5, a similar mechanism is yet to be shown in algae. Despite this uncertainty, increasing evidence from different plant research groups, and microalgae from our own lab, indicate that green light can play an important role in the regulation of photosynthesis and growth. Evidence also suggests that this effect depends on the wavelength combination (e.g. green and white) and intensity. A hypothesis could be that mixed photoperiod of different colours and intensities could resonate better with the circadian clock of microalgae. Indeed, circadian rhythms are known to coordinate aspects of microalgae behaviour, physiology and metabolism6; however, the transcriptional changes that drive these rhythms over a 24hr period have been investigated only in one microalgae6 and only under the typical photoperiods of white light:dark.

Green colour should have a fundamental role in primary production since it is most dominant in coastal systems and together with blue wavelengths penetrates the deepest into the ocean (Figure 2A). However, current underwater lightscapes are bound to shift because of altered terrestrial freshwater inflows and turbidity due to climate change. Moreover, recent studies on artificial light at night have shown that coastal seafloors are particularly susceptible to artificial light within the green range (Figure 2B). Thus, knowledge on the effects of green light generated by this project will directly impact predictions of primary productivity as a result of climate change and expanding artificial light at night, currently at a rate of 2% – 6% per year.

Crucially our results will be highly relevant to the rapidly expanding sector of microalgal production as a sustainable source of high value molecules in biotechnological applications. Our findings will benefit this sector due to the heightened interest of industrial facilities to boost biomass by shifting from external production systems to internal photobioreactors using LED light technology (Figure 2C-D).

The objectives of the proposed project are thus:
Objective 1: Understand the exact conditions causing the stimulatory effect of green light on microalgal growth using a multifactorial experiment that disentangles the effect of mixed photoperiod from that of dim light.
Objective 2: Unravel the molecular mechanism behind the observed growth responses to green light by analysing the changes in genes and metabolic compounds of interest. This will reveal whether the green light response comprises of a novel signalling pathway or rather induces a complementary effect on existing photoreceptors such as cryptochromes or rhodopsin, thus justifying its stimulatory effect4.
Objective 3: Scale up the light settings that were found to be optimal for growth using larger scale photobioreactors as well as alternative solid substrates that increases the rate of CO2 uptake by lowering the mass transfer resistance for gas exchange

References
1. Jaubert, M., J. P. Bouly, M. Ribera d’Alcalà, and A. Falciatore. 2017. Light sensing and responses in marine microalgae. Current Opinion in Plant Biology 37:70–77.
2. Kim, H. H., G. D. Goins, R. M. Wheeler, and J. C. Sager. 2004. Green-light supplementation for enhanced lettuce growth under red-and blue-light-emitting diodes. HortScience 39:1617–1622.
3. Abiusi F, Sampietro G, Marturano G, Biondi N, Rodolfi L, D’Ottavio M, Tredici MR. 2014 Growth, photosynthetic efficiency, and biochemical composition of Tetraselmis suecica F&M-M33 grown with LEDs of different colors. Biotechnol. Bioeng. 111, 956–964.
4. Folta, K. M., and S. A. Maruhnich. 2007. Green light: A signal to slow down or stop. Journal of Experimental Botany 58:3099–3111.
5. Beja, O., L. Aravind, E. V. Koonin, M. T. Suzuki, A. Hadd, L. P. Nguyen, S. B. Jovanovich, C. M. Gates, R. A. Feldman, J. L. Spudich, E. N. Spudich, and E. F. DeLong. 2000. Bacterial rhodopsin: Evidence for a new type of phototrophy in the sea. Science 289:1902–1906.
6. Corellou, F., C. Schwartz, J. P. Motta, E. B. Djouani-Tahri, F. Sanchez, and F. Y. Bougeta. 2009. Clocks in the green lineage: Comparative functional analysis of the circadian architecture of the picoeukaryote Ostreococcus. Plant Cell 21:3436–3449.

Methodology

Objective 1 (Glasgow): Here we will employ a multifactorial experimental design comprising 8 treatments of different combinations of white, green and blue light of either low or high intensity (Figure 3). Key parameters such as the fluence rate, wavelength and time of irradiation are based on optimal experimental set up, already optimised and established in the Spatharis Lab. Each treatment will contain 8 replicate cultures of microalgae species of interest grown in 200ml volumetric cylinders. The effect of green light will be examined based on its role in inhibiting blue light responses in higher plants by repressing cryptochrome photochemistry. Cryptochromes are conserved and are known to promote photosynthesis in microalgae. Five of the 8 replicates will be sampled at selected intervals during a period of 2 weeks to estimate physiological responses such as growth rate, cell density and pigment concentration. The response of phototactic behaviour to the different colour treatments (i.e. photo-tropic versus photo-avoidance) will offer key insights on our physiological and molecular findings underlying growth.

Objective 2 (Glasgow): To unravel the molecular mechanisms underpinning the observed growth responses we will use 3 of the 8 replicates to perform transcriptomics and metabolomics and/or lipidomics analysis at selected time points.

Objective 3 (Newcastle): Upscaling the growth using high intensity green light of optimal photoperiod as identified by the previous experiments. Use of photobioreactors equipped with LED lights as well as experimentation with coating microalgae onto solid substrates. The later method has been shown to increase the rate of CO2 uptake by lowering the mass transfer resistance for gas exchange. The use of green light could enhance this further. The microalgae will be processed to see if the light applied during growth leads to different product compositions of eg lipids.

Project Timeline

Year 1

The scholar will prepare a thorough literature review on microalgae responses to different light wavelengths and periodicities. They will also prepare the laboratory and initiate the microalgae experimentations.

Year 2

The scholar will continue the microalgae experimentations in Glasgow, carry out analysis of cell counts and pigments and perform extractions from cell cultures for subsequent molecular and lipidomics analyses. The second half of the year will continue in Newcastle for the upscaling experiments using phtobioreactors and solid substrates.

Year 3

Finalise sample and data analyses and write up for objective 1-3; attendance of scientific writing courses, attendance of national scientific meeting.

Year 3.5

Attendance of international scientific meeting, completion of manuscripts and submission of thesis.

Training
& Skills

The PhD scholar will obtain skills related to laboratory experimentations using LED lights and microalgae monocultures as well as related sample analyses such as cell counting for abundance and growth rate estimations, spectrophotometric analysis of different pigments and culturing techniques. The scholar will also be trained in extraction of high value molecules eg sterols, fatty acids for subsequent analysis with HPLC by Glasgow Polyomics. The scholar will also obtain skills in data analysis with Generalised Linear Models for understanding the mechanisms driving molecular changes in microalgae under different LED wavelengths.

References & further reading

1. Jaubert, M., J. P. Bouly, M. Ribera d’Alcalà, and A. Falciatore. 2017. Light sensing and responses in marine microalgae. Current Opinion in Plant Biology 37:70–77.
2. Kim, H. H., G. D. Goins, R. M. Wheeler, and J. C. Sager. 2004. Green-light supplementation for enhanced lettuce growth under red-and blue-light-emitting diodes. HortScience 39:1617–1622.
3. Abiusi F, Sampietro G, Marturano G, Biondi N, Rodolfi L, D’Ottavio M, Tredici MR. 2014 Growth, photosynthetic efficiency, and biochemical composition of Tetraselmis suecica F&M-M33 grown with LEDs of different colors. Biotechnol. Bioeng. 111, 956–964.
4. Folta, K. M., and S. A. Maruhnich. 2007. Green light: A signal to slow down or stop. Journal of Experimental Botany 58:3099–3111.
5. Beja, O., L. Aravind, E. V. Koonin, M. T. Suzuki, A. Hadd, L. P. Nguyen, S. B. Jovanovich, C. M. Gates, R. A. Feldman, J. L. Spudich, E. N. Spudich, and E. F. DeLong. 2000. Bacterial rhodopsin: Evidence for a new type of phototrophy in the sea. Science 289:1902–1906.
6. Corellou, F., C. Schwartz, J. P. Motta, E. B. Djouani-Tahri, F. Sanchez, and F. Y. Bougeta. 2009. Clocks in the green lineage: Comparative functional analysis of the circadian architecture of the picoeukaryote Ostreococcus. Plant Cell 21:3436–3449.

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