Genetic engineering is a powerful tool – how reduced shading of microalgae strains can improve algal growth in photobioreactors

By Tim Michelberger

Microalgae are highly interesting organisms for photosynthetic research and industrial applications. These unicellular eukaryotic organisms exhibit the highest photosynthesis and growth rates amongst photosynthetic organisms and produce many interesting compounds such as lipids, pigments, or proteins that find broad application, for example, in the food and feed industries or pharmaceutical products. In addition, they often grow in salt water and can be cultivated in photobioreactors (PBRs) on non-arable land using the sun as sole light source, making them an interesting sustainable crop. However, microalgal strains have not yet been fully domesticated and their cultivation still faces major challenges. Algal cells normally aim to maximize light absorption by increasing their chlorophyll content in the chloroplast. In dense algal cultures, this causes mutual shading of the cells, resulting in light only reaching a small layer in the culture that is close to the light source. In mixed cultures, this makes the algal cells experiencing light fluctuations as they are constantly transported from the illuminated layers into darker areas and vice versa. Under fluctuating light, microalgae cells constantly need to balance photosynthesis and photoprotective mechanisms. This constant switch requires a lot of energy and can still allow photodamage, often resulting in reduced growth.

However, these challenges have also triggered intensive research and the development of technologies for genetic engineering of microalgae to adapt them to the experienced light conditions. Over the last two decades, scientists have created several tools for randomized or targeted genetic deletion, insertion, or mutation. Furthermore, the scientific community is slowly agreeing on roughly 10-20 algal species from different phyla that are commonly used as model organisms and for which most technologies are being developed. Since the discovery of the CRISPR/Cas9 toolkit for genetic engineering in the 2010s years, the development of such technologies has massively accelerated, and we are now equipped with many powerful tools to modify the genomes of several microalgae models fast and simple.

In the laboratory of Prof. Tomas Morosinotto at the University of Padova (Italy), which is part of the Digitalgaesation network, we are working on the optimization of industrially relevant microalgae strains for cultivation in photobioreactors. One strategy to reduce the experienced light fluctuations in PBRs is to reduce the chlorophyll content in the algal cells, allowing light to penetrate deeper into the culture and illuminating more cells at once. Using random mutagenesis, our lab could successfully isolate strains of the microalga Nannochloropsis gaditana that contain less chlorophyll and showed improved growth in small lab-scale systems. However, microalgae are highly adaptive organisms that balance their pigment composition according to the ambient light conditions and other factors such as nutrient availability. Thus, it is important to assess the growth of the newly generated strains in other environments. Testing growth in large industry-scale PBRs requires extensive work and is relatively costly. As part of my PhD project, I therefore aimed to characterize Nannochloropsis gaditana with reduced chlorophyll content in a more sophisticated lab-scale environment.

For this, a selected promising strain was cultivated in a fully automated lab-scale photobioreactor system (Figure 1). The PBR featured automated temperature and light controls. Moreover, dilution with fresh medium was automatically regulated as soon as the culture reached a certain optical density to grow the cells in a constant turbidostatic regime. Optimal pH was guaranteed by on-demand CO2 injection.

We assessed the chlorophyll composition of the strains grown in the PBR and saw that the chlorophyll content was reduced by ~40% (Figure 2, left panel), which is similar to what was observed in lab-scale systems. This suggests that the reduction of the chlorophyll content is stable across different cultivation systems. Investigating the growth performance of the strain, we discovered that the strain with less chlorophyll showed a growth rate that was increased by about 12% compared to the original wild-type (WT) strain (Figure 2, right panel).

Figure 1. Microalgae cultivated in an automated PBR system. The Nannochloropsis strain with reduced chlorophyll content was grown in bottle 2 and 4 and allows more penetration of the light. For the image, all bottles were diluted to the same cell number.

Figure 2. Reduced chlorophyll content can improve growth of N. gaditana in photobioreactors. Left panel: chlorophyll reduction compared to the WT strain. Right panel: Growth rate of N. gaditana with reduced chlorophyll content is increased in photobioreactors. Significance was assessed using a one-tailed Student’s t-test, **p<0.01.

These results could validate that the reduction of chlorophyll represents a valid strategy for improving microalgal growth across diverse cultivation systems. In the next steps, beneficial strains need to be tested in big industry-scale PBRs that in general have more depth, possibly potentiating the advantage of the chlorophyll reduction.

Taken together, we could exemplarily show that genetic engineering is a powerful technology for improving microalgal growth. As we are constantly increasing our knowledge about microalgal cells, the science community is also slowly shifting its focus from enhancing not only the growth but also to exploiting algal cells as biofactories for the production of interesting products. With the development of new technologies in the future, we can then accompany the process of microalgal domestication and usage.