Survival and Development of Photosynthetic Oxygenic Organisms Tolerant to Space Radiation and Production of Compounds with Anti-Oxidant Properties

A Brief Introduction

The unicellular green alga Chlamydomonas reinhardtii

During long-term space flights, such as exploratory missions to Moon and Mars scheduled for the next future or long periods of activity on board the International Space Station, robust biological life support systems are absolutely necessary. These systems, as well as the health of astronauts, can suffer seriously from the combined effects of the space ionizing radiations and other stressors that are present in the planetary environments and the deep space. The , coordinated by Dr. Maria Teresa Giardi of the National Research Council (CNR), was born with the aim to identify new solutions to this problematic, considering as a subject of the study simple photosynthetic organisms.

Biological life support systems, as plants or algae, convert and store solar energy in chemical bonds by the process called photosynthesis that is fundamental for the development and the maintenance of life on Earth. By means of a complex chain of bio-chemical reactions, photosynthetic organisms are able to utilize CO2 from the atmosphere, fixing it as carbohydrate and simultaneously releasing oxygen in the environment. The result of photosynthesis is revitalizing the atmosphere and the organisms by themselves represent organic compounds that can be employed as nutrients. All this aspects make photosynthetic organisms a biological system of great importance to support the astronauts.

Using innovative techniques of bio-engineering applied to the unicellular green alga Chlamydomonas reinhardtii, site-directed and random mutants were produced and used in the experiments. The mutagenesis involved a specific gene encoding the D1 protein which has a fundamental role in the photosynthesis. The strains were tested in space to select the ones showing the higher radioresistance. Following a synergetic approach, a series of experiments were also conducted in ground-based laboratories. A large number of strains were exposed to different ionizing radiations (e.g. neutrons, protons, HZE and gamma rays), simulating part of the complex radiation field present in space. The higher tolerance of the C. reinhardtii strains could be a result of a improved capacity of the organisms to counteract the action of reactive oxygen species (ROS) produced by the space ionizing radiations. This is another topic of interest for the Photo project that is deeply investigated because the strains with increased anti-oxidant properties could be employed in the production of compounds protective for human vision, immune function, and for cancer and heart disease prevention.

The Photo project has been developed in the frame of the MoMa project “From Molecules to Man: Space Research Applied to the improvement of the Quality of Life of the Aging Population on Earth”, funded by the Italian Space Agency. Inside MoMa, Maria Teresa Giardi is coordinating the FAI research line related to the development of anti-aging drugs. The success of the project has been possible thanks to the fruitful work of an international team of young researchers belonging to several research fields (biology, engineering, physics), and of some Italian companies active in the aerospace industry and with expertise in Life Support Systems technologies.

The Aims of the Work

Coltures of C. reinhardtii

The goals pursued in the frame of the Photo project are basically two. The first is to analyze the possibility of some photosynthetic oxygenic microorganisms, as algae, to survive and to develop in space, evaluating the effects of the ionizing radiations. We focused our study on Photosystem II (PSII), one of the main chlorophyll-protein complexes in the chloroplast, where the primary photochemical reactions of the photosynthesis occur. It has great importance for the biological life on our planet. PSII drives the first step of the photosynthetic electron transport chain from which all the photosynthesis depends. It utilizes the light energy to catalyze a series of reactions resulting in the splitting of H2O into O2, protons and electrons. The D1 protein is a subunit involved in the formation of the core complex of PSII and it has a fundamental role in the photosynthetic process. It is an exceptionally stress-susceptible element that could be affected by the space ionizing radiations. This and other stress conditions can damage D1 protein. Testing the radioresistance of different D1-protein-mutants can help us to identify the amino acidic substitutions able to improve the microorganisms’ tolerance to space environment.

The second, basic goal of the project is connected to the content determination of some compounds with anti-oxidant properties, as the carotenoid pigments. One of the key roles of these organic pigments present in the photosynthetic organisms is to protect photosynthesis under different stress conditions. They act as antioxidants preventing the damage induced by the ROS. The xanthophylls are oxygen-containing carotenoids with mains representative zeaxanthin, antheraxanthin, violaxanthin, involved in the photoprotection of the photosynthetic apparatus. Their content is regulated through a cycle that converts a pigment to another one following a specific biosynthetic pathway. When the photosynthetic organisms are illuminated with strong light, which exceed their capacity for photosynthesis, the excess energy can be harmful for the photosynthetic apparatus. Under this conditions the nondissipating pigment violaxanthin is rapidly converted to zeaxanthin (via the intermediate antheraxanthin) that has photoprotective properties, dissipating the energy in excess. This conversion cycle could represent a survival strategy adopted by the photosynthetic organisms even in the space environment. The C. reinhardtii mutants employed in the test have been analyzed to characterize carotenoid pigments content and composition. Organisms which in space could maintain a high level of carotenoids could be used in the production of compounds with anti-oxidant and anti-aging properties which can be used as nutrients for astronauts.

The Experiments in Space

Foton-M3 satellite

The experiments Photo-I and Photo-II, conducted in the frame of the project Photo, flew in Space on board the short-duration missions Foton-M2 and Foton-M3 of the European Space Agency, respectively on June 2005 (for 16 days) and on September 2007 (for 12 days). The Foton satellites, launched from the Russian Cosmodrome in Baikonur (Kazakhstan), were placed in Low-Earth-Orbit and the capsules were then recovered at the end of the missions, carrying back to Earth the many experiments on board.

Artistic view of the Foton satellite while orbiting the Earth (© ESA)

In Photo-I experiment, which flew on 2005, sixteen biological samples –strains of C. reinhardtii– were allocated in a container inside Biopan-5. The Biopan is a shell-like facility mounted outside the satellite capsule that was opened once the satellite was in orbit, exposing the experiment directly to the space environment. After the re-entry of the Foton-M2 capsule on Earth, the samples have been retrieved and then analyzed in laboratory to investigate how they reacted to the extreme conditions in Space.

The Photo-II experiment, which flew on board Foton-M2 and Foton-M3 placed inside the satellite capsule, was composed of an automatic fluorimeter able to monitor in real-time the photosynthetic activity of several micro organisms, and at the same time providing them with the light necessary to their survival.

Foton-M2 capsule back to Earth

Even in Photo-II the organisms employed were strains of the alga C. reinhardtii. The study included site-directed and random mutants of the Photosystem II D1 protein obtained with innovative techniques of bio-engineering, and was finalised to testing and selecting the strains showing the higher radioresistance.
Mounted above the Photo-II fluorimeter was the dosimeter Liulin-Photo, a device able to monitor in real time the variations in the space radiation flux and dose. The coupled set of instruments Photo-II/Liulin-Photo made possible to check during all the mission the response of the samples to the effects of the ionizing radiations, characterizing at the same time the radiation field which induced these effects.