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

The experiment Photo-II on board Foton-M3 satellite

Photo-II and Liulin-Photo dosimeter (on the top) before the integration on the Foton-M3 satellite

The experiment Photo-II integrated in the satellite capsule, few hours before the launch

On 14th September 2007 at 13:00 (CEST), from the Cosmodrome of Baikonour (Kazakhstan), the satellite Foton-M3 was launched with its scientific payload composed of more than 40 experiments in different research fields. The capsule came back to Earth after 12 days in space along an orbit with an inclination of 63° and a mean altitude of ~280 km. Photo-II was one of the experiments: it is an automatic fluorometer placed inside the capsule for testing the tolerance to the space radiation of different simple photosynthetic organisms.

The automatic fluorometer Photo-II

In this section the instrument employed during the Foton-M3 spaceflight will be described. It is an improved, high-tech version of the automatic fluorometer which flew in 2005 on board the Foton-M2 satellite. The technical specifications here reported could be applied also to the previous version of the instrument.
Even if, conceptually, Photo-II is a multifacility suitable for space reseach on cells, proteins, DNA, etc..., for the Foton flights the device was implemented with three basic functions: 1) measuring the chlorophyll a fluorescence induction curve in photosynthetic organisms; 2) recording and storing the data in a flash memory and 3) providing the living conditions to the biological samples by means of day/night cycles produced by white light LEDs. The chlorophyll fluorescence induction, also known as fluorescence transient or Kautsky effect, observed for the first time in 1931 and named after the scientist who discovered it, is a powerful method used in photosynthesis research because it is non-invasive, very sensitive and it conveys a lot of information about the functionality of the photosynthetic apparatus. The Photo-II device implemented this method with success in the space experiments, where it monitored automatically the photosynthetic activity of 24 samples for more than 20 days, measuring hourly the fluorescence curve (also known as OJIP curve) for each sample and determining the main parameters of the curve: F0 and FM (minimal and maximal fluorescence intensity), FV/FM (where FV=FM-F0 is the variable fluorescence) and the Area above the curve.

The Photo-II fluorometer used in Foton-M3 space mission. The main components are showed and highlighted.

Photo-II in Foton-M3 features small dimensions and weight. It is composed of four identical, independent units, each of them powered by two batteries in series. Every unit is composed of two separated modules, each one made up of three optical cells where the fluorescence measurements are carried out. In each cell the measurement system is composed of four red light LEDs and of an optical fluorescence detector. Hourly, the red LEDs provide the exciting light pulse for 6 seconds (intensity peak at λ=660 nm), inducing the chlorophyll fluorescence. The average intensity of the exciting red lights is ~800 µmol m-2 s-1, as measured in the centre of the cells using a quantum radiometer.
A filter is mounted on the top of the optical detector that allows the high-pass transmission of the fluorescence light with wavelengths λ>690 nm. The fluorescence measurements are then digitized and recorded in a serial flash memory. More than 500 measures can be stored, that means one measure per hour for 21 days. Before starting each measurement session, the samples are dark adapted for 15 minutes.

In each measurement cell the living conditions are provided by two white light LEDs, that are switched on continuously for 7 hours during a 24hr period and guarantee the photoperiod of 7/17 hours necessary for the survival of the samples. Using a dedicated software, it is possible to set up the intensity of the survival lights in each module until a maximum of 150 µmol m-2 s-1, as measured over each LED.

Photo-II is also equipped with eight cells where only survival light is provided by means of four white light LEDs, maintaining the 7/17 hours photoperiod. The intensity of the white lights can be set up via software to a maximum of 170 µmol m-2 s-1, as measured in the centre of each cell.

The material used for the containers where the biological samples are housed is Delrin and the transparent window exposed to the light is made of Polycarbonate. Delrin has been chosen because it is compatible with the biological material and the black colour guarantees the optical isolation among the cells. A gasket of silicone provides perfect sealing, thus avoiding any contamination of the biological material after deposition in the container.

A set of eight independent thermometers complete the electronics of Photo-II. They measure the temperature inside the device.

Which organisms did Photo-II monitor on board Foton flights?

Photo-II fluorometer flew in space for the first time on board Foton-M2. That device could host until 10 different samples, having less modules than the new version of the instrument used in Foton-M3. For Foton-M2 flight the choice was 3 strains of the unicellular green alga Chlamydomonas reinhardtii, site-directed mutants in the gene encoding the D1 protein, which has a central role in the photosynthetic apparatus. Each used strain had only one aminoacid changed in the D1 protein aminoacid chain, but even just one substitution implies a great modification in the photosynthetic process. Each strain had its repetition, in order to collect more data concerning the same organism and to take into account the possibility of any malfunctioning in one of the modules.

C. reinhardtii is a motile oxygenic photosynthetic organism that was chosen for the experiments because it is used in biology as a model system, being unicellular, with a quick growth and easy to be transformed with genetic engineering methods.

Photo-II on board Foton-M3 could host a larger number of samples. The same mutants employed in the previous flight were newly selected even for the 2007 mission, in order to collect more data concerning their ability to tolerate the stress induced by the space environment.
Twelve different strains of the unicellular green algae C. reinhardtii were allocated in the 24 measurement cells, so that actually the experiment was repeated twice.
Some of the the samples employed in the experiment on board Foton-M3 are site-directed mutants for the D1 protein, and each mutant is characterized by a single or double substitutions in the aminoacidic chain of the protein. Those strains were obtained in the molecular engineering laboratory of prof. Udo Johanningmeier (Martin-Luther-University, Halle, Germany).
Other strains selected for the experiment on board Foton-M3 are mutants with a particularly high content of xantophylls, that are pigments with a chemical structure which allows the cell to dissipate the energy in excess received by the light and, at the same time, to protect the organism from damages produced by free radicals which form during the process. These mutants, named npq2, lor1, npq2-lor1, together with their wild type cc125, are characterized by having a modified xanthophyll biosynthetic pathway. For example the npq2 mutant is impaired in the conversion of zeaxanthin in antheraxanthin -two of the pigments involved in the xantophyll cycle- and the npq2-lor1 is characterized by an accumulation of zeaxanthin and β-carotene pigments. Testing this strains is particularly interesting, because the ionizing radiation in space could represent a source of energy "in excess" that has to be dissipated from the organism, and a high concentration of protective pigments could counteract the harmful effect of the space stress.
In the survival cells of the fluorometer several colonies of C.reinhardtii were allocated to test their tolerance to the space radiation without performing any measurement during the flight, only providing the light/dark cycle necessary for their survival. Such colonies were represented by random mutants in the D1 protein, i.e. strains obtained using a specific mutagenesis technique generating mutations -not known a priori- in the gene encoding for the protein. Once back to Earth, these colonies were incubated in laboratory for some time, letting them growing up. By sequencing the gene encoding for the D1 protein it is possible to characterize the strains which survived after the flight, then to identify the corresponding aminoacid substitutions.

Some of the strains used in Photo-II experiments were previously selected after irradiation tests conducted in ground-based laboratories. The mutants of C. reinhardtii were exposed to γ-rays, energetic neutrons, protons and heavy ions, and the strains selected for Photo-II were the ones which demonstrated more radioresistance after the treatment.
In Photo-II on Foton-M3 the same mutants tested in Foton-M2 experiment were once again used, in order to collect more data about their ability to tolerate the stress induced by the space environment.

What does Photo-II measure?

Fluorescence curves (OJIP curves) measured by Photo-II fluorometer for three different mutants of the photosynthetic organism C. reinhardtii.

The picture shows a set of fluorescence induction curves measured in space by Photo-II for three different photosynthetic samples. The X-axis reports time (in seconds), while the Y-axis shows the fluorescence intensity in arbitrary units. Fluorescence is measured for 6 seconds, that is the duration of the excitation pulse provided by red light LEDs. Some of the parameters which identify each curve are indicated in the plot: the maximum fluorescence FM, that is directely measured, and the initial fluorescence F0, which is determined extrapolating the curves to t=0. The FV/FM parameter, derived from FM and F0, provides a measure of the photosynthetic efficiency of the organism. When it is constantly monitored for long periods of time, as it was during the Foton flights, the FV/FM parameter gives information on how and how much the photosynthetic performance is changing in response to the space stress. The figure below displays an example of temporal variation of FV/FM, showing the case of an organism which lost its photosynthetic efficiency during the permanence in orbit.
The periodicity observed in the plot is due to the 24-hrs light/dark cycle provided by Photo-II fluorimeter, because the FV/FM parameter changes depend on the illumination state (increasing in presence of light and decreasing when the samples are not illuminated).

Temporal variation of the FV/FM parameter for a C. reinhardtii strain sent in orbit with Foton-M3. The constant decreasing of the paramenter indicates that the organism was not tolerant to the stress induced by the space environment.

The Liulin-Photo spectrum-dosimeter

Liulin-Photo spectrum-dosimeter is an automatic device developed at the Solar-Terrestrial Influences Laboratory of the Bulgarian Academy of Sciences, in Sofia, Bulgaria. The main purpose of Liulin-Photo was to actively monitor in real time the doses and particle fluxes along FM3 orbit, to characterize the radiation environment inside the satellite capsule on the top of Photo-II experiment. In Foton-M3, every 60 seconds Liulin-Photo carried out one measurement of the energy deposited by each particle incident on the silicon detector. The dose and flux are automatically calculated from these data and stored in a memory card. The data were downloaded to the PC when the device was retrieved after the capsule landing. Liulin-Photo is one exemplar of a family of Liulin-type dosimeters employed for 20 years in experiments in space. They successfully flew from 1988 to 1994 on board the Mir space station and since 2001 on board the International Space Station. On 2005, such a device (named R3D-B2) monitored the space radiation environment on board Foton-M2 satellite. The figure below shows an example of what Liulin-Photo measures. The two steps for flux (blue dots) and dose rate (red dots) correspond to measurements performed using standard radioactive sources, with a sampling rate of 10 seconds. The first step is due to 60 keV gamma radiations from 241Am source, while the second one corresponds to a 137Cs source.

Radiation dose rate and flux measured by the Liulin-Photo spectrum-dosimeter during a laboratory test.