Study of the long term and short term photoinhibitory processes to improve the cyanobacteria growth in photobioreactors

This Ph.D. thesis deals with the study of the photoinhibition process in the model cyanobacterium Synechosystis sp. PCC 6803 grown within the commercial flat panel photobioreactor (PBR) FTM 150/1000 under increasing intensities of orange-red light, showing the effects of photodamage in response to long-term and short-term light treatments. The photoinhibition occurs when a high light intensity inhibits the activity of Photosystem II (PSII). This process was studied both in vivo and in vitro systems, aiming to: (i) optimize the growth conditions of Synechocystis in PBR; (ii) better understand the effects of long- (24 h) and short-term (1 h) treatments with increasing intensities of orange-red light on the main protein complexes of the thylakoid membranes in Synechocystis with particular focus on PSII, that is the main target of the photoinhibition process. The photoinhibition under long-term treatment was investigated growing Synechocystis within the FMT150/1000 PBR in semicontinous mode under orange-red light. The following light intensities were tested: 50, 200, 300, 500, 800, 950 and 1460 μmol photons m-2 s-1, adapting cells for 24 h at each intensity. The intensity of 50 μmol photons m-2 s-1 was taken as control light. It was found that for Synechocystis, the saturating orange-red light intensity ranged between 300-500 μmol photons m-2 s-1, and a maximum growth rate was observed at 300 μmol photons m-2 s-1. Symptoms of photoinhibition started to appear at 800 μmol photons m-2 s-1 and reached an apex at 1460 μmol photons m-2 s-1. Upon reverting the light intensity to 200 μmol photons m-2 s-1, Synechocystis showed a remarkable ability to recover from the state of photoinhibition. In order to evaluate the PSII activity of Synechocystis during the long-term treatment, the amount of oxygen evolved by PSII and dissolved in the medium was measured. The highest PSII activity was observed in the range of light intensity between 300 and 500 μmol photons m-2 s-1. Increasing the light intensity to 800 μmol photons m-2 s-1 the amount of oxygen dissolved in the medium decreased, reaching its minimum value at 1460 μmol photons m-2 s-1. When the light was reverted to 200 μmol photons m-2s-1, Synechocystis showed a considerable recover of its PSII activity. In addition, the experimental data of oxygen dissolved in the medium and growth rate were used to generate a model of the PBR by COMSOL 5.3® platform, in order to study the influence of the different light distribution inside the PBR upon variation of the incident light furnished to the system. The long-term photoinhibition was also studied by performing a quantitative proteomic analysis of the changes occurring to the thylakoid proteome of Synechocystis, comparing thylakoids extracted from cells grown under photoinhibitory light intensities of 800, 950 and 1460 μmol photons m-2 s-1 with the control sample grown at 50 μmol photons m-2 s-1. This study revealed several potential strategies adopted by Synechocystis to cope with photoinhibitory growth irradiances among which: 1) a lower number of phycobilisome antennae and an accumulation of the orange carotenoid protein to prevent the excess of light absorption and reduce the oxidative stress from the oxygen reactive species; 2) the accumulation of the PSII repair machinery and of thylakoid-associated ribosomes to increase the turnover of the photodamaged PSII core subunits; 3) an increased cyclic electron flow capacity around PSI to either counterbalance the reduced linear electron flow capacity or sustain the accumulation of supplementary ATP; 4) a differential response of PSI components to sustain cells performance in high light. Preliminary studies of the short-term photoinhibition were conducted to investigate the effect of the strong light irradiation on the integrity of the PSII core. Taking into account the high conservation among the oxygenic photosynthetic organisms of the PSII core D1 protein, which is the main target of photoinhibition, this study was firstly conducted in vitro on PSII core monomers isolated from pea plants. The protein profile of the starting thylakoids used to isolate the PSII core monomers was assessed by SDS-PAGE and compared with the protein composition of the light-treated PSII core monomers, using the untreated counterpart as control. Further, western blot analysis with specific antibodies against the D1 protein revealed that the photodamage occurs at the level of the D1 protein, whose amount was markedly reduced in PSII core monomers treated with high light intensity. In the photoinhibited sample the band of the D1 protein almost disappeared and some bands at lower molecular weight, corresponding to D1 fragments, were clearly detected. Because of the crucial role of the D1 protein for optimal PSII activity in Synechoscystis, the effect of the short-term photoinhibition was further studied in the cyanobacterium in vivo. For this purpose, as starting material we used Synechocystis cells grown under 50 μmol photons m-2 s-1 of orange-red light in the same PBR used for the long-term treatments. The photoinhibitory treatment was conducted at 1460 μmol photons m-2 s-1 for 1 h, either in the absence or presence of the protein synthesis inhibitor lincomycin. Pulse-amplitude modulated (PAM) fluorescence measurements were performed at intervals of ten minutes during the photo-inhibitory treatment to evaluate the quantum yield of PSII photochemistry (φII). Synechocystis growth under strong light intensity and in absence of lincomycin shown a complete inactivation of the PSII after 45 minutes (ɸPSII 00.02) with an almost total recover of the PSII photosynthetic efficiency in 1.30h when the cells were growth under light recovery regime. Conversly, in presence of the lincomycin protein synthesis inhibitor the photosynthetic efficiency of the PSII did not show any recover highlighting the key role of the D1 repair cycle to cope the photoinhibitory condition and promote a prompt reactivation of the PSII.