Taras K. Antal

Production of H2 by sulphur-deprived cells of the unicellular cyanobacteria Gloeocapsa alpicola and Synechocystis sp. PCC 6803 during dark incubation with methane or at various extracellular pH.

Aims:  To examine sulphur (S) deprivation in combination with the presence of methane (CH4) and changes in extracellular pH as a method to enhance in situ hydrogen (H2) generation during fermentation in the unicellular non‐diazotrophic cyanobacteria Gloeocapsa alpicola and Synechocystis PCC 6803.

Acclimation of green algae to sulfur deficiency: underlying mechanisms and application for hydrogen production.

Hydrogen is definitely one of the most acceptable fuels in the future. Some photosynthetic microorganisms, such as green algae and cyanobacteria, can produce hydrogen gas from water by using solar energy. In green algae, hydrogen evolution is coupled to the photosynthetic electron transport in thylakoid membranes via reaction catalyzed by the specific enzyme, (FeFe)-hydrogenase. However, this enzyme is highly sensitive to oxygen and can be quickly inhibited when water splitting is active. A problem of incompatibility between the water splitting and hydrogenase reaction can be overcome by depletion of algal cells of sulfur which is essential element for life. In this review the mechanisms underlying sustained hydrogen photoproduction in sulfur deprived C. reinhardtii and the recent achievements in studying of this process are discussed. The attention is focused on the biophysical and physiological aspects of photosynthetic response to sulfur deficiency in green algae.

Hydrogen production by photoautotrophic sulfur‐deprived Chlamydomonas reinhardtii pre‐grown and incubated under high light

We have previously demonstrated that Chlamydomonas reinhardtii can produce hydrogen under strictly photoautotrophic conditions during sulfur deprivation [Tsygankov et al. (2006); Int J Hydrogen Energy 3:1574–1584]. The maximum hydrogen photoproduction was achieved by photoautotrophic cultures pre‐grown under a low light regime (25 µE m−2 s−1). We failed to establish sustained hydrogen production from cultures pre‐grown under high light (100 µE m−2 s−1). A new approach for sustained hydrogen production by these cultures is presented here. Assuming that stable and reproducible transition to anerobiosis as well as high starch accumulation are important for hydrogen production, the influence of light intensity and dissolved oxygen concentration during the oxygen evolving stage of sulfur deprivation were investigated in cultures pre‐grown under high light. Results showed that light higher than 175 µE m−2 s−1 during sulfur deprivation induced reproducible transition to anerobiosis, although the total amount of starch accumulation and hydrogen production were insignificant. The potential PSII activity measured in the presence of an artificial electron acceptor (DCBQ) and an inhibitor of electron transport (DBMIB) did not change in cultures pre‐grown under 20 µE m−2 s−1 and incubated under 150 µE m−2 s−1 during sulfur deprivation. In contrast, the potential PSII activity decreased in cultures pre‐grown under 100 µE m−2 s−1 and incubated under 420 µE m−2 s−1. This indicates that cultures grown under higher light experience irreversible inhibition of PSII in addition to reversible down regulation. High dissolved O2 content during the oxygen evolving stage of sulfur deprivation has a negative regulatory role on PSII activity. To increase hydrogen production by C. reinhardtii pre‐grown under 100 µE m−2 s−1, cultures were incubated under elevated PFD and decreased oxygen pressure during the oxygen evolving stage. These cultures reproducibly reached anaerobic stage, accumulated significant quantities of starch and produced significant quantities of H2. It was found that elevation of pH from 7.4 to 7.7 during the oxygen producing stage of sulfur deprivation led to a significant increase of accumulated starch. Thus, control of pH during sulfur deprivation is a possible way to further optimize hydrogen production by photoautotrophic cultures. Biotechnol. Bioeng. 2009;102: 1055–1061.

Oxidative stress and photoinhibition can be separated in the cyanobacterium Synechocystis sp. PCC 6803

Roles of oxidative stress and photoinhibition in high light acclimation were studied using a regulatory mutant of the cyanobacterium Synechocystis sp. PCC 6803. The mutant strain ΔsigCDE contains the stress responsive SigB as the only functional group 2 σ factor. The ∆sigCDE strain grew more slowly than the control strain in methyl-viologen-induced oxidative stress. Furthermore, a fluorescence dye detecting H2O2, hydroxyl and peroxyl radicals and peroxynitrite, produced a stronger signal in ∆sigCDE than in the control strain, and immunological detection of carbonylated residues showed more protein oxidation in ∆sigCDE than in the control strain. These results indicate that ∆sigCDE suffers from oxidative stress in standard conditions. The oxidative stress may be explained by the findings that ∆sigCDE had a low content of glutathione and low amount of Flv3 protein functioning in the Mehler-like reaction. Although ∆sigCDE suffers from oxidative stress, up-regulation of photoprotective carotenoids and Flv4, Sll2018, Flv2 proteins protected PSII against light induced damage by quenching singlet oxygen more efficiently in ∆sigCDE than in the control strain in visible and in UV-A/B light. However, in UV-C light singlet oxygen is not produced and PSII damage occurred similarly in the ∆sigCDE and control strains. According to our results, resistance against the light-induced damage of PSII alone does not lead to high light tolerance of the cells, but in addition efficient protection against oxidative stress would be required.

Photosynthesis-related quantities for education and modeling

A quantitative understanding of the photosynthetic machinery depends largely on quantities, such as concentrations, sizes, absorption wavelengths, redox potentials, and rate constants. The present contribution is a collection of numbers and quantities related mainly to photosynthesis in higher plants. All numbers are taken directly from a literature or database source and the corresponding reference is provided. The numerical values, presented in this paper, provide ranges of values, obtained in specific experiments for specific organisms. However, the presented numbers can be useful for understanding the principles of structure and function of photosynthetic machinery and for guidance of future research.

The Relationship between the Photosystem 2 Activity and Hydrogen Production in Sulfur Deprived Chlamydomonas reinhardtiiCells

Previous studies showed that, after 1.5–2 days of adaptation to inorganic S deprivation, the unicellular alga C. reinhardtii is capable of maintaining an intense hydrogen production for several days under actinic light illumination [3, 4]. As was demonstrated earlier, incubation of cells in S-deprived medium during the first 24 h results in a progressive reduction of the photosynthetic rate due to reversible inactivation of PS2 by 85–90% [5]. When the rate of photosynthetic production of O 2 declines below the rate of cell respiration, the cell culture turns into an anaerobic state followed by the onset of H 2 production [3]. However, the actual mechanisms responsible for the change in the PS2 activity during the cell’s transition into the anaerobic state and the role of PS2 in H 2 production in S-deprived cells need to be studied in more detail.

Cultivation of Nannochloropsis for eicosapentaenoic acid production in wastewaters of pulp and paper industry

The eicosapentaenoic acid (EPA) containing marine microalga Nannochloropsis oculata was grown in an effluent from anaerobic digestion of excess activated sludge from a wastewater treatment plant serving a combination of a pulp and a paper mill and a municipality (digester effluent, DE), mixed with the effluent of the same wastewater treatment plant. The maximum specific growth rate and photosynthesis of N. oculata were similar in the DE medium and in artificial sea water medium (ASW) but after 7 days, algae grown in the DE medium contained seven times more triacylglycerols (TAGs) per cell than cells grown in ASW, indicating mild stress in the DE medium. However, the volumetric rate of EPA production was similar in the ASW and DE media. The results suggest that N. oculata could be used to produce EPA, utilizing the nutrients available after anaerobic digestion of excess activated sludge of a pulp and paper mill.

Group 2 Sigma Factor Mutant ΔsigCDE of the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Functionality of Both Carotenoids and Flavodiiron Proteins in Photoprotection of Photosystem II

Adjustment of gene expression during acclimation to stress conditions, such as bright light, in the cyanobacterium Synechocystis sp. PCC 6803 depends on four group 2 σ factors (SigB, SigC, SigD, SigE). A ΔsigCDE strain containing the stress-responsive SigB as the only functional group 2 σ factor appears twice as resistant to photoinhibition of photosystem II (PSII) as the control strain. Microarray analyses of the ΔsigCDE strain indicated that 77 genes in standard conditions and 79 genes in high light were differently expressed compared with the control strain. Analysis of possible photoprotective mechanisms revealed that high carotenoid content and up-regulation of the photoprotective flavodiiron operon flv4-sll0218-flv2 protected PSII in ΔsigCDE, while up-regulation of pgr5-like, hlipB or isiA genes in the mutant strain did not offer particular protection against photoinhibition. Photoinhibition resistance was lost if ΔsigCDE was grown in high CO2, where carotenoid and Flv4, Sll0218, and Flv2 contents were low. Additionally, photoinhibition resistance of the ΔrpoZ strain (lacking the omega subunit of RNA polymerase), with high carotenoid but low Flv4-Sll0218-Flv2 content, supported the importance of carotenoids in PSII protection. Carotenoids likely protect mainly by quenching of singlet oxygen, but efficient nonphotochemical quenching in ΔsigCDE might offer some additional protection. Comparison of photoinhibition kinetics in control, ΔsigCDE, and ΔrpoZ strains showed that protection by the flavodiiron operon was most efficient during the first minutes of high-light illumination.

Study of the effect of reducing conditions on the initial chlorophyll fluorescence rise in the green microalgae Chlamydomonas reinhardtii

Incubation of Chlamydomonas reinhardtii cells under nutrient deficiency results in the faster initial rise in the light-induced chlorophyll fluorescence kinetic curve. We showed that short-term anaerobic incubation of algal cells altered initial fluorescence in a way similar to nutrient starvation, suggesting an important role of the plastoquinones redox state in the observed effect. Bi-component analysis of highly resolved initial fluorescence rise kinetics in sulfur- or oxygen-depleted C. reinhardtii cells suggested that one of the mechanisms underlying the observed phenomenon involves primary closure (photochemical inactivation via Qa reduction) of β-type PSII as compared to α-PSII. Moreover, results of modeling of the fluorescence curve brought us to the conclusion that accumulation of closed centers in α-PSII supercomplexes may also cause a faster initial fluorescence rise. The observed correlations between nutrient supply rate and initial fluorescence rise pattern in green algae can serve to characterize culture nutritional status in vivo.

Multiple regulatory mechanisms in the chloroplast of green algae: relation to hydrogen production

A complex regulatory network in the chloroplast of green algae provides an efficient tool for maintenance of energy and redox balance in the cell under aerobic and anaerobic conditions. In this review, we discuss the structural and functional organizations of electron transport pathways in the chloroplast, and regulation of photosynthesis in the green microalga Chlamydomonas reinhardtii. The focus is on the regulatory mechanisms induced in response to nutrient deficiency stress and anoxia and especially on the role of a hydrogenase-mediated reaction in adaptation to highly reducing conditions and ATP deficiency in the cell.