T. E. Krendeleva

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.

Hydrogen photoproduction in green algae Chlamydomonas reinhardtii under magnesium deprivation

The effect of Mg-deprivation on green algae Chlamydomonas reinhardtii was studied. It resulted in the decrease of photosynthetic activity, increased respiration and accumulation of starch. After 35 hours anaerobic conditions were established and sustained H2 evolution (>7 days) was detected.

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.

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.

Antimycin A effect on the electron transport in chloroplasts of two Chlamydomonas reinhardtii strains

The effects of antimycin A on the redox state of plastoquinone and on electron donation to photosystem I (PS I) were studied in sulfur-deprived Chlamydomonas reinhardtii cells of the strains cc406 and 137c. We found that this reagent suppresses cyclic electron flow around PS I in the cc406 strain, whereas this inhibitory effect was completely absent in the 137c strain. In the latter strain, antimycin A induced rapid reduction of plastoquinone in the dark and considerably enhanced the rate of electron donation to P700+ in the dark. Importantly, neither myxothiazol, an inhibitor of mitochondrial respiration, FCCP, a protonophore, nor propyl gallate, an inhibitor of the plastid terminal oxidase, induced such a strong effect like antimycin A. The results indicate that in the chloroplast of the 137c strain, antimycin A has a site of action outside of the machinery of cyclic electron flow.

Performance index in assessing the physiological state of trees in urban ecosystems

Based on PAM and PEA measurements of fluorescence of bark chloroplasts, we have compared the information capacity of the methods for assessing the physiological state of Tilia cordata Mill. by the maximal quantum efficiency of PS II photochemistry (Fv/Fm) and by the performance index (PI). The measurements were made on annual shoots of linden trees growing in different environs. It was shown that the chlorophyll content in the bark of shoots growing near a busy urban street was twice less than in controls growing out of town. For the trees in the unfavorable environment, a small decrease in (Fv/Fm) was registered, and there was a significant statistical scatter in these values as compared with controls. The PI and its constituent parameters calculated from fluorescence induction curves (PEA method) are more informative and allow recognizing changes in the primary energy conversion processes in PS II when they are still small. Thus, PI can be used as a sensitive, robust, and rapid test to evaluate the physiological state of trees and other plant objects even under minor environmental changes.

Assessment of the effects of methylmercury and copper ions on primary processes of photosynthesis in green microalga Chlamydomonas moewusii by analysis of the kinetic curves of variable chlorophyll fluorescence

The effect of methylmercury and copper ions on the kinetics of light induction and dark relaxation of the variable fluorescence of chlorophyll a has been studied on cultures of unicellular alga Chlamydomonas moewusii. Methylmercury was effective at much lower levels. The toxicants at concentrations that did not decrease the photochemical activity of PS II (Fv/Fm) did affect the electron transport on the acceptor side of PS II, nonphotochemical quenching of excitation in the antenna, and reoxidation of the quinone pool. Our results indicate that this approach can be used for detecting the changes in plant cells at the early stages of toxicant action.

Examination of chlorophyll fluorescence in sulfur-deprived cells of Chlamydomonas reinhardtii

Pulse amplitude modulation fluorimetry was used to assess chlorophyll fluorescence parameters in Chlamydomonas reinhardtii cells during sulfur deprivation. A significant (fourfold) increase in the chlorophyll fluorescence yield (parameters F0 and Fm) normalized to the chlorophyll concentration was shown for deprived cells. The chlorophyll content did not change during the deprivation experiments. An analysis of nonphotochemical quenching of chlorophyll fluorescence indicated a considerable modification of the energy deactivation pathways in photosystem II (PSII) of sulfur-deprived cells. For example, starved cells exhibited a less pronounced pH-dependent quenching of excited states and a higher thermal dissipation of excess light energy in the reaction centers of PSII. It was also shown that the photosynthetic apparatus of starved cells is primarily in state 2 and that back transition to state 1 is suppressed. However, these changes cannot cause the discovered elevation of chlorophyll fluorescence intensity (F0 and Fm) in the cells under sulfur limitation. The observed increase in the chlorophyll fluorescence intensity under sulfur deprivation may be due to partial dissociation of peripheral light-harvesting complexes from the reaction centers of PSII or a malfunction of the dissipative cycle in PSII, involving cytochrome b559.