Molecular Bases of Signaling Processes Regulated by Cryptochrome Sensory Photoreceptors in Plants.

The blue-light sensors, cryptochromes, compose the extensive class of flavoprotein photoreceptors, regulating signaling processes in plants underlying their development, growth, and metabolism. In several algae, cryptochromes may act not only as sensory photoreceptors but also as photolyases, catalyzing repair of the UV-induced DNA lesions. Cryptochromes bind FAD as the chromophore at the photolyase homologous region (PHR) domain and contain the cryptochrome C-terminal extension (CCE), which is absent in photolyases. Photosensory process in cryptochrome is initiated by photochemical chromophore conversions, including formation of the FAD redox forms. In the state with the chromophore reduced to neutral radical (FADH×), the photoreceptor protein undergoes phosphorylation, conformational changes, and disengagement from the PHR domain and CCE with subsequent formation of oligomers of cryptochrome molecules. Photooligomerization is a structural basis of the functional activities of cryptochromes, since it ensures formation of their complexes with a variety of signaling proteins, including transcriptional factors and regulators of transcription. Interactions in such complexes change the protein signaling activities, leading to regulation of gene expression and plant photomorphogenesis. In recent years, multiple papers, reporting novel, more detailed information about the molecular mechanisms of above-mentioned processes were published. The present review mainly focuses on analysis of the data contained in these publications, particularly regarding structural aspects of the cryptochrome transitions into photoactivated states and regulatory signaling processes mediated by the cryptochrome photoreceptors in plants.

Vibrational Spectroscopy as a Tool for Bioanalytical and Biomonitoring Studies.

The review briefly describes various types of infrared (IR) and Raman spectroscopy methods. At the beginning of the review, the basic concepts of biological methods of environmental monitoring, namely bioanalytical and biomonitoring methods, are briefly considered. The main part of the review describes the basic principles and concepts of vibration spectroscopy and microspectrophotometry, in particular IR spectroscopy, mid- and near-IR spectroscopy, IR microspectroscopy, Raman spectroscopy, resonance Raman spectroscopy, Surface-enhanced Raman spectroscopy, and Raman microscopy. Examples of the use of various methods of vibration spectroscopy for the study of biological samples, especially in the context of environmental monitoring, are given. Based on the described results, the authors conclude that the near-IR spectroscopy-based methods are the most convenient for environmental studies, and the relevance of the use of IR and Raman spectroscopy in environmental monitoring will increase with time.

Engineered Polystyrene-Based Microplastics of High Environmental Relevance.

Microplastic (MP) pollution-an emerging environmental challenge of the 21st century-refers to accumulation of environmentally weathered polymer-based particles with potential environmental and health risks. Because of technical and practical challenges when using environmental MPs for risk assessment, most available data are generated using plastic models of limited environmental relevancy (i.e., with physicochemical characteristics inherently different from those of environmental MPs). In this study, we assess the effect of dominant weathering conditions-including thermal, photo-, and mechanical degradation-on surface and bulk characteristics of polystyrene (PS)-based single-use products. Further, we augment the environmental relevance of model-enabled risk assessment through the design of engineered MPs. A set of optimized laboratory-based weathering conditions demonstrated a synergetic effect on the PS-based plastic, which was fragmented into millions of 1-3 µm MP particles in under 16 h. The physicochemical properties of these engineered MPs were compared to those of their environmental counterpart and PS microbeads often used as MP models. The engineered MPs exhibit high environmental relevance with rough and oxidized surfaces and a heterogeneous fragmented morphology. Our results suggest that this top-down synthesis protocol combining major weathering mechanisms can fabricate improved, realistic, and reproducible PS-based plastic models with high levels of control over the particles' properties. Through increased environmental relevancy, our plastic model bolsters the field of risk assessment, enabling more reliable estimations of risk associated with an emerging pollutant of global concern.

Chlorophyll fluorescence induction and relaxation system for the continuous monitoring of photosynthetic capacity in photobioreactors.

The development of high-performance photobioreactors equipped with automatic systems for non-invasive real-time monitoring of cultivation conditions and photosynthetic parameters is a challenge in algae biotechnology. Therefore, we developed a chlorophyll (Chl) fluorescence measuring system for the online recording of the light-induced fluorescence rise and the dark relaxation of the flash-induced fluorescence yield (Qa- - re-oxidation kinetics) in photobioreactors. This system provides automatic measurements in a broad range of Chl concentrations at high frequency of gas-tight sampling, and advanced data analysis. The performance of this new technique was tested on the green microalgae Chlamydomonas reinhardtii subjected to a sulfur deficiency stress and to long-term dark anaerobic conditions. More than thousand fluorescence kinetic curves were recorded and analyzed during aerobic and anaerobic stages of incubation. Lifetime and amplitude values of kinetic components were determined, and their dynamics plotted on heatmaps. Out of these data, stress-sensitive kinetic parameters were specified. This implemented apparatus can therefore be useful for the continuous real-time monitoring of algal photosynthesis in photobioreactors.

Inhibition of cyanobacterial photosynthetic activity by natural ketones.

Microbial volatiles have a significant impact on the physiological functions of prokaryotic and eukaryotic organisms. Various ketones are present in volatile mixtures produced by plants, bacteria, and fungi. Our earlier results demonstrated the inhibitory effects of soil bacteria volatiles, including ketones, on cyanobacteria. In this work, we thoroughly examined the natural ketones, 2-nonanone and 2-undecanone to determine their influence on the photosynthetic activity in Synechococcus sp. PCC 7942. We observed for the first time that the ketones strongly inhibit electron transport through PSII in cyanobacteria cells in vivo. The addition of ketones decreases the quantum yield of primary PSII photoreactions and changes the PSII chlorophyll fluorescence induction curves. There are clear indications that the ketones inhibit electron transfer from QA to QB , electron transport at the donor side of PSII. The ketones can also modify the process of energy transfer from the antenna complex to the PSII reaction center and, by this means, increase both chlorophyll fluorescence quantum yield and the chlorophyll excited state lifetime. At the highest tested concentration (5 mM) 2-nonanone also induced chlorophyll release from Synechococcus cells that strongly indicates the possible role of the ketones as detergents.

Conformational Dynamics of the Single Lipopolysaccharide O-Antigen in Solution.

The O-antigen is the most variable and highly immunogenic part of the lipopolysaccharide molecule that covers the surface of Gram-negative bacteria and makes up the first line of cellular defense. To provide insight into the details of the O-antigen arrangement on the membrane surface, we simulated its behavior in solution by molecular dynamics. We developed the energetically favorable O-antigen conformation by analyzing free-energy distributions for its disaccharide fragments. Starting from this conformation, we simulated the behavior of the O-antigen chain on long timescales. Depending on the force field and temperature, the single molecule can undergo reversible or irreversible coil-to-globule transitions. The mechanism of these transitions is related either to the rotation of the carbohydrate residues around O-glycosidic bonds or to flips of the pyranose rings. We found that the presence of rhamnose in the O-antigen chain crucially increases its conformational mobility.

Orientational ordering of carotenoids in myelin membranes resolved by polarized Raman microspectroscopy.

We study orientational ordering of membrane compounds in the myelinated nerve fiber by means of polarized Raman microspectroscopy. The theory of orientational distribution functions was adapted to live-cell measurements. The obtained orientational distribution functions of carotenoids and lipid acyl chain clearly indicated a predominantly radial-like orientation in membranes of the myelin. Two-dimensional Raman images, made under optimal polarization of incident laser beam, corroborated the proposed carotenoid orientation within the bilayer. Experimental data suggested the tilted orientation of both carotenoid polyenic and lipid acyl chains. The values of maximum tilt angles were similar, with possible implication of carotenoid-induced ordering effect on lipid acyl chains, and hence change of myelin membrane properties. This study stages carotenoids of the nerve as possible mediators of excitation and leverages underlying activity-dependent membrane reordering.

Negligible adsorption and toxicity of microplastic fibers in disinfected secondary effluents.

Wastewater treatment plants play a crucial role in controlling the transport of pollutants to the environment and often discharge persistent contaminants such as synthetic microplastic fibers (MFs) to the ecosystem. In this study, we examined the fate and toxicity of polyethylene terephthalate (PET) MFs fabricated from commercial cloth in post-disinfection secondary effluents by employing conditions that closely mimic disinfection processes applied in wastewater treatment plants. Challenging conventional assumptions, this study illustrated that oxidative treatment by chlorination and ozonation incurred no significant modification to the surface morphology of the MFs. Additionally, experimental results demonstrated that both pristine and oxidized MFs have minimal adsorption potential towards contaminants of emerging concern in both effluents and alkaline water. The limited adsorption was attributed to the inert nature of MFs and low surface area to volume ratio. Slight adsorption was observed for sotalol, sulfamethoxazole, and thiabendazole in alkaline water, where the governing adsorption interactions were suggested to be hydrogen bonding and electrostatic forces. Acute exposure experiments on human cells revealed no immediate toxicity; however, the chronic and long-term consequences of the exposure should be further investigated. Overall, despite the concern associated with MFs pollution, this work demonstrates the overall indifference of MFs in WWTP (i.e., minor effects of disinfection on MFs surface properties and limited adsorption potential toward a mix of trace organic pollutants), which does not change their acute toxicity toward living forms.

Propagation of photoinduced signals with the cytoplasmic flow along Characean internodes: evidence from changes in chloroplast fluorescence and surface pH.

Emerging evidence suggests that cytoplasmic streaming can regulate the plasma-membrane H(+) transport and photosynthetic electron flow. Microfluorometric and surface pH measurements on Chara corallina internodes revealed the transmission of photoinduced signals by the cytoplasmic flow for a distance of few millimeters from the site of stimulus application. When a 30-s pulse of bright light was locally applied, the downstream cell regions responded with either release or enhancement of non-photochemical quenching of chlorophyll fluorescence, depending on the background irradiance of the analyzed cell area. Under dim background irradiance (<20 µmol m(-2) s(-1)), the arrival of the distant signal from the brightly illuminated 400-µm-wide zone elevated the maximal fluorescence F m (') in the analyzed downstream area, whereas at higher background irradiances it induced strong quenching of F m (') . At intermediate irradiances the increase and decrease in F m (') appeared as two successive waves. The transition between the F m (') responses of opposite polarities occurred at a narrow threshold range of irradiances. This indicates that inevitable slight variations in irradiance at the bottom chloroplast layer combined with the cyclosis-transmitted signals may contribute to the formation of a photosynthetic activity pattern. The rapid cyclosis-mediated release of non-photochemical quenching, unlike the delayed response of opposite polarity, was associated with opening of H(+) (OH(-))-conducting plasma membrane channels, as evidenced by the concurrent alkaline pH shift on the cell surface. It is proposed that the initial increase in F m (') after application of a distant photostimulus is determined, among other factors, by the wave of alkaline cytoplasmic pH.

Risk assessment framework for microplastic in marine environments.

Constantly raising microplastic (MP) contamination of water sources poses a direct threat to the gentle balance of the marine environment. This study focuses on a multifactor hazard evaluation of conventional (polyethylene - PE, polypropylene - PP, and polystyrene - PS) and alternative (polyethylene terephthalate with 25 % or 50 % recycled material and polylactic acid) plastics. The risk assessment framework explored included MP abundance, water acidification potential, surface oxidation, fragmentation, and bacterial growth inhibition. Based on MP monitoring campaigns worldwide, we conclude that PE-based plastics are the most abundant MPs in water samples (comprise up to 82 % the MP in those samples). A year-long weathering experiment showed that PS-based and PP-based plastics were oxidized to a higher extent, resulting in the highest water acidification with pH reduction of up to three orders of magnitude. Finally, our laboratory experiments showed that weathered PS was the most fragile plastic during mechanical degradation, while both PP- and PS-based plastic extracts showed a significant growth inhibition toward the marine microorganisms (Bacillus sp. and Pseudoaltermonas sp). Using the examined factors as weighted inputs into our framework, this holistic evaluation of hazards suggest that PP-based plastic products were the most hazardous compared to the other conventional and alternative plastic types.

VII Congress of Russian Biophysicists-2023, Krasnodar, Russia.

This special issue of Biophysical Reviews contains the materials presented at the VII Congress of Biophysicists of Russia, held from 17 to 23 April in Krasnodar. We believe that we have managed to prepare a selection of articles that well reflects the current state of biophysical science in Russia and its place in the world science. The VII Russian Congress on Biophysics was held in Krasnodar in April 2023, continuing the tradition of the series of biophysics conferences held every 4 years. The congress discussed physical principles and mechanisms of biological processes occurring at different life levels-from molecular to cellular and population levels. The results of fundamental and applied research in molecular biophysics, cell biophysics, and biophysics of complex systems were presented at plenary, sectional, and poster sessions. The works in the field of medical biophysics and neurobiology were especially widely presented. The structure and dynamics of biopolymers and fundamental mechanisms underlying the effects of physicochemical factors on biological systems, membrane, and transport processes were actively discussed. Much attention was paid to new experimental methods of biophysical research, methods of bioinformatics, computer, and mathematical modeling as necessary tools of the research at all levels of living systems. Along with fundamental problems of studying biophysical mechanisms of regulation of processes at the molecular, subcellular, and cellular levels, much attention was paid to applied research in the field of biotechnology and environmental monitoring. The Congress has formed the National Committee of Russian biophysicists.

SERS uncovers the link between conformation of cytochrome c heme and mitochondrial membrane potential.

The balance between the mitochondrial respiratory chain activity and the cell's needs in ATP ensures optimal cellular function. Cytochrome c is an essential component of the electron transport chain (ETC), which regulates ETC activity, oxygen consumption, ATP synthesis and can initiate apoptosis. The impact of conformational changes in cytochrome c on its function is not understood for the lack of access to these changes in intact mitochondria. We have developed a novel sensor that uses unique properties of label-free surface-enhanced Raman spectroscopy (SERS) to identify conformational changes in heme of cytochrome c and to elucidate their role in functioning mitochondria. We have verified that molecule bond vibrations assessed by SERS are a reliable indicator of the heme conformation during changes in the inner mitochondrial membrane potential and ETC activity. We have demonstrated that cytochrome c heme reversibly switches between planar and ruffled conformations in response to the inner mitochondrial membrane potential (ΔΨ) and H+ concentration in the intermembrane space. This regulates the efficiency of the mitochondrial respiratory chain, thus, adjusting the mitochondrial respiration to the cell's consumption of ATP and the overall activity. We have found that under hypertensive conditions cytochrome c heme loses its sensitivity to ΔΨ that can affect the regulation of ETC activity. The ability of the proposed SERS-based sensor to track mitochondrial function opens broad perspectives in cell bioenergetics.

Interactions of microplastics and organic compounds in aquatic environments: A case study of augmented joint toxicity.

High levels of persistent contaminants such as microplastics (MPs) and trace organic compounds (TrOCs) in the aquatic environment have become a major threat on the ecosystem and human health. While MP's role as a vector of environmental TrOCs is widely discussed in the literature, the corresponding implications of the interaction between these two compounds on human health (i.e., their joint toxic effect) have not been illustrated. Using a TrOCs model (Triclosan, TCS) and primary MPs (polystyrene microbeads), this work evaluates the sorption and desorption potential of TCS and MPs in simulated environmental and cellular conditions, respectively, and estimates the single and joint toxicity of these interactions toward human cells (Caco-2). Surface functionality of the microbeads highly increased their adsorption capacity of TCS, from 2.3 mg TCS for non-functionalized microbeads to 4.6 mg and 6.1 mg TCS per gram of microbeads for amino- and carboxyl-functionalized MPs, respectively. Using non-functionalized MPs, non-specific "hydrophobic-like" interactions and π-π interactions dominated the sorption mechanism of TCS; however, the addition of hydrogen interactions between functionalized microbeads and TCS increased the microbeads' overall sorption capacity. TCS was desorbed from both functionalized and non-functionalized MPs when changing from environmental conditions to cellular conditions. Desorption was found to be dependent on the matrix complexity and protein content as well as microbead functionality. Finally, toxicity tests suggested that while low concentrations of TCS and MPs (separately) have minor toxic effect toward Caco-2 cells, TCS-sorbed MPs at similar concentrations have an order of magnitude higher toxicity than pristine MPs, potentially associated with the close interaction of both MP and TCS with the cells. Overall, this study not only elucidates the role of MPs as a TrOC vector, but also demonstrates a realistic scenario in which co-presence of these environmental contaminants poses risks to the environment and human health.

Mediterranean microplastic contamination: Israel's coastline contributions.

This study provides an analysis of the current state of microplastic (MP) contamination along the Mediterranean coastline of Israel. Six strategic sites were monitored in this study - each representing a unique coastal environment. We conclude that Tel Aviv and Hadera, both located near stream estuaries, were highly contaminated (18,777 particles/m3) with MP compared to the other locations. The MP detected included both secondary MP and pristine polymeric pellets. In-depth characterization of the MP illustrated a large percentage of both fragmented and film MP morphologies and the most common MP polymers were polyethylene and polypropylene. Further particle analysis showed that MPs were contaminated with biofilm, including microorganisms such as diatoms, as well as metal residues. Through the spatial analysis presented herein we suggest that local rivers are significant contributors to MP contamination along the Mediterranean Sea coastline of Israel and may pose a direct threat to environment and human health.

Slow release of copper from jellyfish-based hydrogels for soil enrichment.

Nanotechnology has shown great potential to increase global food production and enhance food security. However, large-scale application of nano-enabled plant agriculture necessitates careful adjustments in design to overcome barriers associated with targeted nanomaterial delivery and their safety concerns. The research herein proposes the delivery of copper (Cu) from immobilized and non-immobilized copper oxide nanoparticles (Cu2O), an active nanomaterial with antifungal and micro-nutrient properties. A benign and biodegradable jellyfish-based hydrogel was used as a platform during Cu2O delivery to soils. The delivery kinetics and Cu dissolution from the nanocomposite were compared to those obtained with crosslinked ionic Cu in hydrogel, which was found to be a less controlled composite. In addition, changing environmental conditions from DI to soil extracts resulted in a decrease in the Cu dissolution rate (from 0.025 to 0.015 h-1) and an increase in the overall normalized Cu release (0.27 to 0.76 mg g-1). Use of hydrogels from natural sources allowed biodegradability over several months, adding nutrients (in the form of elements such as sulfur, nitrogen, and carbon) back to the environment, which ultimately minimizes nanomaterial required for a given desired nanomaterial yield and enhances the overall performance. Altogether, this work demonstrates the potential of Cu2O embedded hydrogels as a benign composite for Cu slow-release and therefore bolsters the field of nano-enabled plant agriculture and supports its safe deployment at large scales.

An open call for contributions to a special issue of Biophysical Reviews highlighting the research themes of VII Congress of Russian Biophysicists 2023.

We announce a call for contributions to a Special Issue of Biophysical Reviews associated with the VII Congress of Russian Biophysicists (to be held in Krasnodar, Russia, 17-23 April 2023). The Congress is the main biophysical meeting held within Russia and is organized every four years. The Congress will focus on both the physical principles and mechanisms of biological processes occurring at different levels of structural organization, from molecular to cellular to organism and to population levels. The Special Issue will accept reviews on topics from molecular biophysics, structure and dynamics of biopolymers, biophysics of the cell, energy transformation mechanisms, biophotonics, ecological biophysics, and medical biophysics, following the sections of the Congress. The VII Congress of Russian Biophysicists is supported by International Union of Pure and Applied Biophysics (IUPAB). Here we describe main topics and sections of the coming event, the paper types for the journal issue, and the key deadline dates.

Molecular, Brownian, kinetic and stochastic models of the processes in photosynthetic membrane of green plants and microalgae.

The paper presents the results of recent work at the Department of Biophysics of the Biological Faculty, Lomonosov Moscow State University on the kinetic and multiparticle modeling of processes in the photosynthetic membrane. The detailed kinetic models and the rule-based kinetic Monte Carlo models allow to reproduce the fluorescence induction curves and redox transformations of the photoactive pigment P700 in the time range from 100 ns to dozens of seconds and make it possible to reveal the role of individual carriers in their formation for different types of photosynthetic organisms under different illumination regimes, in the presence of inhibitors, under stress conditions. The fitting of the model curves to the experimental data quantifies the reaction rate constants that cannot be directly measured experimentally, including the non-radiative thermal relaxation reactions. We use the direct multiparticle models to explicitly describe the interactions of mobile photosynthetic carrier proteins with multienzyme complexes both in solution and in the biomembrane interior. An analysis of these models reveals the role of diffusion and electrostatic factors in the regulation of electron transport, the influence of ionic strength and pH of the cellular environment on the rate of electron transport reactions between carrier proteins. To describe the conformational intramolecular processes of formation of the final complex, in which the actual electron transfer occurs, we use the methods of molecular dynamics. The results obtained using kinetic and molecular models supplement our knowledge of the mechanisms of organization of the photosynthetic electron transport processes at the cellular and molecular levels.

Questioning the suitability of available microplastics models for risk assessment - A critical review.

The rise of microplastic (MP) pollution in the environment has been bolstering concerns regarding MPs' unknown environmental fate, transport, and potential toxicity toward living forms. However, the use of real environmental plastics for risk assessment is often hindered due to technical and practical challenges such as plastics' heterogeneity and their wide size distribution in the environment. To overcome this issue, most available data in the field is generated using plastic models as surrogates for environmental samples. In this critical review, we describe the gaps in risk assessments drawn from these plastic models. Specifically, we compare physicochemical properties of real environmental plastic particles to synthesized polymeric micro-beads, one of the most commonly used plastic models in current literature. Several surface and bulk characteristics including size, surface chemistry, polymer type, and morphology are shown to not only be inherently different between environmental MP's and synthesized micro-beads, but also drive behavior in fate, transport, and toxicity assays. We highlight the importance of expressing real-world physicochemical characteristics in representative MP models and outline how current state-of-the-art models are limited in this regard. To address this issue, we suggest future areas of research such as combinations of mechanical, photochemical, and thermal degradation processes to simulate real-world weathering, all in an effort to increase realism of plastic modeling and allow more robust and reliable environmental MP risk assessment in the future.

High-Voltage, Pulsed Electric Fields Eliminate Pseudomonas aeruginosa Stable Infection in a Mouse Burn Model.

Objective: The incidence of severe infectious complications after burn injury increases mortality by 40%. However, traditional approaches for managing burn infections are not always effective. High-voltage, pulsed electric field (PEF) treatment shortly after a burn injury has demonstrated an antimicrobial effect in vivo; however, the working parameters and long-term effects of PEF treatment have not yet been investigated. Approach: Nine sets of PEF parameters were investigated to optimize the applied voltage, pulse duration, and frequency or pulse repetition for disinfection of Pseudomonas aeruginosa infection in a stable mouse burn wound model. The bacterial load after PEF administration was monitored for 3 days through bioluminescence imaging. Histological assessments and inflammation response analyses were performed at 1 and 24 h after the therapy. Results: Among all tested PEF parameters, the best disinfection efficacy of P. aeruginosa infection was achieved with a combination of 500 V, 100 µs, and 200 pulses delivered at 3 Hz through two plate electrodes positioned 1 mm apart for up to 3 days after the injury. Histological examinations revealed fewer inflammatory signs in PEF-treated wounds compared with untreated infected burns. Moreover, the expression levels of multiple inflammatory-related cytokines (interleukin [IL]-1α/ß, IL-6, IL-10, leukemia inhibitory factor [LIF], and tumor necrosis factor-alpha [TNF-α]), chemokines (macrophage inflammatory protein [MIP]-1α/ß and monocyte chemoattractant protein-1 [MCP-1]), and inflammation-related factors (vascular endothelial growth factor [VEGF], macrophage colony-stimulating factor [M-CSF], and granulocyte-macrophage colony-stimulating factor [G-CSF]) were significantly decreased in the infected burn wound after PEF treatment. Innovation: We showed that PEF treatment on infected wounds reduces the P. aeruginosa load and modulates inflammatory responses. Conclusion: The data presented in this study suggest that PEF treatment is a potent candidate for antimicrobial therapy for P. aeruginosa burn infections.

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 microE m(-2) s(-1)). We failed to establish sustained hydrogen production from cultures pre-grown under high light (100 microE 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 microE 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 microE m(-2) s(-1) and incubated under 150 microE m(-2) s(-1) during sulfur deprivation. In contrast, the potential PSII activity decreased in cultures pre-grown under 100 microE m(-2) s(-1) and incubated under 420 microE 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 O(2) 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 microE 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 H(2). 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.