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The model green microalga Chlamydomonas reinhardtii is frequently subject to periods of dark and anoxia in its natural environment. Here, by resorting to mutants defective in the maturation of the chloroplastic oxygen-sensitive hydrogenases or in Proton-Gradient Regulation-Like1 (PGRL1)-dependent cyclic electron flow around photosystem I (PSI-CEF), we demonstrate the sequential contribution of these alternative electron flows (AEFs) in the reactivation of photosynthetic carbon fixation during a shift from dark anoxia to light. At light onset, hydrogenase activity sustains a linear electron flow from photosystem II, which is followed by a transient PSI-CEF in the wild type. By promoting ATP synthesis without net generation of photosynthetic reductants, the two AEF are critical for restoration of the capacity for carbon dioxide fixation in the light. Our data also suggest that the decrease in hydrogen evolution with time of illumination might be due to competition for reduced ferredoxins between ferredoxin-NADP(+) oxidoreductase and hydrogenases, rather than due to the sensitivity of hydrogenase activity to oxygen. Finally, the absence of the two alternative pathways in a double mutant pgrl1 hydrogenase maturation factor G-2 is detrimental for photosynthesis and growth and cannot be compensated by any other AEF or anoxic metabolic responses. This highlights the role of hydrogenase activity and PSI-CEF in the ecological success of microalgae in low-oxygen environments.

Comparative analyses of bacterial and archaeal community structures and dynamics in three biogas digesters during start-up and subsequent operation using microwaved, ultrasonicated or untreated waste activated sludge were performed based on 454 pyrosequencing datasets of part of 16S ribosomal RNA sequences and quantitative PCR. The pre-treatment increased the solubility, and thus the availability of the substrate for microbial degradation and significantly affected the succession of the anaerobic community structure over the course of the digestion. Bacteroidetes , Proteobacteria and Firmicutes were the dominant phyla in all digesters throughout operation. Proteobacteria decreased in relative abundance from 23–26 % to 11–13 % in association with enhanced substrate availability. Negative correlations between relative abundance of Alpha -, Beta - and Gammaproteobacteria and the substrate availability and/or biogas production were disclosed in statistical analyses. Clostridiales was the dominant order in Firmicutes , and Clostridiales , Clostridia and Firmicutes relative abundance and richness were shown to positively correlate with substrate availability and biogas generation. Methanogenic communities had a fairly restricted structure, highly dominated by Methanosaeta and Methanobrevibacter phylotypes. A gradual decline in Methanobrevibacter and increased representation of Methanosaeta concilii over time were particularly apparent in the digester receiving untreated waste activated sludge, whereas more diversified archaeal communities were maintained in the pre-treatment digesters. The quantitative PCR analyses revealed a methanogenic community distribution that coincided with the 454 pyrosequencing data.

The effect of substrate (glucose) concentration on the stability and yield of a continuous fermentative process that produces hydrogen was studied. Four anaerobic fluidized bed reactors (AFBRs) were operated with a hydraulic retention time (HRT) from 1 to 8 h and an influent glucose concentration from 2 to 25 g L −1 . The reactors were inoculated with thermally pre-treated anaerobic sludge and operated at a temperature of 30 °C with an influent pH around 5.5 and an effluent pH of about 3.5. The AFBRs with a HRT of 2 h and a feed strength of 2, 4, and 10 g L −1 showed satisfactory H 2 production performance, but the reactor fed with 25 g L −1 of glucose did not. The highest hydrogen yield value was obtained in the reactor with a glucose concentration of 2 g L −1 when it was operated at a HRT of 2 h. The maximum hydrogen production rate value was achieved in the reactor with a HRT of 1 h and a feed strength of 10 g L −1 . The AFBRs operated with glucose concentrations of 2 and 4 g L −1 produced greater amounts of acetic and butyric acids, while AFBRs with higher glucose concentrations produced a greater amount of solvents.

Wheat ( Triticum aestivum L.) seedlings contain four alanine aminotransferase (AlaAT) homologues. Two of them encode AlaAT enzymes, whereas two homologues act as glumate:glyoxylate aminotransferase (GGAT). To address the function of the distinct AlaAT homologues a comparative examination of the changes in transcript level together with the enzyme activity and alanine and glutamate content in wheat seedlings subjected to low oxygen availability, nitrogen and light deficiency has been studied. Shoots of wheat seedlings were more tolerant to hypoxia than the roots as judging on the basis of enzyme activity and transcript level. Hypoxia induced AlaAT1 earlier in roots than in shoots, while AlaAT2 and GGAT were unaffected. The increase in AlaAT activity lagged behind the increase in alanine content. Nitrogen deficiency has little effect on the activity of GGAT. In contrast, lower activity of AlaAT and the level of mRNA for AlaAT1 and AlaAT2 in wheat seedlings growing on a nitrogen-free medium seems to indicate that AlaAT is regulated by the availability of nitrogen. Both AlaAT and GGAT activities were present in etiolated wheat seedlings but their activity was half of that observed in light-grown seedlings. Exposure of etiolated seedlings to light caused an increase in enzyme activities and up-regulated GGAT1. It is proposed that hypoxia-induced AlaAT1 and light-induced peroxisomal GGAT1 appears to be crucial for the regulation of energy availability in plants grown under unfavourable environmental conditions. Key message In young wheat seedlings, both AlaAT and GGAT are down-regulated by nitrogen deficiency, whereas AlaAT1 is upregulated by hypoxia and GGAT1 by light.

The solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect is studied in photosynthetic reaction centers of Heliobacillus mobilis at different magnetic fields by ¹³C MAS (magic-angle spinning) NMR spectroscopy. Two active states of heliobacterial reaction centers are probed: an anaerobic preparation of heliochromatophores (“Braunstoff”, German for “brown substance”) as well as a preparation of cells after exposure to oxygen (“Grünstoff”, “green substance”). Braunstoff shows significant increase of enhanced absorptive (positive) signals toward lower magnetic fields, which is interpreted in terms of an enhanced differential relaxation (DR) mechanism. In Grünstoff, the signals remain emissive (negative) at two fields, confirming that the influence of the DR mechanism is comparably low.

Purple non‐sulfur bacteria (Rhodospirillaceae) have been extensively employed for studying principles of photosynthetic and respiratory electron transport phosphorylation and for investigating the regulation of gene expression in response to redox signals. Here, we use mathematical modeling to evaluate the steady‐state behavior of the electron transport chain (ETC) in these bacteria under different environmental conditions. Elementary‐modes analysis of a stoichiometric ETC model reveals nine operational modes. Most of them represent well‐known functional states, however, two modes constitute reverse electron flow under respiratory conditions, which has been barely considered so far. We further present and analyze a kinetic model of the ETC in which rate laws of electron transfer steps are based on redox potential differences. Our model reproduces well‐known phenomena of respiratory and photosynthetic operation of the ETC and also provides non‐intuitive predictions. As one key result, model simulations demonstrate a stronger reduction of ubiquinone when switching from high‐light to low‐light conditions. This result is parameter insensitive and supports the hypothesis that the redox state of ubiquinone is a suitable signal for controlling photosynthetic gene expression. Synopsis The attractiveness of anoxygenic non‐sulfur photosynthetic bacteria (Rhodospirillaceae) for studying gene regulation in response to cellular redox events is based on their energetic flexibility, which enables growth under a variety of environmental conditions. In the presence of oxygen, electrons are transferred through a respiratory chain comprising NADH (or FADH), ubiquinone (Q), cytochrome bc 1 , cytochrome c 2 and finally to oxygen by cytochrome oxidases or ubiquinol oxidases, thereby enabling phosphorylation of ADP via ATPase complexes. Note the functional homology to the mitochondrial respiratory chain. At low oxygen tensions, the biosynthesis of intracytoplasmic membranes (ICMs) is induced. Specific polypeptide‐bacteriochlorophyll (BChl) components of these membranes––the photosynthetic light‐harvesting complexes (LHCs) and reaction centers (RCs)––capture light energy, which drive a cyclic photophosphorylation system via RC‐Q‐cyt bc 1 ‐cyt c 2 back to RC. It has been stated as early as 1957 that the redox state of components of the respiratory and photosynthetic electron transport chain (ETC) may constitute metabolic signals that govern the expression of LHC‐ and RC‐encoding genes and therefore determine the adaptation of the cells to a given environmental condition (Cohen‐Bazire et al , 1957 ). The repression of ICM synthesis under high oxygen tension and also anaerobically by high light intensities has been studied in detail during the last years and some regulatory systems involved were identified, including the two‐component system RegA/RegB (PrrA/PrrB), the PpsR (CrtJ) repressor and the Fnr‐like transcriptional regulator FnrL (Oh and Kaplan, 2000 ; Bauer et al , 2003 ). However, particularly for light regulation of ICM synthesis, the molecular signals and mechanisms involved in the initiation of signal transduction pathways governing photosynthetic gene expression are still poorly characterized or even unidentified. The high‐light repression effect has been attributed to be dependent on the redox state of the membranous Q pool (Oh and Kaplan, 2000 ). However, to our knowledge, no direct experimental measurements of Q redox states under different light conditions are available so far. Recently, it was demonstrated that oxidized Q directly inhibits the RegB/RegA regulatory system of Rhodobacter capsulatus in vitro (Swem et al , 2006 ), thereby potentially mediating the repression of ICM synthesis also under high oxygen conditions in the dark. The lack of quantitative information about how the redox states of individual ETC components change under different growth conditions limits the verification of the proposed hypotheses of redox regulation and a pure qualitative discussion of the behavior of the ETC is infeasible as too many variables are involved. Here, we present (i) a stoichiometric model of the ETC unambiguously describing the structural and functional capabilities of the ETC of purple non‐sulfur bacteria and (ii) a kinetic model based on thermodynamic constraints of the underlying processes that enables us to simulate the steady‐state behavior of the ETC, in particular the redox states of ETC components, under different environmental conditions. Elementary‐mode analysis (see Schuster et al , 2000 ) of the ETC reveals nine fundamental modes expressing all the potential functional behaviors of the ETC in the steady state. The stoichiometric model reproduces well‐known cycles and pathways for the ETC when operating under photosynthetic (cyclic photosynthesis and reverse electron transport), respiratory (electron transfer from NADH or succinate to ubiquinol oxidase or cytochrome ( cbb 3 ) oxidase) or fermentative (fumarate reduction) conditions. Apart from these well‐known functions, the stoichiometric model revealed two operational modes representing reverse electron flow under respiratory conditions. So far, the functional role of reverse electron flow has been mainly discussed for photosynthetic growth only. In fact, due to thermodynamic constraints, reverse electron flow under aerobic conditions plays only a minor role and as shown by simulations of the kinetic model, the concentration ratios of the metabolites involved have to be at rather extreme levels, to make this state thermodynamically favorable. However, this operation might become important for certain (possibly temporary) metabolic states, for example, if a great imbalance in the current redox potentials of NADH and succinate occurs. The stoichiometric model thus identified––in an unbiased way––the meaningful functions of the ETC; however, it can only show the potential behaviors of the ETC (which are under real conditions normally superimposed). For studying the actual driving forces, electron flows and redox states occurring in the ETC under different environmental conditions, we therefore constructed a kinetic model that also includes known regulatory pathways governing the expression of ETC components. Although in photosynthetic bacteria two types of energy‐converting membranes (ICM and cytoplasmic membrane) are present, which are structurally and functionally different, we set up the model with only one membrane compartment. We justify this approach because we consider in the simulations only scenarios where the model functions either as a pure photosynthetic membrane (ICM model) (i.e. no participation of oxidases) or as a pure respiratory (cytoplasmic) membrane (no participation of photosynthetic RCs). The reaction steps taking place inside the enzyme complexes have been described with rather simple, yet thermodynamically correct kinetic laws based on the differences in the redox potentials of the participating redox couples. As boundary conditions for metabolites directly interacting with the ETC, we fix the concentration ratios of NADH/NAD, succinate/fumarate and ATP/ADP (which can, however, be varied for the different scenarios considered). To our knowledge, it is the most comprehensive model of the ETC in purple bacteria with respect to the components and processes considered. Even though we cannot claim that the model is accurate when absolute quantitative units are considered (uncertainties in parameters and only few available measurements do not permit this), it provides valuable semiquantitative and qualitative insights into the behavior of the ETC, in particular on the redox states of key components which are often difficult to measure. Important results of the steady‐state analysis of the model are shown in Figure 5 . The plots show the dependency of the redox states of the Q and cytochrome c 2 pools ( Q_charge and c2_charge ), of the proton‐motive force ( pmf ) and BChl concentration ( Bchl ; as a measure of ICM) on the cytosolic redox state (represented by NADH/NAD) for four different growth conditions: aerobic and semi‐aerobic in the dark and anaerobic under high‐light and low‐light conditions. The model is able to reproduce the observations about the ICM levels that are repressed during aerobic growth and are relieved from oxygen repression under semi‐aerobic conditions. Importantly, the level of ICM corresponds to the reduction degree of Q. That oxygen‐limiting conditions result in a more reduced Q pool compared to fully aerobic growth can be deduced in a straightforward manner from the operation of a linear respiratory chain. In contrast, the adaptation of the Q redox state when the cyclic photophosphorylation system faces different light intensities is not deducible simply by pure intuition. To our knowledge, the question whether a switch from high‐ to low‐light conditions results in a more reduced (as the physiological regulatory model demands to explain high‐light repression of ICM synthesis) or a more oxidized Q pool is currently not resolved by literature data. Strikingly, the simulation of this situation with the mathematical model yields an unambiguous result: the Q pool is reduced more under low‐light conditions, which is in accordance to its proposed role as a metabolic signal for ICM expression (Swem et al , 2006 ). It is important to point out that this finding is virtually independent of the parameter values employed, instead it is an inherent feature of the system. We could not find a parameter set where the opposite result, that is Q gets reduced more under high‐light conditions, is produced. Another conclusion that can be drawn from Figure 5 is, that (only) under photosynthetic conditions there is an optimal load of the ETC with electrons (and therefore an optimal cytosolic redox state here reflected by the ratio NADH/NAD) resulting in a maximum of Δ p . The model clearly demonstrates that the optimal load is dependent on

Chinese hamster V79 spheroids were stained with a nontoxic fluorescent stain, Hoechst 33342, which penetrates slowly into the spheroids. Single cells from these spheroids were then sorted by a fluorescence-activated cell sorter according to staining intensity (and therefore position in the spheroids). Flow cytometry characterization of the various cell subpopulations indicated that the innermost cells were more radiosensitive than expected on the basis of cell cycle position or cell thiol content. However, comparison of the radiosensitivities of cells sorted from equivalent depths from completely aerobic or anoxic V79 spheroids indicated that the oxygen enhancement ratio remained remarkably constant at 2.7 ± 0.2 through the spheroid.

Gamma-ray-irradiated medium is found to produce an immediate slowing of respiration in cells of Escherichia coli 15 ${\\rm T}^{-}{\\rm L}^{-}$. It also introduces a lag in cell division that can be shortened by the presence of methylene blue and potassium cyanide. The lag is absent if the cells are grown anaerobically. These results suggest that the peroxides produced by irradiating medium block the electron transport system. Since cells concentrate peroxides, the loss of this function induced by bombardment was used to deduce the target molecular weight of the enzyme responsible. It is 73,000.

Differences in the extrapolation number of deeply hypoxic mammalian cells in different laboratories have not yet been satisfactorily explained. If they are due to differences in the efficiency of removal of the last traces of oxygen, then the process by which oxygen is consumed by radiation to give the anoxic radiosensitivity must stop short of complete anoxia. An apparatus to demonstrate the chemical removal of oxygen by radiation at extremely low oxygen concentration has been constructed. The results have shown that in the nanomolar range oxygen is not removed. There is an increase in oxygen concentration with increasing X-ray dose.

Radiotherapy is a crucial cancer treatment, but its outcome is still far from satisfactory. One of the reasons that cancer cells show resistance to ionizing radiation is hypoxia, defined as a low level of oxygenation, which is typical for solid tumors. In the hypoxic environment, cancer cells are 2–3 times more resistant to ionizing radiation than normoxic cells. To overcome this important impediment, radiosensitizers should be introduced to cancer therapy. When modified with an electrophilic substituent, nucleosides may undergo efficient dissociative electron attachment (DEA) that leaves behind nucleoside radicals, which, in secondary reactions, are able to induce DNA damage, leading to cancer cell death. We report the radiosensitizing effect of one of the best-known DEA-type radiosensitizers—5-bromo-2′-deoxyuridine (BrdU)—on breast (MCF-7) and prostate (PC3) cancer cells under both normoxia and hypoxia. MCF-7 and PC3 cells were treated with BrdU to investigate the effect of hypoxia on cell proliferation, incorporation into DNA and radiosensitivity. While the oxygen concentration did not significantly affect the efficiency of BrdU incorporation into DNA or the proliferation of tumor cells, the radiosensitizing effect of BrdU on hypoxic cells was more evident than on normoxic cells. Further mechanistic studies performed with the use of flow cytometry showed that under hypoxia, BrdU increased the level of histone H2A.X phosphorylation after X-ray exposure to a greater extent than under normal oxygenation conditions. These results confirm that the formation of double-strand breaks in hypoxic BrdU-treated cancer cells is more efficient. In addition, by performing stationary radiolysis of BrdU solution in the presence of an ●OH radical scavenger, we compared the degree of its electron-induced degradation under aerobic and anaerobic conditions. It was determined that radiodegradation under anaerobic conditions was almost twice as high as that under aerobic conditions.