Syngas obtained by microwave pyrolysis of household wastes as feedstock for polyhydroxyalkanoate production in Rhodospirillum rubrum.

The massive production of urban and agricultural wastes has promoted a clear need for alternative processes of disposal and waste management. The potential use of municipal solid wastes (MSW) as feedstock for the production of polyhydroxyalkanoates (PHA) by a process known as syngas fermentation is considered herein as an attractive bio-economic strategy to reduce these wastes. In this work, we have evaluated the potential of Rhodospirillum rubrum as microbial cell factory for the synthesis of PHA from syngas produced by microwave pyrolysis of the MSW organic fraction from a European city (Seville). Growth rate, uptake rate, biomass yield and PHA production from syngas in R. rubrum have been analysed. The results revealed the strong robustness of this syngas fermentation where the purity of the syngas is not a critical constraint for PHA production. Microwave-induced pyrolysis is a tangible alternative to standard pyrolysis, because it can reduce cost in terms of energy and time as well as increase syngas production, providing a satisfactory PHA yield.

Fluorescence relaxation in intact cells of photosynthetic bacteria: donor and acceptor side limitations of reopening of the reaction center.

The dark relaxation of the yield of variable BChl fluorescence in the 10(-5)-10 s time range is measured after laser diode (808 nm) excitation of variable duration in intact cells of photosynthetic bacteria Rba. sphaeroides, Rsp. rubrum, and Rvx. gelatinosus under various treatments of redox agents, inhibitors, and temperature. The kinetics of the relaxation is complex and much wider extended than a monoexponential function. The longer is the excitation, the slower is the relaxation which is determined by the redox states, sizes, and accessibility of the pools of cytochrome [Formula: see text] and quinone for donor and acceptor side-limited bacterial strains, respectively. The kinetics of fluorescence decay reflects the opening kinetics of the closed RC. The relaxation is controlled preferentially by the rate of re-reduction of the oxidized dimer by mobile cytochrome [Formula: see text] in Rba. sphaeroides and Rsp. rubrum and by the rate constant of the [Formula: see text] interquinone electron transfer, (350 µs)(-1) and/or the quinol/quinone exchange at the acceptor side in Rvx. gelatinosus. The commonly used acceptor side inhibitors (e.g., terbutryn) demonstrate kinetically limited block of re-oxidation of the primary quinone. The observations are interpreted in frame of a minimum kinetic and energetic model of electron transfer reactions in bacterial RC of intact cells.

Dark excited States of carotenoid regulated by bacteriochlorophyll in photosynthetic light harvesting.

In photosynthesis, carotenoids play important roles in light harvesting (LH) and photoprotective functions, which have been described mainly in terms of two singlet excited states of carotenoids: S(1) and S(2). In addition to the "dark" S(1) state, another dark state, S*, was recently identified and its involvement in photosynthetic functions was determined. However, there is no consistent picture concerning its nature or the mechanism of its formation. One particularly anomalous behavior obtained from femtosecond transient absorption (TA) spectroscopy is that the S*/S(1) population ratio depends on the excitation intensity. Here, we focus on the effect of nearby bacteriochlorophyll (BChl) on the relaxation dynamics of carotenoid in the LH complex. We performed femtosecond TA spectroscopy combined with pre-excitation of BChl in the reconstituted LH1 complex from Rhodospirillum rubrum S1. We observed that the energy flow from S(1), including its vibrationally excited hot states, to S* occurs only when nearby BChl is excited into Q(y), resulting in an increase in S*/S(1). We also examined the excitation-intensity dependence of S*/S(1) by conventional TA spectroscopy. A comparison between the pre-excitation effect and excitation-intensity dependence shows a strong correlation of S*/S(1) with the number of BChls excited into Q(y). In addition, we observed an increase in triplet formation as the S* population increased, indicating that S* is an electronic excited state that is the precursor to triplet formation. Our findings provide an explanation for observed spectroscopic features, including the excitation-intensity dependences debated so far, and offer new insights into energy deactivation mechanisms inherent in the LH antenna.

Excitation energy pathways in the photosynthetic units of reaction center LM- and H-subunit deletion mutants of Rhodospirillum rubrum.

Light-induced reaction dynamics of isolated photosynthetic membranes obtained from wild-type (WT) and reaction center (RC)-subunit deletion strains SPUHK1 (an H-subunit deletion mutant) and SK Delta LM (an (L+M) deletion mutant) of the purple non-sulphur bacterium Rhodospirillum rubrum have been investigated by femtosecond transient absorption spectroscopy. Upon excitation of the spirilloxanthin (Spx) S(2) state at 546 nm, of the bacteriochlorophyll Soret band at 388 nm and probing spectral regions, which are characteristic for carotenoids, similar dynamics in the SPUHK1, SK Delta LM and WT strains could be observed. The excitation of Spx S(2) is followed by the simultaneous population of the lower singlet excited states S(1) and S* which decay with lifetimes of 1.4 and 5 ps, respectively for the mutants, and 1.4 and 4 ps, respectively, for the wild-type. The excitation of the BChl Soret band is followed by relaxation into BChl lower excited states which compete with excitation energy transfer BChl-to-Spx. The deexcitation pathway BChl(Soret) --> Spx(S(2)) --> Spx(S(1)) occurs with the same transition rate for all investigated samples (WT, SPUHK1 and SK Delta LM). The kinetic traces measured for the Spx S(1) --> S(N) transition display similar behaviour for all samples showing a positive signal which increases within the first 400 fs (i.e. the time needed for the excitation energy to reach the Spx S(1) excited state) and decays with a lifetime of about 1.5 ps. This suggests that the Spx excited state dynamics in the investigated complexes do not differ significantly. Moreover, a longer excited state lifetime of BChl for SPUHK1 in comparison to WT was observed, consistent with a photochemical quenching channel present in the presence of RC. For long delay times, photobleaching of the RC special pair and an electrochromic blue shift of the monomeric BChl a can be observed only for the WT but not for the mutants. The close similarity of the excited state decay processes of all strains indicates that the pigment geometry of the LH1 complex in native membranes is unaffected by the presence of an RC and allows us to draw a model representation of the WT, SK Delta LM and SPUHK1 PSU complexes.

Nitrogenase switch-off and regulation of ammonium assimilation in response to light deprivation in Rhodospirillum rubrum are influenced by the nitrogen source used during growth.

Nitrogen fixation and ammonium assimilation in Rhodospirillum rubrum are regulated in response to changes in light availability, and we show that the response in terms of glutamine synthetase activity and P(II) modification is dependent on the nitrogen source used for growth, N(2) or glutamate, although both lead to nitrogenase derepression.

Experimental design and environmental parameters affect Rhodospirillum rubrum S1H response to space flight.

In view of long-haul space exploration missions, the European Space Agency initiated the Micro-Ecological Life Support System Alternative (MELiSSA) project targeting the total recycling of organic waste produced by the astronauts into oxygen, water and food using a loop of bacterial and higher plant bioreactors. In that purpose, the alpha-proteobacterium, Rhodospirillum rubrum S1H, was sent twice to the International Space Station and was analyzed post-flight using a newly developed R. rubrum whole genome oligonucleotide microarray and high throughput gel-free proteomics with Isotope-Coded Protein Label technology. Moreover, in an effort to identify a specific response of R. rubrum S1H to space flight, simulation of microgravity and space-ionizing radiation were performed on Earth under identical culture set-up and growth conditions as encountered during the actual space journeys. Transcriptomic and proteomic data were integrated and permitted to put forward the importance of medium composition and culture set-up on the response of the bacterium to space flight-related environmental conditions. In addition, we showed for the first time that a low dose of ionizing radiation (2 mGy) can induce a significant response at the transcriptomic level, although no change in cell viability and only a few significant differentially expressed proteins were observed. From the MELiSSA perspective, we could argue the effect of microgravity to be minimized, whereas R. rubrum S1H could be more sensitive to ionizing radiation during long-term space exploration mission.

Functions of carotenoids in xanthorhodopsin and archaerhodopsin, from action spectra of photoinhibition of cell respiration.

The recent discovery of a carotenoid light-harvesting antenna in xanthorhodopsin, a retinal-based proton pump in Salinibacter ruber, made use of photoinhibition of respiration in whole cells to obtain action spectra [Balashov et al. Science 309, (2005) 2061-2064]. Here we provide further details of this phenomenon, and compare action spectra in three different systems where carotenoids have different functions or efficiencies of light-harvesting. The kinetics of light-induced inhibition of respiration in Salinibacter ruber was determined with single short flashes, and the photochemical cross section of the photoreaction was estimated. These measurements confirm that the xanthorhodopsin complex includes no more than a few, and most likely only one, carotenoid molecule, which is far less than the core complex antenna of photosynthetic bacteria. Although the total cross-section of light absorption in the purple bacterium Rhodospirillum rubrum greatly exceeds that in Salinibacter, the cross-sections are roughly equivalent in the shared wavelength range. We show further that despite interaction of bacterioruberin with archaerhodopsin, another retinal-based proton pump, there is no significant energy transfer from this carotenoid. This emphasizes the uniqueness of the salinixanthin-retinal interaction in xanthorhodopsin, and indicates that bacterioruberin in Halorubrum species has a structural or photoprotective rather than energetic role.

Tributyl phosphate degradation by Rhodopseudomonas palustris and other photosynthetic bacteria.

Tributyl phosphate (TBP) is widely used in nuclear fuel processing and other waste generating chemical industries. Although TBP is bacteriostatic, some microbes are resistant to it and may degrade it. Under dark aerobiosis, purple non-sulfur photosynthetic bacteria degraded up to 0.6 mM TBP, initially present at 2 mM, within 3 weeks and under photosynthetic conditions, Rhodopseudomonas palustris degraded 1.6 mM TBP within 3 weeks. The curing of the Rhodopseudomonas palustris endogenous plasmid demonstrated that the genes involved in the TBP degradation are chromosomal.

Modelling continuous culture of Rhodospirillum rubrum in photobioreactor under light limited conditions.

Rhodospirillum rubrum was grown continuously and photoheterotrophically under light limitation using a cylindrical photobioreactor in which the steady state biomass concentration was varied between 0.4 to 4 kg m(-3) at a constant radiant incident flux of 100 W m(-2). Kinetic and stoichiometric models for the growth are proposed. The biomass productivities, acetate consumption rate and the CO2 production rate can be quantitatively predicted to a high level of accuracy by the proposed model calculations.

Electron transport dynamics at the quinone acceptor site of bacterial photosynthetic reaction centers as probed using fast temperature changes.

Methods of laser-induced temperature jumps and fast freezing were used for testing the rates of thermoinduced conformational transitions of reaction center (RC) complexes in chromatophores and isolated RC preparations of various photosynthesizing purple bacteria. An electron transfer reaction from primary to secondary quinone acceptors was used as a probe of electron transport efficiency. The thermoinduced transition of the acceptor complex to the conformational state facilitating electron transfer to the secondary quinone acceptor was studied. To investigate the dynamics of spontaneous decay of the RC state induced by the thermal pulse, the thermal pulse was applied either before or during photoinduced activation of electron transport reactions in the RC acceptor complex. The maximum effect was observed if the thermal pulse was applied against the background of steady-state photoactivation of the RC. It was shown that neither the characteristic time of the thermoinduced transition within the temperature range 233-253 K nor the characteristic time of spontaneous decay of this state at 253 K exceeded several tens of milliseconds. Independent support of the estimates was obtained from experiments with varied cooling rates of the samples tested.

[Dark metabolism of acetate in Rhodospirillum rubrum cells, grown under photoheterotropic conditions]. / Issledovanie temnovogo metabolizma atsetata u kletok Rhodospirillum rubrum, vyrosshikh v fotogeterotrofnykh usloviiakh.

The mechanism of the dark assimilation of acetate in the photoheterotrophically grown nonsulfur bacterium Rhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation in Rsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C4-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle in Rsp. rubrum cells grown aerobically in the dark can function as an anaplerotic pathway. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO2 in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicative that citramalate and mesaconate are intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts of Rsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function in Rsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.

Induction of carbon monoxide dehydrogenase to facilitate redox balancing in a ribulose bisphosphate carboxylase/oxygenase-deficient mutant strain of Rhodospirillum rubrum.

A ribulose-1,5-bisphosphate carboxylase/oxygenase-deficient mutant strain (strain I-19) of Rhodospirillum rubrum was capable of growth under photoheterotrophic conditions in the absence of exogenous electron acceptors. These results suggested that alternative means of removing reducing equivalents have been acquired that allow this strain to remove reducing equivalents in the absence of a functional Calvin-Benson-Bassham reductive pentose phosphate pathway. Previously, the proton-reducing activity of the dinitrogenase complex was implicated in helping to maintain redox balance. However, since considerable amounts of CO2 were still fixed in this strain, the complete profile of enzymes involved in alternative CO2 fixation schemes was assessed. A specific and substantial induction of carbon monoxide dehydrogenase (CO dehydrogenase) synthesis was found in the mutant strain; although none of the other CO2 fixation pathways or enzyme activities were altered. These results suggested that CO dehydrogenase contributes to the photoheterotrophic success of strain I-19. Furthermore, the data implicate interacting and complex regulatory processes required to maintain the proper redox balance of this organism and other nonsulfur purple bacteria.

A perturbed two-level model for exciton trapping in small photosynthetic systems.

The study of exciton trapping in photosynthetic systems provides significant information about migration kinetics within the light harvesting antenna (LHA) and the reaction center (RC). We discuss two random walk models for systems with weakly coupled pigments, with a focus on the application to small systems (10-40 pigments/RC). Details of the exciton transfer to and from the RC are taken into consideration, as well as migration within the LHA and quenching in the RC. The first model is obtained by adapting earlier local trap models for application to small systems. The exciton lifetime is approximated by the sum of three contributions related to migration in the LHA, trapping by the RC, and quenching within the RC. The second model is more suitable for small systems and regards the finite rate of migration within the LHA as a perturbation of the simplified model, where the LHA and the RC are each represented by a single pigment level. In this approximation, the exciton lifetime is the sum of a migration component and a single nonlinear expression for the trapping and quenching of the excitons. Numerical simulations demonstrate that both models provide accurate estimates of the exciton lifetime in the intermediate range of 20-50 sites/RC. In combination, they cover the entire range of very small to very large photosynthetic systems. Although initially intended for regular LHA lattices, the models can also be applied to less regular systems. This becomes essential as more details of the structure of these systems become available. Analysis with these models indicates that the excited state decay in LH1 is limited by the average rate at which excitons transfer to the RC from neighboring sites in the LHA. By comparing this to the average rate of transfer within the LHA, various structural models that have been proposed for the LH1 core antenna are discussed.

Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense.

Reversible ADP ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase activating glycohydrolase (DRAG) regulatory system, has been characterized in both Rhodospirillum rubrum and Azospirillum brasilense. Although the general functions of DRAT and DRAG are very similar in these two organisms, there are a number of interesting differences, e.g., in the timing and extent of the regulatory response to different stimuli. In this work, the basis of these differences has been studied by the heterologous expression of either draTG or nifH from A. brasilense in R. rubrum mutants that lack these genes, as well as the expression of draTG from R. rubrum in an A. brasilense draTG mutant. In general, these hybrid strains respond to stimuli in a manner similar to that of the wild-type parent of the recipient strain rather than the wild-type source of the introduced genes. These results suggest that the differences seen in the regulatory response in these organisms are not primarily a result of different properties of DRAT, DRAG, or dinitrogenase reductase. Instead, the differences are likely the result of different signal pathways that regulate DRAG and DRAT activities in these two organisms. Our results also suggest that draT and draG are cotranscribed in A. brasilense.

Changes in the NAD(P)H concentration caused by addition of nitrogenase 'switch-off' effectors in Rhodospirillum rubrum G-9, as measured by fluorescence.

The effect of nitrogenase 'switch-off' effectors on the concentration of NAD(P)H in Rhodospirillum rubrum G-9 was investigated by fluorescence. A rapid decrease in fluorescence was observed when cells, either N2-grown or nitrogen-starved, were subjected to the effectors, but not when sodium chloride or Tris buffer was added. No effects on the fluorescence were observed in non-nitrogen fixing cultures except when NAD+ was added. The results strongly indicate that the redox state of the pyridine nucleotide pool affects the control of the regulation of nitrogenase activity in R. rubrum.

Energy migration and trapping in a spectrally and spatially inhomogeneous light-harvesting antenna.

In this paper, we analyze the process of excitation energy migration and trapping by reaction centres in photosynthesis and discuss the mechanisms that may provide an overall description of this process in the photosynthetic bacterium Rhodospirillum (Rs.) rubrum and related organisms. A wide range of values have been published for the pigment to pigment transfer rate varying from less than 1 ps up to 10 ps. These differences occur because the interpretation of trapping measurements depend on the assumptions made regarding the organization of the photosynthetic system. As we show, they can be reconciled by assuming a spatially inhomogeneous model where the distance of the reaction center to its surrounding pigments is larger than the pigment-pigment distances within the antenna. We estimate their ratio to be 1.7-1.8. The observed spectral inhomogeneity (at low temperature) of the photosynthetic antenna has resulted in various models. We demonstrate that the excitation kinetics can be modelled at all temperatures by assuming an inhomogeneous distribution of spectral shifts for each pigment. A transition temperature can be distinguished where the effects of spectral inhomogeneity become apparent and we discuss the ranges above (e.g., room temperature), around (e.g., 77K) and below (e.g., 4K) this temperature. Although the basic model is the same in all cases, the dominant mechanism differs in each range. We present explicit expressions for the exciton lifetime in the first two cases and demonstrate that at both temperatures the transfer rate from the light-harvesting antenna to the special pair of the reaction center is the rate-limiting step. Furthermore we demonstrate that at all temperatures a finite number of functional "levels" can be distinguished in the spectral distribution. At high temperature all pigments can be considered spectrally identical and only one level is needed. In the intermediate range a blue-shifted fraction is necessary. At low temperature a third redshifted fraction must be introduced.

Comparison of the contribution from different energy-linked reactions to the function of a membrane potential in photosynthetic bacteria.

The steady-state membrane potentials generated by light, PP(i), ATP or the reverse transhydrogenase reaction were studied in chromatophores from two different phototrophic bacteria, Rhodospirillum rubrum and Rhodopseudomonas viridis. The membrane potentials generated by the different energy-linked reactions were evaluated by a tetraphenylboron(TPB(-)) ion-selective electrode. The generated by light was estimated to be 110 mV and 50 mV in R. rubrum and Rps. viridis chromatophores, respectively. In the dark, PP (i), ATP and reversed transhydrogenase generated membrane potentials in R. rubrum and Rps. viridis chromatophores 50, 60 and 35 mV, and 14, 35 and 25 mV,respectively. The effect of magnesium ion on the membrane potential generated by different energy-linked reactions was also studied. The induced by different energy-generating reactions in R. rubrum and Rps. viridis chromatophores and the possible relationship to the chromatophore structures are discussed.

Pyruvate-dependent diauxic growth of Rhodospirillum rubrum in light.

When Rhodospirillum rubrum mutant C was first exposed to radiant energy after long-term anaerobic dark growth, the cells often exhibited a diauxic growth response. This happened with pyruvate in the medium and when cultures were exposed to a less-than-growth-saturating white light intensity of about 6,460 lx. Under the growth-saturating light condition, mutant C photometabolized and growth was not affected by Na hypophosphite, an inhibitor of pyruvate fermentation. In lower intensity light, in which diauxie occurred, initial (phase I) growth occurred by fermentation of Na pyruvate and was sensitive to Na hypophosphite inhibition. Once pyruvate was depleted, phase I growth stopped, the bacteriochlorophyll content of the cells began to increase from about 3 nmol/mg of protein, and growth finally resumed phototrophically (phase II). The lag period and phase II growth were influenced by radiant energy. By changing the white light intensity from 2,150 to 753 lx between experiments, the duration of both the lag period and the generation time of cells in phase II growth increased. Diauxic growth was pyruvate dependent. It occurred with pyruvate even if malate, a photometabolizable substrate, was added to the growth medium. Moreover, the biphasic growth response was reversible. It was observed not only with R. rubrum mutant C grown cells photosynthetically, but also when other strains of R. rubrum were placed in pyruvate medium under lowered light conditions. Only R. rubrum S1 did not exhibit the typical pyruvate-dependent diauxic growth response.

Effect of light intensity and inhibitors of nitrogen assimilation on NH4+ inhibition of nitrogenase activity in Rhodospirillum rubrum and Anabaena sp.

Nitrogenase activity in Rhodospirillum rubrum was inhibited by NH4+ more rapidly in low light than in high light. Furthermore, the nitrogenase of cells exposed to phosphorylation uncouplers was inhibited by NH4+ more rapidly than was the nitrogenase of controls without an uncoupler. These observations suggest that high levels of photosynthate inhibit the nitrogenase inactivation system. L-Methionine-DL-sulfoximine, a glutamine synthetase inhibitor, prevented NH4+ from inhibiting nitrogenase activity, which suggests that NH4+ must be processed at least to glutamine for inhibition to occur. An inhibitor of glutamate synthase activity, 6-diazo-5-oxo-L-norleucine, inhibited nitrogenase activity in the absence of NH4+, but only in cells exposed to low light. The mechanism of 6-diazo-5-oxo-L-norleucine inhibition appeared to be the same as that induced by NH4+, because nitrogenase activity could be restored in vitro by activating enzyme and Mn2+. The inhibitor data suggest that the glutamine pool or a molecule that responds to it activates the Fe protein-modifying (or protein-inactivating) system and that the accumulation of this (unidentified) molecule is retarded when the cells are exposed to high light. It was confirmed here that Anabaena nitrogenase is also inhibited by NH4+, but only when the cells are incubated under low light. This inhibition, however, unlike that in R. rubrum, could be completely reversed in high light, suggesting that the mechanisms of nitrogenase inhibition by NH4+ in these two phototrophs are different.