Enhanced carotenoid production by Rhodopseudomonas palustris ATCC 17001 under low light conditions.

Carotenoids (CD) are biological pigments produced for commercial purposes. Therefore, it is necessary to study and determine the optimal conditions for increased CD production. There is no consensus in the literature about the conditions that increase CD production. Some authors stated that CD will be preferentially produced at low light intensities, at this adverse condition, microorganism will increase CD production as a survival response mechanism to get more energy. Other authors have mentioned that CD concentrations increase as the light intensity supplied increases, to avoid the overexposure of light and in turn photo-inhibition. Additionally, to increase the specific CD production is also necessary to increase the amount of biomass. In this work, the ammonium concentration (high (HAC) and low (LAC)) and the low light (LL) intensity effect on the CD production was evaluated. Data showed that a high CD-specific concentration of 8.8 mg/gcell was obtained by using R. palustris ATCC 17001 under HAC and LL intensity. CD production was similar at HAC and LAC, suggesting that the light intensity has a greater effect on the specific CD concentration than the nitrogen concentration. In general, the results showed a low biomass production compared to the literature, with high CD synthesis.

Redox Regulation of a Light-Harvesting Antenna Complex in an Anoxygenic Phototroph.

The purple nonsulfur bacterium Rhodopseudomonas palustris is a model for understanding how a phototrophic organism adapts to changes in light intensity because it produces different light-harvesting (LH) complexes under high light (LH2) and low light intensities (LH3 and LH4). Outside of this change in the composition of the photosystem, little is understood about how R. palustris senses and responds to low light intensity. On the basis of the results of transcription analysis of 17 R. palustris strains grown in low light, we found that R. palustris strains downregulate many genes involved in iron transport and homeostasis. The only operon upregulated in the majority of R. palustris exposed to low light intensity was pucBAd, which encodes LH4. In previous work, pucBAd expression was shown to be modulated in response to light quality by bacteriophytochromes that are part of a low-light signal transduction system. Here we found that this signal transduction system also includes a redox-sensitive protein, LhfE, and that its redox sensitivity is required for LH4 synthesis in response to low light. Our results suggest that R. palustris upregulates its LH4 system when the cellular redox state is relatively oxidized. Consistent with this, we found that LH4 synthesis was upregulated under high light intensity when R. palustris was grown semiaerobically or under nitrogen-fixing conditions. Thus, changes in the LH4 system in R. palustris are not dependent on light intensity per se but rather on cellular redox changes that occur as a consequence of changes in light intensity.IMPORTANCE An essential aspect of the physiology of phototrophic bacteria is their ability to adjust the amount and composition of their light-harvesting apparatus in response to changing environmental conditions. The phototrophic purple bacterium R. palustris adapts its photosystem to a range of light intensities by altering the amount and composition of its peripheral LH complexes. Here we found that R. palustris regulates its LH4 complex in response to the cellular redox state rather than in response to light intensity per se Relatively oxidizing conditions, including low light, semiaerobic growth, and growth under nitrogen-fixing conditions, all stimulated a signal transduction system to activate LH4 expression. By understanding how LH composition is regulated in R. palustris, we will gain insight into how and why a photosynthetic organism senses and adapts its photosystem to multiple environmental cues.

Modeling the Interplay between Photosynthesis, CO2 Fixation, and the Quinone Pool in a Purple Non-Sulfur Bacterium.

Rhodopseudomonas palustris CGA009 is a purple non-sulfur bacterium that can fix carbon dioxide (CO2) and nitrogen or break down organic compounds for its carbon and nitrogen requirements. Light, inorganic, and organic compounds can all be used for its source of energy. Excess electrons produced during its metabolic processes can be exploited to produce hydrogen gas or biodegradable polyesters. A genome-scale metabolic model of the bacterium was reconstructed to study the interactions between photosynthesis, CO2 fixation, and the redox state of the quinone pool. A comparison of model-predicted flux values with available Metabolic Flux Analysis (MFA) fluxes yielded predicted errors of 5-19% across four different growth substrates. The model predicted the presence of an unidentified sink responsible for the oxidation of excess quinols generated by the TCA cycle. Furthermore, light-dependent energy production was found to be highly dependent on the quinol oxidation rate. Finally, the extent of CO2 fixation was predicted to be dependent on the amount of ATP generated through the electron transport chain, with excess ATP going toward the energy-demanding Calvin-Benson-Bassham (CBB) pathway. Based on this analysis, it is hypothesized that the quinone redox state acts as a feed-forward controller of the CBB pathway, signaling the amount of ATP available.

Light-induced complex formation of bacteriophytochrome RpBphP1 and gene repressor RpPpsR2 probed by SAXS.

Bacteriophytochrome proteins (BphPs) are molecular light switches that enable organisms to adapt to changing light conditions through the control of gene expression. Canonical type 1 BphPs have histidine kinase output domains, but type 3 RpBphP1, in the bacterium Rhodopseudomonas palustris (Rps. palustris), has a C terminal PAS9 domain and a two-helix output sensor (HOS) domain. Type 1 BphPs form head-to-head parallel dimers; however, the crystal structure of RpBphP1ΔHOS, which does not contain the HOS domain, revealed pseudo anti-parallel dimers. HOS domains are homologs of Dhp dimerization domains in type 1 BphPs. We show, by applying the small angle X-ray scattering (SAXS) technique on full-length RpBphP1, that HOS domains fulfill a similar role in the formation of parallel dimers. On illumination with far-red light, RpBphP1 forms a complex with gene repressor RpPpsR2 through light-induced structural changes in its HOS domains. An RpBphP1:RpPpsR2 complex is formed in the molecular ratio of 2 : 1 such that one RpBphP1 dimer binds one RpPpsR2 monomer. Molecular dimers have been modeled with Pfr and Pr SAXS data, suggesting that, in the Pfr state, stable dimeric four α-helix bundles are formed between HOS domains, rendering RpBphP1functionally inert. On illumination with light of 760 nm wavelength, four α-helix bundles formed by HOS dimers are disrupted, rendering helices available for binding with RpPpsR2.

Different Metabolomic Responses to Carbon Starvation between Light and Dark Conditions in the Purple Photosynthetic Bacterium, Rhodopseudomonas palustris.

Purple photosynthetic bacteria utilize light energy for growth. We previously demonstrated that light energy contributed to prolonging the survival of multiple purple bacteria under carbon-starved conditions. In order to clarify the effects of illumination on metabolic states under carbon-starved, non-growing conditions, we herein compared the metabolic profiles of starved cells in the light and dark using the purple bacterium, Rhodopseudomonas palustris. The metabolic profiles of starved cells in the light were markedly different from those in the dark. After starvation for 5 d in the light, cells showed increases in the amount of ATP and the NAD+/NADH ratio. Decreases in the amounts of most metabolites related to glycolysis and the TCA cycle in energy-rich starved cells suggest the active utilization of these metabolites for the modification of cellular components. Starvation in the dark induced the consumption of cellular compounds such as amino acids, indicating that the degradation of these cellular components produced ATP in order to maintain viability under energy-poor conditions. The present results suggest that intracellular energy levels alter survival strategies under carbon-starved conditions through metabolism.

Adaptation of Rhodopseudomonas acidophila strain 7050 to growth at different light intensities: what are the benefits to changing the type of LH2?

Typical purple bacterial photosynthetic units consist of light harvesting one/reaction centre 'core' complexes surrounded by light harvesting two complexes. Factors such as the number and size of photosynthetic units per cell, as well as the type of light harvesting two complex that is produced, are controlled by environmental factors. In this paper, the change in the type of LH2 present in the Rhodopsuedomonas acidophila strain 7050 is described when cells are grown at a range of different light intensities. This species contains multiple pucBA genes that encode the apoproteins that form light-harvesting complex two, and a more complex mixture of spectroscopic forms of this complex has been found than was previously thought to be the case. Femto-second time resolved absorption has been used to investigate how the energy transfer properties in the membranes of high-light and low-light adapted cells change as the composition of the LH2 complexes varies.

Acclimation strategy of Rhodopseudomonas palustris to high light irradiance.

The ability of Rhodopseudomonas palustris cells to rapidly acclimate to high light irradiance is an essential issue when cells are grown under sunlight. The aim of this study was to investigate the photo-acclimation process in Rhodopseudomonas palustris 42OL under different culturing conditions: (i) anaerobic (AnG), (ii) aerobic (AG), and (iii) under H2-producing (HP) conditions both at low (LL) and high light (HL) irradiances. The results obtained clearly showed that the photosynthetic unit was significantly affected by the light irradiance at which Rp. palustris 42OL was grown. The synthesis of carotenoids was affected by both illumination and culturing conditions. At LL, lycopene was the main carotenoid synthetized under all conditions tested, while at HL under HP conditions, it resulted the predominant carotenoid. Oppositely, under AnG and AG at HL, rhodovibrin was the major carotenoid detected. The increase in light intensity produced a deeper variation in light-harvesting complexes (LHC) ratio. These findings are important for understanding the ecological distribution of PNSB in natural environments, mostly characterized by high light intensities, and for its growth outdoors.

Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium.

Nitrogenase is an ATP-requiring enzyme capable of carrying out multielectron reductions of inert molecules. A purified remodeled nitrogenase containing two amino acid substitutions near the site of its FeMo cofactor was recently described as having the capacity to reduce carbon dioxide (CO2) to methane (CH4). Here, we developed the anoxygenic phototroph, Rhodopseudomonas palustris, as a biocatalyst capable of light-driven CO2 reduction to CH4 in vivo using this remodeled nitrogenase. Conversion of CO2 to CH4 by R. palustris required constitutive expression of nitrogenase, which was achieved by using a variant of the transcription factor NifA that is able to activate expression of nitrogenase under all growth conditions. Also, light was required for generation of ATP by cyclic photophosphorylation. CH4 production by R. palustris could be controlled by manipulating the distribution of electrons and energy available to nitrogenase. This work shows the feasibility of using microbes to generate hydrocarbons from CO2 in one enzymatic step using light energy.

A novel PSB-EDI system for high ammonia wastewater treatment, biomass production and nitrogen resource recovery: PSB system.

A novel process coupling photosynthetic bacteria (PSB) with electrodeionization (EDI) treatment was proposed to treat high ammonia wastewater and recover bio-resources and nitrogen. The first stage (PSB treatment) was used to degrade organic pollutants and accumulate biomass, while the second stage (EDI) was for nitrogen removal and recovery. The first stage was the focus in this study. The results showed that using PSB to transform organic pollutants in wastewater into biomass was practical. PSB could acclimatize to wastewater with a chemical oxygen demand (COD) of 2,300 mg/L and an ammonia nitrogen (NH4(+)-N) concentration of 288-4,600 mg/L. The suitable pH was 6.0-9.0, the average COD removal reached 80%, and the biomass increased by an average of 9.16 times. The wastewater COD removal was independent of the NH4(+)-N concentration. Moreover, the PSB functioned effectively when the inoculum size was only 10 mg/L. The PSB-treated wastewater was then further handled in an EDI system. More than 90% of the NH4(+)-N was removed from the wastewater and condensed in the concentrate, which could be used to produce nitrogen fertilizer. In the whole system, the average NH4(+)-N removal was 94%, and the average NH4(+)-N condensing ratio was 10.0.

A polymorphism in the oxygen-responsive repressor PpsR2 confers a growth advantage to Rhodopseudomonas palustris under low light.

The purple nonsulfur bacterium Rhodopseudomonas palustris grows aerobically using oxidative phosphorylation or anaerobically using photophosphorylation. The oxygen-responsive transcription regulator, PpsR2, regulates the transition to a phototrophic lifestyle by repressing transcription of photosynthesis genes during aerobic growth. Whereas most R. palustris strains have an arginine (Arg) at position 439 in the helix-turn-helix DNA-binding domain of this protein, some strains, including the well-studied strain CGA009, have a cysteine (Cys) at this position. Using allelic exchange, we found that the Cys439 in PpsR2 resulted in increased pigmentation and photosynthetic gene expression under both aerobic and anaerobic conditions. The Cys439 substitution also conferred a growth advantage to R. palustris at low light intensities. This indicates that variation in the PpsR2 protein results in R. palustris strains that have two different thresholds for derepressing photosynthesis genes in response to oxygen and light.

H2 production in Rhodopseudomonas palustris as a way to cope with high light intensities.

The ability of coping with the damaging effects of high light intensity represents an essential issue when purple non-sulfur bacteria (PNSB) are grown under direct sunlight for photobiological hydrogen production. This study was aimed at investigating whether H2 photo-evolution could represent, for Rhodopseudomonas palustris 42OL, a safety valve to dissipate an excess of reducing power generated under high light intensities. The physiological status of this strain was assessed under anaerobic (AnG) and aerobic (AG) growing conditions and under H2-producing (HP) conditions at low and high light intensities. The results obtained clearly showed that Fv/Fm ratio was significantly affected by the light intensity under which R. palustris 42OL cells were grown, under either AnG or AG conditions, while, under HP, it constantly remained at its highest value. The increase in light intensity significantly increased the H2 production rate, which showed a positive correlation with the maximum electron transfer rate (rETRmax). These findings are important for optimization of hydrogen production by PNSB under solar light.

Light-enhanced bioaccumulation of molybdenum by nitrogen-deprived recombinant anoxygenic photosynthetic bacterium Rhodopseudomonas palustris.

As molybdenum (Mo) is an indispensable metal for plant nitrogen metabolisms, accumulation of dissolved Mo into bacterial cells may connect to the development of bacterial fertilizers that promote plant growth. In order to enhance Mo bioaccumulation, nitrogen removal and light illumination were examined in anoxygenic photosynthetic bacteria (APB) because APB possess Mo nitrogenase whose synthesis is strictly regulated by ammonium ion concentration. In addition, an APB, Rhodopseudomonas palustris, transformed with a gene encoding Mo-responsive transcriptional regulator ModE was constructed. Mo content was most markedly enhanced by the removal of ammonium ion from medium and light illumination while their effects on other metal contents were limited. Increases in contents of trace metals including Mo by the genetic modification were observed. Thus, these results demonstrated an effective way to enrich Mo in the bacterial cells by the culture conditions and genetic modification.

Biomass and pigments production in photosynthetic bacteria wastewater treatment: Effects of photoperiod.

This study aimed at enhancing the bacterial biomass and pigments production in together with pollution removal in photosynthetic bacteria (PSB) wastewater treatment via using different photoperiods. Different light/dark cycles and light/dark cycle frequencies were examined. Results showed that PSB had the highest biomass production, COD removal and biomass yield, and light energy efficiency with light/dark cycle of 2h/1h. The corresponding biomass, COD removal and biomass yield reached 2068mg/L, 90.3%, and 0.38mg-biomass/mg-COD-removal, respectively. PSB showed higher biomass production and biomass yield with higher light/dark cycle frequency. Mechanism analysis showed within a light/dark cycle from 1h/2h to 2h/1h, the carotenoid and bacteriochlorophyll production increased with an increase in light/dark cycle. Moreover, the pigment contents were much higher with lower frequency of 2-4 times/d.

Biomass and pigments production in photosynthetic bacteria wastewater treatment: effects of light sources.

This study is aimed at enhancing biomass and pigments production together with pollution removal in photosynthetic bacteria (PSB) wastewater treatment via different light sources. Red, yellow, blue, white LED and incandescent lamp were used. Results showed different light sources had great effects on the PSB. PSB had the highest biomass production, COD removal and biomass yield with red LED. The corresponding biomass, COD removal and biomass yield reached 2580 mg/L, 88.6% and 0.49 mg-biomass/mg-COD-removal, respectively. The hydraulic retention time of wastewater treatment could be shortened to 72 h with red LED. Mechanism analysis showed higher ATP was produced with red LED than others. Light sources could significantly affect the pigments production. The pigments productions were greatly higher with LED than incandescent lamp. Yellow LED had the highest pigments production while red LED produced the highest carotenoid/bacteriochlorophyll ratio. Considering both efficiency and energy cost, red LED was the optimal light source.

Enhancement of cell production in photosynthetic bacteria wastewater treatment by low-strength ultrasound.

The present study is aimed at enhanced cell production in together with pollution removal in photosynthetic bacteria wastewater treatment through low-strength ultrasound stimulation. The ultrasound strength was 0.3W/cm(2) with 40kHz frequency. Results showed 1-10min sonication significantly improved the cell production. The optimal sonication time was 2min. When the irradiation period was over 10min, a strong mechanical damage occurred. These phenomena could be explained by the changes of PSB dehydrogenase activity. Sonication stimulated the initial dehydrogenase activity; moderate sonication increased the cell activity while too-long sonication led to quick enzyme activity decrease. The optimal sonication strategy was 1min at the 0th hour and 1min at the 27th hour. The corresponding cell production and cell yield increased by 110% and 93% respectively, and the COD removal reached 98%. The cost of low-strength ultrasound was only 1% of the incremental sales value of cells.

Broadband 2D electronic spectroscopy reveals a carotenoid dark state in purple bacteria.

Although the energy transfer processes in natural light-harvesting systems have been intensively studied for the past 60 years, certain details of the underlying mechanisms remain controversial. We performed broadband two-dimensional (2D) electronic spectroscopy measurements on light-harvesting proteins from purple bacteria and isolated carotenoids in order to characterize in more detail the excited-state manifold of carotenoids, which channel energy to bacteriochlorophyll molecules. The data revealed a well-resolved signal consistent with a previously postulated carotenoid dark state, the presence of which was confirmed by global kinetic analysis. The results point to this state's role in mediating energy flow from carotenoid to bacteriochlorophyll.

Preservation of H2 production activity in nanoporous latex coatings of Rhodopseudomonas palustris†CGA009 during dry storage at ambient temperatures.

To assess the applicability of latex cell coatings as an 'off-the-shelf' biocatalyst, the effect of osmoprotectants, temperature, humidity and O2 on preservation of H2 production in Rhodopseudomonas palustris coatings was evaluated. Immediately following latex coating coalescence (24 h) and for up to 2 weeks of dry storage, rehydrated coatings containing different osmoprotectants displayed similar rates of H2 production. Beyond 2 weeks of storage, sorbitol-treated coatings lost all H2 production activity, whereas considerable H2 production was still detected in sucrose- and trehalose-stabilized coatings. The relative humidity level at which the coatings were stored had a significant impact on the recovery and subsequent rates of H2 production. After 4 weeks storage under air at 60% humidity, coatings produced only trace amounts of H2 (0-0.1% headspace accumulation), whereas those stored at < 5% humidity retained 27-53% of their H2 production activity after 8 weeks of storage. When stored in argon at < 5% humidity and room temperature, R. palustris coatings retained full H2 production activity for 3 months, implicating oxidative damage as a key factor limiting coating storage. Overall, the results demonstrate that biocatalytic latex coatings are an attractive cell immobilization platform for preservation of bioactivity in the dry state.

Bioconversion characteristics of Rhodopseudomonas palustris CQK 01 entrapped in a photobioreactor for hydrogen production.

The performance of the entrapped-cell photobioreactor during H2 production was assessed by using glucose as substrate in a continuous operation mode. The maximal hydrogen production rate and light conversion efficiency, 2.61 mmol/L/h and 82.3%, were obtained at a HRT of 11.4 h, an substrate loading rate of 4.2 mmol/h and an illumination of 590 nm and 6000 lux, the corresponding hydrogen yield and total energy efficiency were 0.62 mmol H2/(mmol glucose) and 4.8%, respectively. The results indicate the H2 production system illuminated at 590 nm wavelength engaged in energy storage for H2 production due to more ATP synthesized in primary reaction center, and was of higher energy recovery capacity. Furthermore, the total energy efficiency was far lower than the corresponding light conversion efficiency due to intermediates production.

Hydrogen photoproduction by Rhodopseudomonas palustris 42OL cultured at high irradiance under a semicontinuous regime.

The main goal of this study was to increase the hydrogen production rate improving the culture technique and the photobioreactor performances. Experiments were carried out at a constant culture temperature of 30°C and at an average irradiance of 480 W m(-2) using a cylindrical photobioreactor (4.0 cm, internal diameter). The culture technique, namely, the semicontinuous regime for growing Rhodopseudomonas palustris 42OL made it possible to achieve a very high daily hydrogen production rate of 594 ± 61 mL (H(2)) L(-1) d(-1). This value, never reported for this strain, corresponds to about 25 mL (H(2)) L(-1) h(-1), and it was obtained when the hydraulic retention time (HRT) was of 225 hours. Under the same growth conditions, a very high biomass production rate (496 ± 45 mg (dw) L(-1) d(-1)) was also achieved. Higher or lower HRTs caused a reduction in both the hydrogen and the biomass production rates. The malic-acid removal efficiency (MA(re)) was always higher than 90%. The maximal hydrogen yield was 3.03 mol H(2) mol MA(-1) at the HRT of 360 hours. The highest total energy conversion efficiency was achieved at the HRT of 225 hours.

Factors influencing the production of hydrogen by the purple non-sulphur phototrophic bacterium Rhodopseudomonas acidophila KU001.

Rhodopseudomonas acidophila KU001 was isolated from leather industry effluents and the effect of different cultural conditions on hydrogen production was studied. Anaerobic light induced more hydrogen production than anaerobic dark conditions. Growing cells produced more amounts of hydrogen between 96 and 144 h of incubation. Resting and growing cells preferred a pH of 6.0 ± 0.24 for hydrogen production. Succinate was the most preferred carbon source for the production of hydrogen while citrate was a poor source of carbon. Acetate and malate were also good carbon sources for hydrogen production under anaerobic light. Among the nitrogen sources, R. acidophila preferred ammonium chloride followed by urea for production of hydrogen. L-tyrosine was the least preferred nitrogen source by both growing and resting cells.