Growth of Rhodopseudomonas palustris under phototrophic and non-phototrophic conditions and its CO-dependent H2 production

A new hydrogen producing bacterium, Rhodopseudomonas palustris P4, originally isolated under an anaerobic/phototrophic condition, grew well under aerobic/chemoheterotrophic or anaerobic/chemoheterotrophic conditions and showed CO-dependent, H₂ production activity when transferred to anaerobic conditions. Cell growth was best under an aerobic/chemoheterotrophic condition as the doubling time of 1 h, while the H₂ production activity was highest in the cells grown under an aerobic/chemoheterotrophic condition at 20 mmol g^–1 cell^–1 h^–1.     

Effects of growth conditions of acetate utilization byRhodopseudomonas palustris isolated from a freshwater lake

Rhodopseudomonas palustris, a purple non-sulfur bacterium, was recently found throughout the water column in Lake Kinneret. It was demonstrated to be of a versatile nature, growing under both aerobic and anaerobic conditions at different light intensities. A comparison of C-acetate uptake byR. palustris andChlorobium phaeobacterioides, a green sulfur bacterium, showed that, under identical growth conditions, C-acetate assimilation byR. palustris was greater. Furthermore, C-acetate uptake forR. palustris was greater than C−CO₂ uptake at all light intensities. Depending on the prevailing conditions, acetate can be used byR. palustris as both an electron donor and carbon source. Malate synthase was used as an indicator of activity of the glyoxylic acid cycle. It was found that enzyme activity was higher (i.e., acetate was used mainly as a carbon source) under anaerobic conditions, in the dark, or in the absence of HCO ₃ ⁻ . Acetate was used preferably as an electron donor under photosynthetic microaerophillic conditions.     

Genes essential for phototrophic growth by a purple alphaproteobacterium

Tn‐seq was used to identify genes essential for phototrophic growth by the purple bacterium Rhodopseudomonas palustris. About 167 genes required for anaerobic growth on acetate in light were identified, 35 of which are annotated as photosynthesis genes. The essentiality of many of these genes by analysing the phenotypes of independently generated mutants that had altered pigmentation was verified. Three genes were identified, two possibly involved in biogenesis of the membrane‐bound photosynthetic apparatus and one for phosphatidylcholine biosynthesis, that were not known to be essential for phototrophic growth. Site‐directed mutagenesis was used to show that the NADH:quinone oxidoreductase complex I_E was essential for phototrophic growth under strictly anaerobic conditions and appeared to play a role in reverse electron transport to generate NADH. A homologous NADH:quinone oxidoreductase complex I_A likely operates in the opposite direction to oxidize NADH. The operation of the two enzymes in opposition would allow R. palustris to maintain redox balance. As a complement to the genetic data, proteomics experiments were carried out in which it was found that 408 proteins were present in significantly higher amounts in cells grown anaerobically in light compared with aerobically. Among these were proteins encoded by subset of the phototrophic growth‐essential genes.     

Growth‐independent cross‐feeding modifies boundaries for coexistence in a bacterial mutualism

Nutrient cross‐feeding can stabilize microbial mutualisms, including those important for carbon cycling in nutrient‐limited anaerobic environments. It remains poorly understood how nutrient limitation within natural environments impacts mutualist growth, cross‐feeding levels and ultimately mutualism dynamics. We examined the effects of nutrient limitation within a mutualism using theoretical and experimental approaches with a synthetic anaerobic coculture pairing fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris. In this coculture, E. coli and R. palustris resemble an anaerobic food web by cross‐feeding essential carbon (organic acids) and nitrogen (ammonium) respectively. Organic acid cross‐feeding stemming from E. coli fermentation can continue in a growth‐independent manner during nitrogen limitation, while ammonium cross‐feeding by R. palustris is growth‐dependent. When ammonium cross‐feeding was limited, coculture trends changed yet coexistence persisted under both homogenous and heterogenous conditions. Theoretical modelling indicated that growth‐independent fermentation was crucial to sustain cooperative growth under conditions of low nutrient exchange. In contrast to stabilization at most cell densities, growth‐independent fermentation inhibited mutualistic growth when the E. coli cell density was adequately high relative to that of R. palustris. Thus, growth‐independent fermentation can conditionally stabilize or destabilize a mutualism, indicating the potential importance of growth‐independent metabolism for nutrient‐limited mutualistic communities.     

Degradation of 3-Chlorobenzoate under Low-Oxygen Conditions in Pure and Mixed Cultures of the Anoxygenic Photoheterotroph Rhodopseudomonas palustris DCP3 and an Aerobic Alcaligenes Species

The presence or absence of molecular oxygen has been shown to play a crucial role in the degradability of haloaromatic compounds. In the present study, it was shown that anaerobic phototrophic 3-chlorobenzoate (3CBA) metabolism by Rhodopseudomonas palustris DCP3 is oxygen tolerant up to a concentration of 3 μM O₂. Simultaneous oxidation of an additional carbon source permitted light-dependent anaerobic 3CBA degradation at oxygen input levels which, in the absence of such an additional compound, would result in inhibition of light-dependent dehalogenation. Experiments under the same experimental conditions with strain DCP3 in coculture with an aerobic 3CBA-utilizing heterotroph, Alcaligenes sp. strain L6, revealed that light-dependent dehalogenation of 3CBA did not occur. Under both oxygen limitation (O₂ < 0.1 μM) and low oxygen concentrations (3 μM O₂), all the 3CBA was metabolized by the aerobic heterotroph. These data suggest that biodegradation of (halo)aromatics by photoheterotrophic bacteria such as R. palustris DCP3 may be restricted to anoxic photic environments.     

Characterization of the Photosynthetic Apparatus and Proteome of Roseobacter denitrificans

The phototrophic capacity of aerobic anoxygenic phototrophic bacteria endows them with a selective advantage over other heterotrophic bacteria in the oligotrophic ocean. Here, we reported the phototrophic features and proteome of an aerobic phototrophic bacterium Roseobacter denitrificans under starvation stress. The fluorescence induction and relaxation measurements suggested that the photosynthetic capacity in R. denitrificans was preserved but was lower than in the photoautotrophic bacterium Rhodobacter sphaeroides. The existence of light-harvesting complexes (LH1 and LH2) and the reaction center (RC) in the native membrane were demonstrated through atomic force microscopy image analysis as direct evidence of their phototrophy. The homology-based LH1–RC complex structure was proposed in which RC was the Rb. sphaeroides homolog structure surrounded by the LH1. Moreover, the protein expression profiles of cells in the stationary phase under heterotrophic and mixotrophic conditions show that light enhanced or activated some proteins such as carbon monoxide dehydrogenase and NifU to cope with the low levels of amino acids and carbon sources under starvation conditions.     

Anaerobic and aerobic biodegradation of chlorophenols using UASB and ASG bioreactors

Chlorophenol degradation was studied by combined anaerobic–aerobic treatments as a single or multi-substrate system. 2,4-Dichlorophenol (2,4-DCP) was degraded to the extent of 52 and 78% in up-flow anaerobic sludge blanket (UASB) and aerobic suspended growth (ASG) reactors respectively, at organic loading rates of 0.18kg/m³/day and hydraulic retention time of 26.4h in the presence of glucose. The UASB represents the dominating facultative anaerobic microbial population. When the effluent from the anaerobic reactor (UASB) was subjected to aerobic treatment on the ASG reactor, 2,4-DCP and COD removals of 86 and 95% respectively were achieved. Aerobic degradation of chlorophenol by acclimated mixed bacterial isolates was found to be sequential: 2-Chlorophenol (2-CP) and 4-CP were degraded first, followed by 2,4-DCP and 2,4,6-Trichlorophenol (2,4,6-TCP) while the contrary was obtained in anaerobic degradation. In anaerobic degradation by acclimated mixed bacterial cells, 2,4-DCP and 2,4,6-TCP were degraded first followed by mono-chlorophenols. The anaerobic/aerobic bioreactors were most efficient when operated in sequence (series) rather than in parallel.     

Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.     

Removal of chlorophenols in sequential anaerobic-aerobic reactors

Combination of upflow anaerobic sludge blanket (UASB) and aerobic rotating biological contactor (RBC) reactors having higher biomass concentration and higher sludge retention time (SRT) was applied for the sequential treatment of priority pollutant chlorophenol containing wastewater. Target compounds 2-chlorophenol (2-CP) and 2,4-dichlorophenol (2,4-DCP) present in two simulated wastewaters at a concentration of 30 mg/l each individually were sequentially treated in continuous mode by combined UASB-I, RBC-I and combined UASB-II, RBC-II reactors, respectively after the acclimation of their biomass with the corresponding chlorophenol. Reactor combinations took 190 and 215 days for acclimation with 30 mg/l of 2-CP and 2,4-DCP respectively. Hydraulic retention time (HRT) studies showed that 12h HRT of UASB-I and 23 h HRT of RBC-I as well as 12h HRT of UASB-II and 28.8h HRT of RBC-II were the optimum combinations for the treatment of simulated wastewater containing 2-CP and 2,4-DCP respectively. Optimum HRT combinations produced 2-CP and 2,4-DCP effluent having corresponding chlorophenol concentration of below detectable limit (BDL) and 0.1 mg/l respectively. Half velocity coefficients (Ks) for 2-CP and 2,4-DCP biodegradation in UASB reactors were determined to be 5.07 mg 2-CP/l and 6.49 mg 2,4-DCP/l. Optimum ratio of substrate (chlorophenol): co-substrate (sodium acetate) was 1:100.     

The purple non‐sulfur bacterium Rhodopseudomonas palustris produces novel petrobactin‐related siderophores under aerobic and anaerobic conditions

Many bacteria produce siderophores to bind and take up Fe(III), an essential trace metal with extremely low solubility in oxygenated environments at circumneutral pH. The purple non‐sulfur bacterium Rhodopseudomonas palustris str. CGA009 is a metabolically versatile model organism with high iron requirements that is able to grow under aerobic and anaerobic conditions. Siderophore biosynthesis has been predicted by genomic analysis, however, siderophore structures were not identified. Here, we elucidate the structure of two novel siderophores from R. palustris: rhodopetrobactin A and B. Rhodopetrobactins are structural analogues of the known siderophore petrobactin in which the Fe chelating moieties are conserved, including two 3,4‐dihydroxybenzoate and a citrate substructure. In the place of two spermidine linker groups in petrobactin, rhodopetrobactins contain two 4,4′‐diaminodibutylamine groups of which one or both are acetylated at the central amine. We analyse siderophore production under different growth modes and show that rhodopetrobactins are produced in response to Fe limitation under aerobic as well as under anaerobic conditions. Evaluation of the chemical characteristics of rhodopetrobactins indicates that they are well suited to support Fe acquisition under variable oxygen and light conditions.     

Complete assimilation of cysteine by a newly isolated non-sulfur purple bacterium resembling Rhodovulum sulfidophilum (Rhodobacter sulfidophilus)

A rod-shaped, motile, phototrophic bacterium, strain SiCys, was enriched and isolated from a marine microbial mat, with cysteine as sole substrate. During phototrophic anaerobic growth with cysteine, sulfide was produced as an intermediate, which was subsequently oxidized to sulfate. The molar growth yield with cysteine was 103 g mol^–1, in accordance with complete assimilation of electrons from the carbon and the sulfur moiety into cell material. Growth yields with alanine and serine were proportionally lower. Thiosulfate, sulfide, hydrogen, and several organic compounds were used as electron donors in the light, whereas cystine, sulfite, or elemental sulfur did not support phototrophic anaerobic growth. Aerobic growth in the dark was possible with fructose as substrate. Cultures of strain SiCys were yellowish-brown in color and contained bacteriochlorophyll a, spheroidene, spheroidenone, and OH-spheroidene as major photosynthetic pigments. Taking the morphology, photosynthetic pigments, aerobic growth in the dark, and utilization of sulfide for phototrophic growth into account, strain SiCys was assigned to the genus Rhodovulum (formerly Rhodobacter) and tentatively classified as a strain of R. sulfidophilum. In cell-free extracts in the presence of pyridoxal phosphate, cysteine was converted to pyruvate and sulfide, which is characteristic for cysteine desulfhydrase activity (l-cystathionine γ-lyase, EC 4.4.1.1). Received: 15 December 1995 / Accepted: 1 April 1996     

Marine sponges as habitats of anaerobic phototrophic bacteria

Enrichment cultures were prepared with different media for phototrophic bacteria from four species of marine sponges, collected from oxic coastal waters near Split (Yugoslavia). We obtained pure cultures of six strains ofChromatiaceae and two strains ofRhodospirillaceae by agar shake dilution. TheRhodospirillaceae were identified asRhodopseudomonas sulfidophila and a marine form ofRhodopseudomonas palustris. TheChromatiaceae were identified asChromatium vinosum, Chromatium gracile, Chromatium minutissimum. Ectothiorhodospira mobilis, and a Chromatium species, which in some respects resemblesChromatium minus. The occurrence of strictly anaerobic phototrophic bacteria in aerobic sponges is discussed with respect to nutrition and possible syntrophism.     

Neotabrizicola shimadae gen. nov., sp. nov., an aerobic anoxygenic phototrophic bacterium harbouring photosynthetic genes in the family Rhodobacteraceae,isolated from a terrestrial hot spring

A bacteriochlorophyll-containing bacterium, designated as strain N10^T, was isolated from a terrestrial hot spring in Nagano Prefecture, Japan. Gram-stain-negative, oxidase- and catalase-positive and ovoid to rod-shaped cells showed the features of aerobic anoxygenic phototrophic bacteria, i.e., strain N10^T synthesised bacteriochlorophylls under aerobic conditions and could not grow anaerobically even under illumination. Genome analysis found genes for bacteriochlorophyll and carotenoid biosynthesis, light-harvesting complexes and type-2 photosynthetic reaction centre in the chromosome. Phylogenetic analyses based on the 16S rRNA gene sequence and 92 core proteins revealed that strain N10^T was located in a distinct lineage near the type species of the genera Tabrizicola and Xinfangfangia and some species in the genus Rhodobacter (e.g., Rhodobacter blasticus). Strain N10^T shared?<?97.1% 16S rRNA gene sequence identity with those species in the family Rhodobacteraceae. The digital DNA–DNA hybridisation, average nucleotide identity and average amino acid identity values with the relatives, Tabrizicola aquatica RCRI19^T (an aerobic anoxygenic phototrophic bacterium), Xinfangfangia soli ZQBW^T and R. blasticus ATCC 33485^T were 19.9–20.7%, 78.2–79.1% and 69.1–70.1%, respectively. Based on the phenotypic features, major fatty acid and polar lipid compositions, genome sequence and phylogenetic position, a novel genus and species are proposed for strain N10^T, to be named Neotabrizicola shimadae (=?JCM 34381^T?=?DSM 112087T). Strain N10^T which is phylogenetically located among aerobic anoxygenic phototrophic bacteria (Tabrizicola), bacteriochlorophyll-deficient bacteria (Xinfangfangia) and anaerobic anoxygenic phototrophic bacteria (Rhodobacter) has great potential to promote studies on the evolution of photosynthesis in Rhodobacteraceae.

     

Phototrophic bacterial production of oleic acid in an illuminated upflow anaerobic sludge blanket reactor

Phototrophic bacterial cells in the effluent from a lighted upflow anaerobic sludge blanket reactor supplied with a medium containing 142 mg S (as SO₄ ^2–) l^–1 accumulated a 6.8% w/w oleic acid content in cells and 19 mg cell-bound oleic acid l^–1 in the effluent. Pure cultures of Rhodopseudomonas palustris and Blastochloris sulfoviridis isolated from the effluent also accumulated 5.1 and 6.4% w/w oleic acid contents in cells, respectively. The oleic acid content in the cells recovered from the LUASB reactor effluent was related to the phototrophic bacterial population in the LUASB reactor. The inverse relationship was observed in the LUASB reactor between phototrophic bacterial growth and sulfate concentration in the influent.     

Experimental and Computational Investigation of Biofilm Formation by Rhodopseudomonas palustris Growth under Two Metabolic Modes

We examined biofilms formed by the metabolically versatile bacterium Rhodopseudomonas palustris grown via different metabolic modes. R. palustris was grown in flow cell chambers with identical medium conditions either in the presence or absence of light and oxygen. In the absence of oxygen and the presence of light, R. palustris grew and formed biofilms photoheterotrophically, and in the presence of oxygen and the absence of light, R. palustris grew and formed biofilms heterotrophically. We used confocal laser scanning microscopy and image analysis software to quantitatively analyze and compare R. palustris biofilm formation over time in these two metabolic modes. We describe quantifiable differences in structure between the biofilms formed by the bacterium grown heterotrophically and those grown photoheterotrophically. We developed a computational model to explore ways in which biotic and abiotic parameters could drive the observed biofilm architectures, as well as a random-forest machine-learning algorithm based on structural differences that was able to identify growth conditions from the confocal imaging of the biofilms with 87% accuracy. Insight into the structure of phototrophic biofilms and conditions that influence biofilm formation is relevant for understanding the generation of biofilm structures with different properties, and for optimizing applications with phototrophic bacteria growing in the biofilm state.     

2-Ketocyclohexanecarboxyl Coenzyme A Hydrolase, the Ring Cleavage Enzyme Required for Anaerobic Benzoate Degradation by Rhodopseudomonas palustris

2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 μmol min⁻¹ mg of protein⁻¹. Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.     

Cysteine and S-sulfocysteine biosynthesis in phototrophic bacteria

Forteen species (17 strains) of phototrophic bacteria as well as one strain of Thiobacillus denitrificans were tested for cysteine synthase and S-sulfocysteine synthase. All strains contain cysteine synthase active with O-acetylserine; only the Chromatiaceae, two species of the Rhodospirillaceae and T. denitrificans contain S-sulfocysteine synthase. In six species repression by different sulfur compounds in the medium was studied. In Chromatium vinosum, cysteine synthase was found to be constitutive, while in the Rhodospirillaceae tested the enzyme is repressed by sulfide. Thiosulfate had a derepressive effect in Rhodopseudomonas globiformis but strongly repressed cysteine synthase in R. sulfidophila and R. palustris. Cysteine had only moderate effects with the species tested.     

Photocatabolism of Aromatic Compounds by the Phototrophic Purple Bacterium Rhodomicrobium vannielii

The phototrophic purple non-sulfur bacterium Rhodomicrobium vannielii grew phototrophically (illuminated anaerobic conditions) on a variety of aromatic compounds (in the presence of CO₂). Benzoate was universally photocatabolized by all five strains of R. vannielii examined, and benzyl alcohol was photocatabolized by four of the five strains. Catabolism of benzyl alcohol by phototrophic bacteria has not been previously reported. Other aromatic substrates supporting reasonably good growth of R. vannielii strains were the methoxylated benzoate derivatives vanillate (4-hydroxy-3-methoxybenzoate) and syringate (4-hydroxy-3,5-dimethoxybenzoate). However, catabolism of vanillate and syringate led to significant inhibition of bacteriochlorophyll synthesis in R. vannielii cells, eventually causing cultures to cease growing. No such effect on photopigment synthesis in cells grown on benzoate or benzyl alcohol was observed. Along with a handful of other species of anoxygenic phototrophic bacteria, the ability of the species R. vannielii to photocatabolize aromatic compounds indicates that this organism may also be ecologically significant as a consumer of aromatic derivatives in illuminated anaerobic habitats in nature.     

Identification and characterization of a pigment-producing denitrifying bacterium

Herein, a denitrifying bacterium that produced greenish fluorescent pigment under aerobic conditions was accidentally isolated from municipal sewage sludge. Using 16S-rDNA sequence analysis, we identified the isolate as Pseudomonas aeruginosa R12, with 100% similarity. We achieved the highest pigment production rate (1.36 mg/L/h) in a 1-L bioreactor under aerobic conditions, using the optimal culture parameters determined in this study: 37°C, pH 8.0, 200 rpm, 5 wm aeration, and medium containing succinate and (NH₄)₂SO₄. The pigment was not a secondary metabolite and had no antibacterial activity on its co-isolates. Under anaerobic conditions, the isolate produced mainly N₂ and behaved as a strong denitrifier, displaying synergistic denitrification with co-isolated denitrifiers. To our knowledge, herein we have described the first instance in which P. aeruginosa R12 produces a fluorescent pigment under aerobic conditions. This newly-isolated strain therefore shows potential as a commercial resource for natural pigment.     

Accumulation of unusual carotenoids in the spheroidene pathway,demethylspheroidene and demethylspheroidenone,in an alkaliphilic purple nonsulfur bacterium Rhodobaca bogoriensis

Carotenoids extracted from cells of a novel alkaliphilic purple nonsulfur bacterium Rhodobaca bogoriensis strain LBB1 included unusual carotenoids in the spheroidene pathway; demethylspheroidene, demethylspheroidenone, neurosporene and spheroidenone. Spheroidene was present in only small amounts, and the demethyl-carotenoids demethylspheroidene and demethylspheroidenone predominated in phototrophic cultures. Furthermore, the keto-carotenoids spheroidenone and demethylspheroidenone constituted nearly half of the total carotenoids, even in strict anaerobic phototrophic cultures. Spheroidenone was, however, the sole carotenoid in aerobic cultures. Phototrophic cultures of Rbc. bogoriensis were yellow in colour and quite distinct from the brown-red colour of cultures of Rhodobacter species. The carotenogenesis pathways of Rhodobaca and Rhodobacter species are compared with special reference to two key enzymes of the spheroidene pathway, CrtA and CrtF, whose activities are thought to be responsible for the unusual carotenoid composition of Rhodobaca. This bacterium also contained bacteriochlorophyll a _p and ubiquinone-10. This revised version was published online in June 2006 with corrections to the Cover Date.