Isolation and pigment composition of the antenna system of four species of green sulfur bacteria

New and rapid procedures were developed for the isolation of chlorosomes and FMO-protein from the green sulfur bacteria Prosthecochloris (P.) aestuarii, Chlorobium (Cb.) phaeovibrioides, Cb. tepidum and Cb. vibrioforme. The resulting preparations were free from contaminating pigments and proteins as was shown by absorption spectroscopy, pigment analysis and SDS-PAGE. Two spectrally different types of FMO-protein were found. The first type, present in P. aestuarii and Cb. vibrioforme, has a main absorption band at 6 K at 815 nm, whereas the second type, isolated from Cb. tepidum and Cb. phaeovibrioides, has a strong band at 806 nm. In contrast to what was recently suggested (Tronrud DE and Matthews BW (1993) In: Deisenhofer J and Norris J (eds) The Photosynthetic Reaction Center, Vol 1, pp 13–21. Academic Press, San Diego, CA) the FMO-proteins contained no polar BChl a homologue. The isolated chlorosomes showed a small blue-shift of the QY absorption maximum with respect to intact cells. For the different species, grown under the same light conditions, the homologue composition of BChls c and d was approximately identical whereas for the BChl e in Cb. phaeovibrioides the relative amounts of homologues with larger alkyl substituents at position 8 were considerably larger. Baseplate BChl a was present in all chlorosomes and comprised 1–2% of the chlorosomal BChl. Its QY absorption band was located at about 802 nm and was clearly separated from the major QY absorption band at 6 K. The predominant esterifying alcohol of BChl a in the chlorosomes as well as in the FMO-proteins was phytol, but both antenna complexes also contained small amounts of BChl a esterified with the metabolic intermediates geranylgeraniol, dihydrogeranylgeraniol and tetrahydrogeranylgeraniol, like most purple bacteria. Since the esterifying alcohols of the chlorosomal BChl a and of the main chlorosomal pigments (BChls c, d and e) are different, esterification, and perhaps also the synthesis, of the BChls in the interior of the chlorosome and of the BChls in the baseplate must be spatially and genetically separated processes.     

Hole burning study of excited state structure and energy transfer dynamics of bacteriochlorophyll c in chlorosomes of green sulphur photosynthetic bacteria

Results of low temperature fluorescence and spectral hole burning experiments with whole cells and isolated chlorosomes of the green sulfur bacterium Chlorobium limicola containing BChl c are reported. At least two spectral forms of BChl c (short-wavelength and long-wavelength absorbing BChl c) were identified in the second derivative fluorescence spectra. The widths of persistent holes burned in the fluorescence spectrum of BChl c are determined by excited state lifetimes due to fast energy transfer. Different excited state lifetimes for both BChl c forms were observed. A site distribution function of the lowest excited state of chlorosomal BChl c was revealed. The excited state lifetimes are strongly influenced by redox conditions of the solution. At anaerobic conditions the lifetime of 5.3 ps corresponds to the rate of energy transfer between BChl c clusters. This time shortens to 2.6 ps at aerobic conditions. The shortening may be caused by introducing a quencher. Spectral bands observed in the fluorescence of isolated chlorosomes were attributed to monomeric and lower state aggregates of BChl c. These forms are not functionally connected with the chlorosome.Abbreviations BChl bacteriochlorophyll - EET electronic energy transfer - FWHM full width at half maximum - SDF site distribution function - RC reaction centre     

Structural investigation of oxidized chlorosomes from green bacteria using multifrequency electron paramagnetic resonance up to 330 GHz

Chemical oxidation of the chlorosomes from Chloroflexus aurantiacus and Chlorobium tepidum green bacteria produces bacteriochlorophyll radicals, which are characterized by an anomalously narrow EPR signal compared to in vitro monomeric BChl c ^.+ [Van Noort PI, Zhu Y, LoBrutto R and Blankenship RE (1997) Biophys J 72: 316–325]. We have performed oxidant concentration and temperature-dependent X-band EPR measurements in order to elucidate the line narrowing mechanism. The linewidth decreases as the oxidant concentration is increased only for Chloroflexus indicating that for this system Heisenberg spin exchange is at least partially responsible for the EPR spectra narrowing. For both species the linewidth is decreasing on increasing the temperature. This indicates that temperature-activated electron transfer is the main narrowing mechanism for BChl radicals in chlorosomes. The extent of the electron transfer process among different BChl molecules has been evaluated and a comparison between the two species representative of the two green bacteria families has been made. In parallel, high frequency EPR experiments have been performed on the oxidized chlorosomes of Chloroflexus and Chlorobium at 110 and 330 GHz in the full temperature range investigated at X-band. The g-tensor components obtained from the simulation of the 330 GHz EPR spectrum from Chlorobium show the same anisotropy as those of monomeric Chl a ^.+ [Bratt PJ, Poluektov OG, Thurnauer MC, Krzystek J, Brunel LC, Schrier J, Hsiao YW, Zerner M and Angerhofer A (2000) J Phys Chem B 104: 6973–6977]. The spectrum of Chloroflexus has a nearly axial g-tensor with reduced anisotropy compared to Chlorobium and monomeric Chl a in vitro. g-tensor values and temperature dependence of the linewidth have been discussed in terms of the differences in the local structure of the chlorosomes of the two families.This revised version was published online in October 2005 with corrections to the Cover Date.     

A comparative study of the optical characteristics of intact cells of photosynthetic green sulfur bacteria containing bacteriochlorophyll c,d or e

Energy transfer and pigment arrangement in intact cells of the green sulfur bacteria Prosthecochloris aestuarii, Chlorobium vibrioforme and chlorobium phaeovibrioides, containing bacteriochlorophyll (BChl) c, d or e as main light harvesting pigment, respectively, were studied by means of absorption, fluorescence, circular dichroism and linear dichroism spectroscopy at low temperature. The results indicate a very similar composition of the antenna in the three species and a very similar structure of main light harvesting components, the chlorosome and the membrane-bound BChl a protein. In all three species the Q_y transition dipoles of BChl c, d or e are oriented approximately parallel to the long axis of the chlorosome. Absorption and fluorescence excitation spectra demonstrate the presence of at least two BChl c-e pools in the chlorosomes of all three species, long-wavelength absorbing BChls being closest to the membrane. In C. phaeovibrioides, energy from BChl e is transferred with an efficiency of 25% to the chlorosomal BChl a at 6 K, whereas the efficiency of transfer from BChl e to the BChl a protein is 10%. These numbers are compatible with the hypothesis that the chlorosomal BChl a is an intermediary in the energy transfer from the chlorosome to the membrane.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - CD circular dichroism - LD linear dichroism     

Tubular exciton models for BChl c antennae in chlorosomes from green photosynthetic bacteria

Exciton calculations on tubular pigment aggregates similar to recently proposed models for BChl c/d/e antennae in light-harvesting chlorosomes from green photosynthetic bacteria yield electronic absorption spectra that are super-impositions of linear J-aggregate spectra. While the electronic spectroscopy of such antennae differs considerably from that of linear J-aggregates, tubular exciton models (which may be viewed as cross-coupled J-aggregates) may be constructed to yield spectra that resemble that of the BChl c antenna in the green bacterium Chloroflexus aurantiacus. Highly symmetric tubular models yield absorption spectra with dipole strength distributions essentially identical to that of a J-aggregate; strong symmetry-breaking is needed to simulate the absorption spectrum of the BChl c antenna.Abbreviations BChl bacteriochlorophyll - [E,M] BChl c _S bacteriochlorophyll c with ethyl and methyl substituents in the 8- and 12-positions, and with stearol as the esterifying alcohol     

Excitation energy transfer in chlorosomes of Chlorobium phaeobacteroides strain CL1401: the role of carotenoids

The role of carotenoids in chlorosomes of the green sulfur bacterium Chlorobium phaeobacteroides, containing bacteriochlorophyll (BChl) e and the carotenoid (Car) isorenieratene as main pigments, was studied by steady-state fluorescence excitation, picosecond single-photon timing and femtosecond transient absorption (TA) spectroscopy. In order to obtain information about energy transfer from Cars in this photosynthetic light-harvesting antenna with high spectral overlap between Cars and BChls, Car-depleted chlorosomes, obtained by inhibition of Car biosynthesis by 2-hydroxybiphenyl, were employed in a comparative study with control chlorosomes. Excitation spectra measured at room temperature give an efficiency of 60–70% for the excitation energy transfer from Cars to BChls in control chlorosomes. Femtosecond TA measurements enabled an identification of the excited state absorption band of Cars and the lifetime of their S₁ state was determined to be 10 ps. Based on this lifetime, we concluded that the involvement of this state in energy transfer is unlikely. Furthermore, evidence was obtained for the presence of an ultrafast (>100 fs) energy transfer process from the S₂ state of Cars to BChls in control chlorosomes. Using two time-resolved techniques, we further found that the absence of Cars leads to overall slower decay kinetics probed within the Q_y band of BChl e aggregates, and that two time constants are generally required to describe energy transfer from aggregated BChl e to baseplate BChl a.This revised version was published online in October 2005 with corrections to the Cover Date.     

Separation of bacteriochlorophyll homologues from green photosynthetic sulfur bacteria by reversed-phase HPLC

A reversed-phase High Performance Liquid Cromatography (HPLC) method has been developed to accurately separate bacteriochlorophyllsc, d ande homologues in a reasonably short run time of 60 minutes. By using this method, two well-defined groups of bacteriochlorophyll homologue peaks can be discriminated. The first one consists of 4 peaks (min 24 to 30), which corresponds to the four main farnesyl homologues. The second peak subset is formed by a cluster of up to 10 minor peaks (min 33 to 40). These peaks can be related with series of several alcohol esters of the different chlorosome chlorophylls. The number of homologues was, however, quite variable depending on both, the bacteriochlorophyll and the bacterial species. The method hereby described, also provides a good separation of other photosynthetic pigments, either bacterial (Bacteriochlorophylla, chlorobactene, isorenieratene and okenone) or algal ones (Chlorophylla, Pheophytina and -carotene). A preliminary screening of the homologue composition of several green photosynthetic bacterial species and isolates, has revealed different relative quantitative patterns. These differences seem to be related to physiological aspects rather than to taxonomic ones. The application of the method to the study of natural populations avoids the typical drawbacks on the pigment identification of overlapping eukaryotic and prokaryotic phototrophic microorganisms, giving further information about their physiological status.     

Energy transfer from carotenoid and FMO-protein in subcellular preparations from green sulfur bacteria. Spectroscopic characterization of an FMO-reaction center core complex at low temperature

The Fenna-Matthews-Olson (FMO)-protein and the FMO-reaction center (RC) core complex from the green sulfur bacterium Chlorobium tepidum were examined at 6 K by absorption and fluorescence spectroscopy. The absorption spectrum of the RC core complex was obtained by a subtraction method and found to have fiye peaks in the Q_Y region, at 797, 808, 818, 834 and 837 nm. The efficiency of energy transfer from carotenoid to bacteriochlorophyll a in the RC core complex was 23% at 6 K, and from the FMO-protein to the core it was 35%. Energy transfer from the FMO-protein to the core complex was also measured in isolated membranes of Prosthecochloris aestuarii from the action spectra of charge separation. Again, a low efficiency of energy transfer was obtained, both at 6 K and at room temperature.Abbreviations BChl- bacteriochlorophyll - P840- primary electron donor - RC- reaction center - FMO-protein- Fenna-Matthews-Olson-protein     

Rapid one-step purification of the BChl-a containing FMO-protein from the green sulfur bacterium Chlorobium tepidum using a high efficiency immunomatrix

A new and rapid procedure has been developed for the isolation of the bacteriochlorophyll a-containing Fenna—Matthews—Olson (FMO)-protein from green sulfur bacteria. Polyclonal antibodies raised against the FMO-protein of Chlorobium (Chl.) tepidum were employed in the preparation of an antibody column utilizing immobilized protein A as the matrix. The antibody column afforded essentially a one-step purification process, resulting in preparations that were free from contaminating pigments and proteins. This was evidenced by absorption spectroscopy, SDS—PAGE, and fluorescence emission.This revised version was published online in October 2005 with corrections to the Cover Date.     

Rearrangement of light harvesting bacteriochlorophyll homologues as a response of green sulfur bacteria to low light intensities

The pigment composition of two species of green-colored BChl c-containing green sulfur bacteria (Chlorobium limicola and C. chlorovibrioides) and two species of brown-colored BChl e-containing ones (C. phaeobacteroides and C. phaeovibrioides) incubated at different light intensities have been studied. All species responded to the reduction of light intensity from 50 to 1 Einstein(E) m^–2 s^–1 by an increase in the specific content of light harvesting pigments, bacteriochlorophylls and carotenoids. At critical light intensities (0.5 to 0.1 E m^–2 s^–1) only brown-colored chlorobia were able to grow, though at low specific rates (0.002 days^–1 mg prot^–1). High variations in the relative content of farnesyl-bacteriochlorophyll homologues were found, in particular BChl e ₁ and BChl e ₄, which were tentatively identified as [M, E] and [I, E] BChl_F e, respectively. The former was almost completely lost upon reduction of light intensity from 50 to 0.1 E m^–2 s^–1, whereas the latter increased from 7.2 to 38.4% and from 13.6 to 42.0% in C. phaeobacteroides and C. phaeovibrioides, respectively. This increase in the content of highly alkylated pigment molecules inside the chlorosomes of brown species is interpreted as a physiological mechanism to improve the efficiency of energy transfer towards the reaction center. This study provides some clues for understanding the physiological basis of the adaptation of brown species to extremely low light intensities.Abbreviations BChl bacteriochlorophyll - [M, E] BChl_F e 8-methyl, 12-ethyl BChl e, esterified with farnesol (F). Analogously: I - isobutyl - Pr propyl - Car carotenoids - Chlb chlorobactene - HPLC high performance liquid chromatography - Isr isorenieratene - LHP light harvesting pigments - PDA photodiode array detector - RC reaction center - RCH relative content of homologues     

Quinones in chlorosomes of green sulfur bacteria and their role in the redox-dependent fluorescence studied in chlorosome-like bacteriochlorophyll c aggregates

The light-harvesting chlorosome antennae of anaerobic, photosynthetic green sulfur bacteria exhibit a highly redox-dependent fluorescence such that the fluorescence intensity decreases under oxidizing conditions. We found that chlorosomes from Chlorobium tepidum contain three isoprenoid quinone species (chlorobiumquinone, menaquinone-7, and an unidentified quinone that probably is a chlorobiumquinone derivative) at a total concentration of approximately 0.1 mol per mol bacteriochlorophyll c. Most of the cellular chlorobiumquinone was found in the chlorosomes and constituted about 70% of the total chlorosome quinone pool. When the quinones were added to artificial, chlorosome-like bacteriochlorophyll c aggregates in an aqueous solution, a high redox dependency of the fluorescence was observed. Chlorobiumquinones were most effective in this respect. A lesser redox dependency of the fluorescence was still observed in the absence of quinones, probably due to another unidentified redox-active component. These results suggest that quinones play a significant, but not exclusive role in controlling the fluorescence and in inhibiting energy transfer in chlorosomes under oxic conditions. Chlorosomes from Chloroflexus aurantiacus contained menaquinone in an amount similar to that of total quinone in Chlorobium tepdium chlorosomes, but did not contain chlorobiumquinones. This may explain the much lower redox-dependent fluorescence observed in Chloroflexus chlorosomes. Received: 4 November 1996 / Accepted: 18 February 1997     

Low-temperature fluorescence from single chlorosomes, photosynthetic antenna complexes of green filamentous and sulfur bacteria

Fluorescence spectra of single chlorosomes isolated from a green filamentous bacterium (Chloroflexus (Cfl.) aurantiacus) and a green sulfur bacterium (Chlorobium (Cb.) tepidum) were measured by using a confocal laser microscope at 13 K. Chlorosomes were frozen either in a liquid solution (floating chlorosome) or on a quartz plate after being adsorbed (adsorbed chlorosome). Fluorescence peak wavelengths were shorter for the adsorbed single chlorosomes than for the floating ones. Single floating Cfl. chlorosomes showed a distribution of fluorescence peak positions having a center at 759.0 nm with a full width at half maximum of 6.3 nm. Single floating Cb. chlorosomes showed a 782.7 nm center with a full width at half-maximum of 3.4 nm. The distribution shifted to the blue and became wider with increasing temperature, especially in Cb. chlorosomes, suggesting a large excitonic density of states just above the lowest level. Energy transfer from BChl-c aggregates to BChl-a molecules in the baseplate proteins was observed in the floating chlorosomes but not in the adsorbed ones. A positive correlation was found between the peak wavelength of BChl-c fluorescence and the intensity of BChl-a fluorescence in single Cfl. chlorosomes. The results suggest that the BChl-c aggregates with longer wavelengths of the fluorescence peaks have a more efficient F?rster-type energy transfer to the baseplate BChl-a.     

Occurrence, biosynthesis and function of isoprenoid quinones

Isoprenoid quinones are one of the most important groups of compounds occurring in membranes of living organisms. These compounds are composed of a hydrophilic head group and an apolar isoprenoid side chain, giving the molecules a lipid-soluble character. Isoprenoid quinones function mainly as electron and proton carriers in photosynthetic and respiratory electron transport chains and these compounds show also additional functions, such as antioxidant function. Most of naturally occurring isoprenoid quinones belong to naphthoquinones or evolutionary younger benzoquinones. Among benzoquinones, the most widespread and important are ubiquinones and plastoquinones. Menaquinones, belonging to naphthoquinones, function in respiratory and photosynthetic electron transport chains of bacteria. Phylloquinone K₁, a phytyl naphthoquinone, functions in the photosynthetic electron transport in photosystem I. Ubiquinones participate in respiratory chains of eukaryotic mitochondria and some bacteria. Plastoquinones are components of photosynthetic electron transport chains of cyanobacteria and plant chloroplasts. Biosynthetic pathway of isoprenoid quinones has been described, as well as their additional, recently recognized, diverse functions in bacterial, plant and animal metabolism.     

Chlorosomes of green sulfur bacteria: Pigment composition and energy transfer

The pigment composition and energy transfer pathways in isolated chlorosomes ofChlorobium phaeovibrioides andChlorobium vibrioforme were studied by means of high performance liquid chromatography (HPLC) and picosecond absorbance difference spectroscopy. Analysis of pigment extracts of the chlorosomes revealed that they contain small amounts of bacteriochlorophyll (BChl)a esterified with phytol, whereas the BChlsc, d ande are predominantly esterified with farnesol. The chlorosomal BChla content inC. phaeovibrioides andC. vibrioforme was found to be 1.5% and 0.9%, respectively. The time resolved absorbance difference spectra showed a bleaching shifted to longer wavelengths as compared to the Q_y absorption maxima and in chlorosomes ofC. vibrioforme also an absorbance increase at shorter wavelengths was observed. These spectral features were ascribed to excitation of oligomers of BChle and BChlc/d, respectively. One-color and two-color pump-probe kinetics ofC. phaeovibrioides showed rapid energy transfer to long-wavelength absorbing BChle oligomers, followed by trapping of excitations by BChla with a time constant of about 60 ps. Time resolved anisotropy measurements inC. vibrioforme showed randomization of excitations among BChla molecules with a time constant of about 20 ps, indicating that BChla in the baseplate is organized in clusters. One-color and two-color pump-probe measurements inC. vibrioforme showed rapid energy transfer from short-wavelength to long-wavelength absorbing oligomers with a time constant of about 11 ps. Trapping of excitations by BChla in this species could not be resolved unambiguously due to annihilation processes in the BChla clusters, but may occur with time constants of 15, 70 and 200 ps.     

Identification of the major chlorosomal bacteriochlorophylls of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides; their function in lateral energy transfer

The chlorosomal bacteriochlorophyll (BChl) composition of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides was investigated by means of normal-phase high-performance liquid chromatography. From both species a number of homologues was isolated, which were identified by absorption and ²⁵²Cf-plasma desorption mass spectroscopy. Besides BChl d, C. vibrioforme contained a significant amount of BChl c, which may provide an explanation for the previous observation of at least two spectrally different pools of BChl in the chlorosomes of green sulfur bacteria (Otte et al. 1991). C. phaeovibrioides contained various homologues of BChl e only. Absorption spectra in acetone of BChl c, d and e, as well as bacteriopheophytin e are presented. No systematic differences were found for the various homologues of each pigment. In addition to farnesol, the mass spectra revealed the presence of various minor esterifying alcohols in both species, including phytol, oleol, cetol and 4-undecyl-2-furanmethanol, as well as an alcohol of low molecular mass, which is tentatively assumed to be decenol.Abbreviations BChl bacteriochlorophyll - BPh bacteriopheophytin (used as a general name for the Mg-free compound, irrespective of the esterifying alcohol) - HPLC high-performance liquid chromatography     

Glycolipids and the structure of chlorosomes in green bacteria

1. The cellular content of galactolipids in Chlorobium and Chloroflexus is not related to bacteriochlorophyll content nor to the total amount of chlorosome material in the cells.
2. Chlorosomes of both bacteria were agglutinated by Ricinus lectin and the agglutination was increased after treatment of the chlorosomes with trypsin.
3. When cell free preparations of both bacteria were treated with trypsin prior to centrifugation on sucrose gradients, the resulting chlorosome fractions were less contaminated with material derived from the cytoplasmic membrane than when trypsin was not employed.

     

Supramolecular organization of chlorosomes (chlorobium vesicles) and of their membrane attachment sites in Chlorobium Limicola

The photosynthetic green bacterium Chlorobium limicola 6230 has been examined by freeze-fracture electron microscopy to investigate the size, form, distribution and supramolecular architecture of its chlorosomes (chlorobium vesicles) as well as the chlorosome attachment sites on the cytoplasmic membrane. The oblong chlorosomes that underlie the cytoplasmic membrane show a considerable variation in size from about 40 × 70 nm to 100 × 260 nm and exhibit no particular orientation. The chlorosome core, which appears to be hydrophobic in nature, contains between 10 and 30 rod-shaped elements (approx. 10 nm in diameter) surrounded by an unetchable matrix. The rod elements are closely packed and extend the full length of the chlorosome. Separating the chlorosome core from the cytoplasm is a approx. 3 nm thick lipid-like envelope layer, which exhibits no substructure. A 5–6 nm thick, crystalline baseplate connects the chlorosome to the cytoplasmic membrane. The ridges of the baseplate lattice make an angle of between 40° and 60° with the longitudinal axis of the chlorosome and have a repeating distance of approx. 6 nm. In addition, each ridge exhibits a granular substructure with a periodicity of approx. 3.3 nm. The cytoplasmic membrane regions adjacent to the baseplates are enriched in large (greater than 9 nm) intramembrane particles, most of which belong to approx. 10 nm and approx. 12.5 nm particle size categories. Each chlorosome attachment site contains between 20 and 30 very large (greater than 12.0 nm diameter) intramembrane particles.The following interpretive model of a chlorosome is discussed in terms of biophysical, biochemical and structural information reported by others: it is proposed that the bacteriochlorophyll c (BChl c; chlorobium chlorophyll) is located in the rod elements of the core and that it is complexed with specific proteins. The cytoplasm-associated envelope layer is depicted as consisting of a monolayer of galactosyl diacylglycerol molecules. BChl a-protein complexes in a planar lattice configuration most likely make up the crystalline baseplate. The greater than 12-nm particles in the chlorosome attachment sites of the cytoplasmic membrane, finally, may correspond to complexes containing a reaction center and non-crystalline light-harvesting BChl a. The crystalline nature of the baseplate is consistent with the notion that it serves two functions: besides transferring excitation energy to the reaction centers it could also function as a distributor of this energy amongst the reaction centers.     

Pigment interactions in chlorosomes of various green bacteria

Resonance Raman experiments were performed on different green bacteria. With blue excitation, i.e. under Soret resonance or preresonance conditions, resonance Raman contributions were essentially arising from the chlorosome pigments. By comparing these spectra and those of isolated chlorosomes, it is possible to evaluate how the latter retain their native structure during the isolation procedures. The structure of bacteriochlorophyll oligomers in chlorosomes was interspecifically compared, in bacteriochlorophyllc- and bacteriochlorophylle- synthesising bacteria. It appears that interactions assumed by the 9-keto carbonyl group are identical inChlorobium limicola, Chlorobium tepidum, andChlorobium phaeobacteroides. In the latter strain, the 3-formyl carbonyl group of bacteriochlorophylle is kept free from intermolecular interactions. By contrast, resonance Raman spectra unambiguously indicate that the structure of bacteriochlorophyll oligomers is slightly different in chlorosomes fromChloroflexus auranticus, either isolated or in the whole bacteria.     

Photoproduction of H2 from acetate by syntrophic cocultures of green sulfur bacteria and sulfur-reducing bacteria

The marine green sulfur bacterium Chlorobium vibrioforme strain 1930 produced H₂ and elemental sulfur from sulfide or thiosulfate under N limitation in the light. H₂ production depended on nitrogenase and occurred only in the absence of ammonia. Methionine sulfoximine, an inhibitor of glutamine synthetase, prevented the switch-off by ammonia. In defined syntrophic cocultures of the acetate-oxidizing, sulfur-reducing bacterium Desulfuromonas acetoxidans with green sulfur bacteria, H₂ was produced from acetate via a light-driven sulfur cycle. The sulfur-reducing bacterium could not be replaced by sulfate-reducing bacteria in these experiments. In a coculture of the marine Chlorobium vibrioforme strain 1930 and the sulfur-reducing bacterium Desulfuromonas acetoxidans strain 5071, optimum long-term H₂ production from acetate was obtained with molecular nitrogen as N source, at low light intensity (110 mol · m^-2 · s^-1), in sulfide-reduced mineral medium (2 mM Na₂S) at pH 6.8. Traces of sulfide (10 M) were sufficient to keep the sulfur cycle running. The coculture formed no poly--hydroxyalkanoates (PHA), but 20%–40% polysaccharide per cell dry mass. Per mol acetate added, the coculture formed 3.1 mol of H₂ (78% of the theoretical maximum). Only 8% of the reducing equivalents was incorporated into biomass. The maximum rate of H₂ production was 1300 ml H₂ per day and g cell dry mass.Non-standard abbrevations MOPS 2-(N-morpholino) propane sulfonic acid - MSX Methionine sulfoximine - PHA poly--hydroxyalkanoates     

Growth yields of green sulfur bacteria in mixed cultures with sulfur and sulfate reducing bacteria

1. Dry weight yields from mixed cultures ofProsthecochloris aestuarii orChlorobium limicola with the sulfur reducingDesulfuromonas acetoxidans were determined on different growth limiting amounts of acetate, ethanol or propanol. The obtained yields agreed well with values predicted from stoichiometric calculations. 2. From mixed cultures of twoChlorobium limicola strains withDesulfovibrio desulfuricans orD. gigas on ethanol as the growth limiting substrate, dry weight yields were obtained as calculated for the complete utilization of the ethanol by the mixed cultures. 3. Dry weight yield determinations for two pure cultures ofChlorobium limicola with different growth limiting amounts of sulfide in the absence and presence of excess acetate confirmed that acetate is incorporated byChlorobium in a fixed proportion to sulfide; compared to the yield in the absence of acetate the yield is increased two to threefold in the presence of acetate. 4. The lowest possible sulfide concentrations necessary for optimal growth of mixed cultures of eitherProsthecochloris orChlorobium withDesulfuromonas on acetate were 7–8 mg H₂S per liter of medium. 5. Doubling times at the growth rate limiting light intensities of 5, 10, 20, 50, 100 and 200 lux were determined under optimal growth conditions for the following phototrophic bacteria:Prosthecochloris aestuarii, Chlorobium phaeovibriodes, Chromatium vinosum andRhodopseudomonas capsulata. Reasonably good growth was still obtained withProsthecochloris at 10 and 5 lux light intensity at which no growth of the purple bacteria could be observed.