RESUMEN

Populations of genetically identical cells can display marked variation in phenotypic traits; such variation is termed phenotypic heterogeneity. Here, we investigate the effect of substrate and electron donor limitation on phenotypic heterogeneity in N2 and CO2 fixation in the green sulphur bacterium Chlorobium phaeobacteroides. We grew populations in chemostats and batch cultures and used stable isotope labelling combined with nanometer-scale secondary ion mass spectrometry (NanoSIMS) to quantify phenotypic heterogeneity. Experiments in H2 S (i.e. electron donor) limited chemostats show that varying levels of NH4+ limitation induce heterogeneity in N2 fixation. Comparison of phenotypic heterogeneity between chemostats and batch (unlimited for H2 S) populations indicates that electron donor limitation drives heterogeneity in N2 and CO2 fixation. Our results demonstrate that phenotypic heterogeneity in a certain metabolic activity can be driven by different modes of limitation and that heterogeneity can emerge in different metabolic processes upon the same mode of limitation. In conclusion, our data suggest that limitation is a general driver of phenotypic heterogeneity in microbial populations.

Asunto(s)

Chlorobium/metabolismo , Sulfuro de Hidrógeno/metabolismo , Azufre/metabolismo , Chlorobium/clasificación , Chlorobium/genética , Chlorobium/aislamiento & purificación , Transporte de Electrón , Fijación del Nitrógeno , Fenotipo , Espectrometría de Masa de Ion Secundario

RESUMEN

Anoxygenic photoautotrophic bacteria which use light energy and electrons from Fe(II) for growth, so-called photoferrotrophs, are suggested to have been amongst the first phototrophic microorganisms on Earth and to have contributed to the deposition of sedimentary iron mineral deposits, i.e. banded iron formations. To date only two isolates of marine photoferrotrophic bacteria exist, both of which are closely related purple non-sulfur bacteria. Here we present a novel green-sulfur photoautotrophic Fe(II) oxidizer isolated from a marine coastal sediment, Chlorobium sp. strain N1, which is closely related to the freshwater green-sulfur bacterium Chlorobium luteolum DSM273 that is incapable of Fe(II) oxidation. Besides Fe(II), our isolated strain grew phototrophically with other inorganic and organic substrates such as sulfide, hydrogen, lactate or yeast extract. Highest Fe(II) oxidation rates were measured at pH 7.0-7.3, the temperature optimum was 25°C. Mössbauer spectroscopy identified ferrihydrite as the main Fe(III) mineral and fluorescence and helium-ion microscopy revealed cell-mineral aggregates without obvious cell encrustation. In summary, our study showed that the new isolate is physiologically adapted to the conditions of its natural habitat but also to conditions as proposed for early Earth and is thus a suitable model organism for further studies addressing phototrophic Fe(II) oxidation on early Earth.

Asunto(s)

Chlorobium , Compuestos Férricos/metabolismo , Sedimentos Geológicos/microbiología , Chlorobium/clasificación , Chlorobium/aislamiento & purificación , Chlorobium/metabolismo , Compuestos Ferrosos/metabolismo , Agua Dulce/microbiología , Hierro/metabolismo , Luz , Oxidación-Reducción , Azufre/metabolismo , Temperatura

RESUMEN

A natural planktonic bloom of a brown-pigmented photosynthetic green sulfur bacteria (GSB) from the disphotic zone of karstic Lake Banyoles (NE Spain) was studied as a natural enrichment culture from which a nearly complete genome was obtained after metagenomic assembly. We showed in situ a case where horizontal gene transfer (HGT) explained the ecological success of a natural population unveiling ecosystem-specific adaptations. The uncultured brown-pigmented GSB was 99.7% identical in the 16S rRNA gene sequence to its green-pigmented cultured counterpart Chlorobium luteolum DSM 273T. Several differences were detected for ferrous iron acquisition potential, ATP synthesis and gas vesicle formation, although the most striking trait was related to pigment biosynthesis strategy. Chl. luteolum DSM 273T synthesizes bacteriochlorophyll (BChl) c, whereas Chl. luteolum CIII incorporated by HGT a 18-kbp cluster with the genes needed for BChl e and specific carotenoids biosynthesis that provided ecophysiological advantages to successfully colonize the dimly lit waters. We also genomically characterized what we believe to be the first described GSB phage, which based on the metagenomic coverage was likely in an active state of lytic infection. Overall, we observed spread HGT and we unveiled clear evidence for virus-mediated HGT in a natural population of photosynthetic GSB.

Asunto(s)

Chlorobium/metabolismo , Transferencia de Gen Horizontal , Lagos/microbiología , Azufre/metabolismo , Proteínas Bacterianas/metabolismo , Bacterioclorofilas/metabolismo , Chlorobium/clasificación , Chlorobium/genética , Chlorobium/aislamiento & purificación , Ecosistema , Metagenómica , Fotosíntesis , ARN Ribosómico 16S/genética , España

RESUMEN

BACKGROUND: Chlorobium tepidum and Pelodictyon phaeoclathratiforme are organisms within the green sulphur bacteria family, Chlorobiaceae, occupying very different habitats. It has recently been proposed that the genera Chlorobium and Pelodictyon are synonymous. RESULTS: To investigate generic boundaries for the two species, protein families were predicted computationally based on sequence similarity across the genome-wide protein sets of Chlorobium tepidum TLS and Pelodictyon phaeoclathratiforme BU-1. The distribution of the resulting protein families across the two species was summarized. The largest number of families exhibited 1:1 putative orthology between the two species (1468 families). Of families unique to one of the species, the largest number was unique to P. phaeoclathratiforme (113 families), of which the largest family contained pentapeptide repeat proteins (16 proteins). Families unique to P. phaeoclathratiforme also included a family of gas vesicle synthesis proteins (four proteins). Although only seven families were identified as containing paralogous proteins in both species (with two or more proteins in each species), this group included families of major biochemical importance. One such family, with three members in each species, contained magnesium chelatase, an enzyme involved in the chlorophyll biosynthetic pathway. CONCLUSION: The unique protein family groups in both C. tepidum and P. phaeoclathratiforme mirror the occupancy of different environments, while key shared family groups provide evidence for a common origin for the species, as previously suggested in the literature. The current study only uses sequence similarity-based protein families for the two species. This, alone, does not permit a firm conclusion to be drawn on the taxonomic question, of whether the two species belong in one genus or two.

Asunto(s)

Proteínas Bacterianas/genética , Chlorobi/genética , Chlorobium/genética , Genoma Bacteriano , Liasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Chlorobi/clasificación , Chlorobi/metabolismo , Chlorobium/clasificación , Chlorobium/metabolismo , Biología Computacional , Ecosistema , Expresión Génica , Liasas/metabolismo , Redes y Vías Metabólicas/genética , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Chlorosomes, the major antenna complexes in green sulphur bacteria, filamentous anoxygenic phototrophs, and phototrophic acidobacteria, are attached to the cytoplasmic side of the inner cell membrane and contain thousands of bacteriochlorophyll (BChl) molecules that harvest light and channel the energy to membrane-bound reaction centres. Chlorosomes from phototrophs representing three different phyla, Chloroflexus (Cfx.) aurantiacus, Chlorobaculum (Cba.) tepidum and the newly discovered "Candidatus (Ca.) Chloracidobacterium (Cab.) thermophilum" were analysed using PeakForce Tapping atomic force microscopy (PFT-AFM). Gentle PFT-AFM imaging in buffered solutions that maintained the chlorosomes in a near-native state revealed ellipsoids of variable size, with surface bumps and undulations that differ between individual chlorosomes. Cba. tepidum chlorosomes were the largest (133×57×36nm; 141,000nm(3) volume), compared with chlorosomes from Cfx. aurantiacus (120×44×30nm; 84,000nm(3)) and Ca. Cab. thermophilum (99×40×31nm; 65,000nm(3)). Reflecting the contributions of thousands of pigment-pigment stacking interactions to the stability of these supramolecular assemblies, analysis by nanomechanical mapping shows that chlorosomes are highly stable and that their integrity is disrupted only by very strong forces of 1000-2000pN. AFM topographs of Ca. Cab. thermophilum chlorosomes that had retained their attachment to the cytoplasmic membrane showed that this membrane dynamically changes shape and is composed of protrusions of up to 30nm wide and 6nm above the mica support, possibly representing different protein domains. Spectral imaging revealed significant heterogeneity in the fluorescence emission of individual chlorosomes, likely reflecting the variations in BChl c homolog composition and internal arrangements of the stacked BChls within each chlorosome.

Asunto(s)

Bacterioclorofilas/química , Estructuras de la Membrana Celular/química , Chlorobium/clasificación , Chlorobium/fisiología , Citoplasma/metabolismo , Estructuras de la Membrana Celular/ultraestructura , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente

RESUMEN

At two stations surveyed in Nitinat Lake, a approximately 200-m-deep anoxic tidal fjord, sulfide was detected as close as 15 m from the surface. Biological characterization, determined from small subunit ribosomal RNA gene sequencing, of the chemocline and anaerobic zone revealed many sequences related to sulfur-oxidizing bacteria, suggesting that sulfur cycling is a dominant process. gamma- and epsilon-Proteobacteria related to thiotrophic symbionts, as well as Chlorobium sp., dominated the transition zone. These are expected to play a role in dark and phototrophic CO(2) fixation, respectively. epsilon-Proteobacteria phylotype abundance increased with depth, eventually comprising 69-97% of all sequences recovered from the anoxic zone. The vast majority (74%) of these phylotypes were affiliated with a novel Acrobacter sp. group (NITEP5). Quantification of NITEP5 revealed that up to 2.8 x 10(5) cells ml(-1) were present in the anoxic zone. Surprisingly, although sequences related to known sulfate-reducing bacteria were recovered from the transition zone, quantification of the dsr gene and (35)SO(4)(2-) uptake tests suggest that sulfate-reduction within the water column is negligible. Overall, sequence diversity between different vertical zones was high, although the spatial segregation of gamma-Proteobacteria, Chlorobi, and epsilon-Proteobacteria did not appear to vary significantly between seasons.

Asunto(s)

Bacterias/clasificación , Biodiversidad , Agua Dulce/microbiología , Filogenia , Bacterias/genética , Bacterias/aislamiento & purificación , Canadá , Chlorobium/clasificación , Chlorobium/genética , Chlorobium/aislamiento & purificación , Epsilonproteobacteria/clasificación , Epsilonproteobacteria/aislamiento & purificación , Gammaproteobacteria/clasificación , Gammaproteobacteria/genética , Gammaproteobacteria/aislamiento & purificación , Genes Bacterianos , Variación Genética , Geografía

RESUMEN

The first committed step in the biosynthesis of (bacterio-)chlorophyll is the insertion of Mg2+ into protoporphyrin IX by Mg-chelatase. In all known (B)Chl-synthesizing organisms, Mg-chelatase is encoded by three genes that are homologous to bchH, bchD, and bchI of Rhodobacter spp. The genomes of all sequenced strains of green sulfur bacteria (Chlorobi) encode multiple bchH paralogs, and in the genome of Chlorobaculum tepidum, there are three bchH paralogs, denoted CT1295 (bchT), CT1955 (bchS), and CT1957 (bchH). Cba. tepidum mutants lacking one or two of these paralogs were constructed and characterized. All of the mutants lacking only one of these BchH homologs, as well as bchS bchT and bchH bchT double mutants, which can only produce BchH or BchS, respectively, were viable. However, attempts to construct a bchH bchS double mutant, in which only BchT was functional, were consistently unsuccessful. This result suggested that BchT alone is unable to support the minimal (B)Chl synthesis requirements of cells required for viability. The pigment compositions of the various mutant strains varied significantly. The BChl c content of the bchS mutant was only approximately 10% of that of the wild type, and this mutant excreted large amounts of protoporphyrin IX into the growth medium. The observed differences in BChl c production of the mutant strains were consistent with the hypothesis that the three BchH homologs function in end product regulation and/or substrate channeling of intermediates in the BChl c biosynthetic pathway.

Asunto(s)

Proteínas Bacterianas/genética , Chlorobium/genética , Chlorobium/metabolismo , Clorofila/biosíntesis , Proteínas Bacterianas/metabolismo , Chlorobium/clasificación , Clorofila/química , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , Liasas/genética , Liasas/metabolismo , Modelos Genéticos , Estructura Molecular , Mutación , Filogenia , Protoporfirinas/química , Protoporfirinas/metabolismo

RESUMEN

The nitrogen cycling of Lake Cadagno was investigated by using a combination of biogeochemical and molecular ecological techniques. In the upper oxic freshwater zone inorganic nitrogen concentrations were low (up to approximately 3.4 microM nitrate at the base of the oxic zone), while in the lower anoxic zone there were high concentrations of ammonium (up to 40 microM). Between these zones, a narrow zone was characterized by no measurable inorganic nitrogen, but high microbial biomass (up to 4 x 10(7) cells ml(-1)). Incubation experiments with (15)N-nitrite revealed nitrogen loss occurring in the chemocline through denitrification (approximately 3 nM N h(-1)). At the same depth, incubations experiments with (15)N(2)- and (13)C(DIC)-labelled bicarbonate, indicated substantial N(2) fixation (31.7-42.1 pM h(-1)) and inorganic carbon assimilation (40-85 nM h(-1)). Catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and sequencing of 16S rRNA genes showed that the microbial community at the chemocline was dominated by the phototrophic green sulfur bacterium Chlorobium clathratiforme. Phylogenetic analyses of the nifH genes expressed as mRNA revealed a high diversity of N(2) fixers, with the highest expression levels right at the chemocline. The majority of N(2) fixers were related to Chlorobium tepidum/C. phaeobacteroides. By using Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS), we could for the first time directly link Chlorobium to N(2) fixation in the environment. Moreover, our results show that N(2) fixation could partly compensate for the N loss and that both processes occur at the same locale at the same time as suggested for the ancient Ocean.

Asunto(s)

Agua Dulce/microbiología , Fijación del Nitrógeno , Nitrógeno/análisis , Dióxido de Carbono/análisis , Chlorobium/clasificación , Chlorobium/aislamiento & purificación , Chlorobium/metabolismo , Chromatium/aislamiento & purificación , Chromatium/metabolismo , Agua Dulce/química , Hibridación in Situ , Nitritos/análisis , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Filogenia , Compuestos de Amonio Cuaternario/análisis , ARN Ribosómico 16S/metabolismo , Suiza

RESUMEN

The anoxygenic phototrophic bacterial community of the high-altitude meromictic Lake Gek-Gel (Azerbaijan) was investigated in September 2003. The highest concentration of bacteriochlorophyll e (48 microg/l) was detected at a depth of 30 m; the peak of bacteriochlorophyll a (4.5 microg/l) occurred at 29 m. Phylogenetic analysis revealed that brown-colored green sulfur bacteria Chlorobium phaeobacteroides predominated in the lake. Nonsulfur purple bacteria phylogenetically close to Blastochloris sulfoviridis were found in insignificant amounts; these organisms have not been previously reported in Lake Gek-Gel.

Asunto(s)

Chlorobium/clasificación , Agua Dulce/microbiología , Hyphomicrobiaceae/clasificación , Anaerobiosis , Azerbaiyán , Chlorobium/aislamiento & purificación , Chlorobium/metabolismo , Chlorobium/ultraestructura , Recuento de Colonia Microbiana , Hyphomicrobiaceae/aislamiento & purificación , Hyphomicrobiaceae/metabolismo , Hyphomicrobiaceae/ultraestructura , Microscopía Electrónica , Fotosíntesis , Filogenia

RESUMEN

The Black Sea is the largest extant anoxic water body on Earth. Its oxic-anoxic boundary is located at a depth of 100 m and is populated by a single phylotype of marine green sulfur bacteria. This organism, Chlorobium sp. strain BS-1, is extraordinarily low light adapted and can therefore serve as an indicator of deep photic zone anoxia (A. K. Manske, J. Glaeser, M. M. M. Kuypers, and J. Overmann, Appl. Environ. Microbiol. 71:8049-8060, 2005). In the present study, two sediment cores were retrieved from the bottom of the Black Sea at depths of 2,006 and 2,162 m and were analyzed for the presence of subfossil DNA sequences of BS-1 using ancient-DNA methodology. Using optimized cultivation media, viable cells of the BS-1 phylotype were detected only at the sediment surface and not in deeper layers. In contrast, green sulfur bacterial 16S rRNA gene fragments were amplified from all the sediment layers investigated, including turbidites. After separation by denaturing gradient gel electrophoresis and sequencing, 14 different sequence types were distinguished. The sequence of BS-1 represented only a minor fraction of the amplification products and was found in 6 of 22 and 4 of 26 samples from the 2,006- and 2,162-m stations, respectively. Besides the sequences of BS-1, three additional phylotypes of the marine clade of green sulfur bacteria were detected. However, the majority of sequences clustered with groups from freshwater habitats. Our results suggest that a considerable fraction of green sulfur bacterial chemofossils did not originate in a low-light marine chemocline environment and therefore were likely to have an allochthonous origin. Thus, analysis of subfossil DNA sequences permits a more differentiated interpretation and reconstruction of past environmental conditions if specific chemofossils of stenoec species, like Chlorobium sp. strain BS-1, are employed.

Asunto(s)

Chlorobi/clasificación , Fósiles , ARN Ribosómico 16S/genética , Agua de Mar/microbiología , Análisis de Secuencia de ADN , Chlorobi/genética , Chlorobi/crecimiento & desarrollo , Chlorobium/clasificación , Chlorobium/genética , Chlorobium/crecimiento & desarrollo , ADN Bacteriano/análisis , ADN Bacteriano/aislamiento & purificación , Genes de ARNr , Sedimentos Geológicos/microbiología , Datos de Secuencia Molecular , Filogenia

RESUMEN

A symbiotic green sulfur bacterium, strain CaD, was isolated from an enrichment culture of the phototrophic consortium "Chlorochromatium aggregatum". The capability of the epibiont to grow in pure culture indicates that it is not obligately symbiotic. Cells are Gram-negative, nonmotile, rod-shaped and contain chlorosomes. Strain CaD is obligately anaerobic and photolithoautotrophic, using sulfide as electron donor. Acetate and peptone are photoassimilated in the presence of sulfide and hydrogencarbonate. Photosynthetic pigments contain bacteriochlorophylls a and c, and gamma-carotene and OH-gamma-carotene glucoside laurate as the dominant carotenoids. In cells from pure cultures, chlorosomes are equally distributed along the inner face of the cytoplasmic membrane. In contrast, the distribution of the chlorosomes in symbiotic epibiont cells is uneven, with chlorosomes being entirely absent at the site of attachment to the central bacterium. The symbiotic epibiont cells display a conspicuous additional layered structure at the attachment site. The G + C content of genomic DNA of strain CaD is 46.7 mol%. On the basis of 16S rRNA sequence comparison, the strain is distantly related to Chlorobium species within the green sulfur bacteria phylum (

Asunto(s)

Chlorobium/clasificación , Chlorobium/fisiología , Sulfuros/metabolismo , Simbiosis , Microbiología del Agua , Ácido Acético/metabolismo , Anaerobiosis , Proteínas Bacterianas/análisis , Bacterioclorofila A/análisis , Bacterioclorofilas/análisis , Composición de Base , Bicarbonatos/metabolismo , Carotenoides/análisis , Chlorobium/citología , Chlorobium/aislamiento & purificación , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Violeta de Genciana , Locomoción , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Orgánulos/ultraestructura , Peptonas/metabolismo , Fenazinas , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico

RESUMEN

Population analyses in water samples obtained from the chemocline of crenogenic, meromictic Lake Cadagno, Switzerland, in October for the years 1994 to 2003 were studied using in situ hybridization with specific probes. During this 10-year period, large shifts in abundance between purple and green sulfur bacteria and among different populations were obtained. Purple sulfur bacteria were the numerically most prominent phototrophic sulfur bacteria in samples obtained from 1994 to 2001, when they represented between 70 and 95% of the phototrophic sulfur bacteria. All populations of purple sulfur bacteria showed large fluctuations in time with populations belonging to the genus Lamprocystis being numerically much more important than those of the genera Chromatium and Thiocystis. Green sulfur bacteria were initially represented by Chlorobium phaeobacteroides but were replaced by Chlorobium clathratiforme by the end of the study. C. clathratiforme was the only green sulfur bacterium detected during the last 2 years of the analysis, when a shift in dominance from purple sulfur bacteria to green sulfur bacteria was observed in the chemocline. At this time, numbers of purple sulfur bacteria had decreased and those of green sulfur bacteria increased by about 1 order of magnitude and C. clathratiforme represented about 95% of the phototrophic sulfur bacteria. This major change in community structure in the chemocline was accompanied by changes in profiles of turbidity and photosynthetically available radiation, as well as for sulfide concentrations and light intensity. Overall, these findings suggest that a disruption of the chemocline in 2000 may have altered environmental niches and populations in subsequent years.

Asunto(s)

Chlorobi/clasificación , Chlorobi/crecimiento & desarrollo , Chromatiaceae/clasificación , Chromatiaceae/crecimiento & desarrollo , Ecosistema , Agua Dulce/microbiología , Chlorobi/genética , Chlorobium/clasificación , Chlorobium/genética , Chlorobium/crecimiento & desarrollo , Chromatiaceae/genética , Chromatium/clasificación , Chromatium/genética , Chromatium/crecimiento & desarrollo , Recuento de Colonia Microbiana/métodos , Sondas de ADN , ADN Bacteriano/análisis , ADN Ribosómico/análisis , Agua Dulce/química , Hibridación in Situ , Datos de Secuencia Molecular , Fotosíntesis , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Azufre/metabolismo , Suiza , Factores de Tiempo

RESUMEN

The tendencies in the study of anoxygenic photosynthesizing bacteria (APB) are considered in the review in the historical aspect, since APB discovery till present day. The contribution made by the researchers of the Institute of Microbiology, Russian Academy of Sciences, to the study of the phylogeny, ecology, and morphophysiological diversity of APB is noted. At present, the molecular-biological approaches play a decisive role in ecology and taxonomy. The most important task at the modem stage of the development of microbiology is to maintain the continuity of the historically formed classical approaches in the study of APB diversity.

Asunto(s)

Bacterias Anaerobias , Fotosíntesis , Bacterias Anaerobias/clasificación , Bacterias Anaerobias/metabolismo , Chlorobium/clasificación , Chlorobium/metabolismo , Microbiología Ambiental , Filogenia , Federación de Rusia

RESUMEN

The light-dependent composition change of light harvesting bacteriochlorophyll(BChl)s in the present culture of a green sulfur photosynthetic bacterium Chlorobium (Chl.) vibrioforme f. sp. thiosulfatophilum strain NCIB 8327 was investigated by visible absorption spectroscopy and HPLC analyses. When the culture was repeatedly grown in liquid media under a low light condition, both the Soret and Qy absorption bands of the in vivo spectrum were shifted to longer wavelengths. Analysis of the extracted pigments by HPLC revealed that the ratio of the amount of BChl-c to that of BChl-d molecules gradually increased during repeated cultivation. In contrast, when the culture grown under a low light intensity was transferred to a high light condition and continued to be grown, the absorption bands were shifted to shorter wavelengths and the ratio of BChls-c/d decreased finally to the almost original value. Colonies were prepared on solid agar media from the liquid culture containing both BChls-c and d, which was grown under a low light intensity. Each colony obtained was found to contain either BChl-c or d, but not both of them. Two types of cells isolated in this study were derived from the same clone, judged from their genetic analyses. The variation of pigment composition in our liquid culture observed here could be ascribed to the difference of growth rates between two substrains containing BChl-c and BChl-d, respectively, depending on light conditions.

Asunto(s)

Proteínas Bacterianas/análisis , Bacterioclorofilas/análisis , Chlorobium/química , Chlorobium/aislamiento & purificación , Separación Celular , Chlorobium/clasificación , Cromatografía Líquida de Alta Presión , Luz , Análisis Espectral