Heribert Cypionka

Oxidation of H2, organic compounds and inorganic sulfur compounds coupled to reduction of O2 or nitrate by sulfate-reducing bacteria

All of fourteen sulfate-reducing bacteria tested were able to carry out aerobic respiration with at least one of the following electron donors: H2, lactate, pyruvate, formate, acetate, butyrate, ethanol, sulfide, thiosulfate, sulfite. Generally, we did not obtain growth with O2 as electron acceptor. The bacteria were microaerophilic, since the respiration rates increased with decreasing O2 concentrations or ceased after repeated O2 additions. The amounts of O2 consumed indicated that the organic substrates were oxidized incompletely to acetate; only Desulfobacter postgatei oxidized acetate with O2 completely to CO2. Many of the strains oxidized sulfite (completely to sulfate) or sulfide (incompletely, except Desulfobulbus propionicus); thiosulfate was oxidized only by strains of Desulfovibrio desulfuricans; trithionate and tetrathionate were not oxidized by any of the strains. With Desulfovibrio desulfuricans CSN and Desulfobulbus propionicus the oxidation of inorganic sulfur compounds was characterized in detail. D. desulfuricans formed sulfate during oxidation of sulfite, thiosulfate or elemental sulfur prepared from polysulfide. D. propionicus oxidized sulfite and sulfide to sulfate, and elemental sulfur mainly to thiosulfate. A novel pathway that couples the sulfur and nitrogen cycles was detected: D. desulfuricans and (only with nitrite) D. propionicus were able to completely oxidize sulfide coupled to the reduction of nitrate or nitrite to ammonia. Cell-free extracts of both strains did not oxidize sulfide or thiosulfate, but formed ATP during oxidation of sulfite (37 nmol per 100 nmol sulfite). This, and the effects of AMP, pyrophosphate and molybdate on sulfite oxidation, suggested that sulfate is formed via the (reversed) sulfate activation pathway (involving APS reductase and ATP sulfurylase). Thiosulfate oxidation with O2 probably required a reductive first step, since it was obtained only with energized intact cells.

Cyclic AMP and Acyl Homoserine Lactones Increase the Cultivation Efficiency of Heterotrophic Bacteria from the Central Baltic Sea

ABSTRACT The effect of signal molecules on the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea was investigated. Numbers of cultivated cells were determined by the most-probable-number (MPN) technique. Artificial brackish water supplemented with different carbon substrates at low concentrations (200 μM each) was employed as the growth medium. Compared to the results of previous studies, this approach yielded significantly higher cultivation efficiencies (up to 11% in fluid media). A further and pronounced increase in cultivation success was accomplished by the addition of cyclic AMP (cAMP), N-butyryl homoserine lactone, or N-oxohexanoyl-dl-homoserine lactone at a low concentration of 10 μM. The most effective inducer was cAMP, which led to cultivation efficiencies of up to 100% of total bacterial counts. From the highest positive dilutions of these latter MPN series, several strains were isolated in pure culture and one strain (G100) was used to study the physiological effect of cAMP. Dot blot hybridization revealed, however, that strain G100 represented only a small fraction of the total bacterial community. This points towards an inherent limitation of the MPN approach, which does not necessarily recover abundant species from highly diverse communities. Bacterial cells of strain G100 that were starved for 6 weeks attained a higher growth rate and a higher biomass yield when resuscitated in the presence of cAMP instead of AMP.

The complete genome sequence of the algal symbiont Dinoroseobacter shibae: a hitchhiker's guide to life in the sea.

Dinoroseobacter shibae DFL12T, a member of the globally important marine Roseobacter clade, comprises symbionts of cosmopolitan marine microalgae, including toxic dinoflagellates. Its annotated 4 417 868 bp genome sequence revealed a possible advantage of this symbiosis for the algal host. D. shibae DFL12T is able to synthesize the vitamins B1 and B12 for which its host is auxotrophic. Two pathways for the de novo synthesis of vitamin B12 are present, one requiring oxygen and the other an oxygen-independent pathway. The de novo synthesis of vitamin B12 was confirmed to be functional, and D. shibae DFL12T was shown to provide the growth-limiting vitamins B1 and B12 to its dinoflagellate host. The Roseobacter clade has been considered to comprise obligate aerobic bacteria. However, D. shibae DFL12T is able to grow anaerobically using the alternative electron acceptors nitrate and dimethylsulfoxide; it has the arginine deiminase survival fermentation pathway and a complex oxygen-dependent Fnr (fumarate and nitrate reduction) regulon. Many of these traits are shared with other members of the Roseobacter clade. D. shibae DFL12T has five plasmids, showing examples for vertical recruitment of chromosomal genes (thiC) and horizontal gene transfer (cox genes, gene cluster of 47 kb) possibly by conjugation (vir gene cluster). The long-range (80%) synteny between two sister plasmids provides insights into the emergence of novel plasmids. D. shibae DFL12T shows the most complex viral defense system of all Rhodobacterales sequenced to date.

Growth with hydrogen, and further physiological characteristics of Desulfotomaculum species

Growth of Desulfotomaculum orientis, D. ruminis, D. nigrificans and the Desulfotomaculum strains TEP, TWC and TWP, that were newly isolated with sulfate and fatty acids, was studied using defined mineral media. Four of these strains grew with hydrogen plus sulfate as the only energy source. Under these conditions the growth yield of D. orientis in batch culture was 7.5 g cell dry mass per mol sulfate reduced. Growth on methanol with growth yields of about 6 g cell dry mass per mol sulfate was obtained with D. orientis and strain TEP. All strains tested grew slowly with formate as electron donor. Fatty acids from propionate to palmitate were utilized by the strains TEP, TWC and TWP. D. orientis and the strains TEP and TWC were able to utilize the methoxyl groups of trimethoxybenzoate for growth. D. orientis was found to grow chemoautotrophically with hydrogen, carbon dioxide and sulfate; during growth with C1-compounds no additional organic carbon source was required. Furthermore, D. orientis was able to grow slowly in sulfate-free medium with formate, methanol, ethanol lactate, pyruvate or trimethoxybenzoate. Under these conditions acetate was excreted, indicating the function of carbon dioxide as electron acceptor in a homoacetogenic process. A growth-promoting effect of pyrophosphate added to the medium of Desulfotomaculum species was not observed. The results show a high catabolic and anabolic versatility among Desulfotomaculum species, and indicate that electron transport to sulfate can be the sole energy conserving process in this genus.

Microbial Diversity in Coastal Subsurface Sediments: a Cultivation Approach Using Various Electron Acceptors and Substrate Gradients

ABSTRACT Microbial communities in coastal subsurface sediments are scarcely investigated and have escaped attention so far. But since they are likely to play an important role in biogeochemical cycles, knowledge of their composition and ecological adaptations is important. Microbial communities in tidal sediments were investigated along the geochemical gradients from the surface down to a depth of 5.5 m. Most-probable-number (MPN) series were prepared with a variety of different carbon substrates, each at a low concentration, in combination with different electron acceptors such as iron and manganese oxides. These achieved remarkably high cultivation efficiencies (up to 23% of the total cell counts) along the upper 200 cm. In the deeper sediment layers, MPN counts dropped significantly. Parallel to the liquid enrichment cultures in the MPN series, gradient cultures with embedded sediment subcores were prepared as an additional enrichment approach. In total, 112 pure cultures were isolated; they could be grouped into 53 different operational taxonomic units (OTU). The isolates belonged to the Proteobacteria, “Bacteroidetes,” “Fusobacteria,” Actinobacteria, and “Firmicutes.” Each cultivation approach yielded a specific set of isolates that in general were restricted to this single isolation procedure. Analysis of the enrichment cultures by PCR and denaturing gradient gel electrophoresis revealed an even higher diversity in the primary enrichments that was only partially reflected by the culture collection. The majority of the isolates grew well under anoxic conditions, by fermentation, or by anaerobic respiration with nitrate, sulfate, ferrihydrite, or manganese oxides as electron acceptors.

Influence of oxygen on sulfate reduction and growth of sulfate-reducing bacteria

The ambivalent relations of sulfate-reducing bacteria to molecular O2 have been studied with ten freshwater and marine strains. Generally, O2 was reduced prior to sulfur compounds and suppressed the reduction of sulfate, sulfite or thiosulfate to sulfide. Three strains slowly formed sulfide at O2 concentrations of below 15 μM (6% air saturation). In homogeneously aerated cultures, two out of seven strains tested, Desulfovibrio desulfuricans and Desulfobacterium autotrophicum, revealed weak growth with O2 as electron acceptor (up to one doubling of protein). However, O2 was concomitantly toxic. Depending on its concentration cell viability and motility decreased with time. In artificial oxygen-sulfide gradients with sulfide-containing agar medium and also in sulfide-free agar medium under an oxygen-containing gas phase, sulfate reducers grew in bands close to the oxic/anoxic interface. The specific O2 tolerance and respiration capacity of different strains led to characteristically stratified gradients. The maximum O2 concentration at the surface of a bacterial band (determined by means of microelectrodes) was 9 μM. The specific rates of O2 uptake per cell were in the same order of magnitude as the sulfate reduction rates in pure cultures. The bacteria stabilized the gradients, which were rapidly oxidized in the absence of cells or after killing the cells by formaldehyde. The motile strain Desulfovibrio desulfuricans CSN slowly migrated in the gradients in response to changing O2 concentrations in the gas phase.

Phospholipid analysis as a tool to study complex microbial communities in marine sediments.

To complement information on microbial communities in marine sediments that can be obtained using microbiological methods, we developed an analytical procedure to trace microbial lipids in environmental samples. We focused on analyzing intact phospholipids as these membrane constituents are known to be biomarkers for viable cells. Analysis of intact phospholipids from a fractionated and preconcentrated sediment extract was achieved using liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). The combined analysis of phospholipid types and their fatty acid substituents allowed a differentiation between various groups of microorganisms living in the sediment. For comparison three strains of marine sulfate-reducing bacteria (SRB) were analysed for their lipid content.

Microbial Communities in the Chemocline of a Hypersaline Deep-Sea Basin (Urania Basin, Mediterranean Sea)

ABSTRACT The Urania basin is a hypersaline sulfidic brine lake at the bottom of the eastern Mediterranean Sea. Since this basin is located at a depth of ∼3,500 m below the sea surface, it receives only a small amount of phytoplankton organic carbon. In the present study, the bacterial assemblages at the interface between the hypersaline brine and the overlaying seawater were investigated. The sulfide concentration increased from 0 to 10 mM within a vertical interval of 5 m across the interface. Within this chemocline, the total bacterial cell counts and the exoenzyme activities were elevated. Employing 11 cultivation methods, we isolated a total of 70 bacterial strains. The 16S ribosomal DNA sequences of 32 of the strains were identical to environmental sequences detected in the chemocline by culture-independent molecular methods. These strains were identified as flavobacteria, Alteromonas macleodii, and Halomonas aquamarina. All 70 strains could grow chemoorganoheterotrophically under oxic conditions. Sixty-six strains grew on peptone, casein hydrolysate, and yeast extract, whereas only 15 strains did not utilize polymeric carbohydrates. Twenty-one of the isolates could grow both chemoorganotrophically and chemolithotrophically. While the most probable numbers in most cases ranged between 0.006 and 4.3% of the total cell counts, an unsually high value of 54% was determined above the chemocline with media containing amino acids as the carbon and energy source. Our results indicate that culturable bacteria thriving at the oxic-anoxic interface of the Urania basin differ considerably from the chemolithoautotrophic bacteria typical of other chemocline habitats.

Specific Bacterial, Archaeal, and Eukaryotic Communities in Tidal-Flat Sediments along a Vertical Profile of Several Meters

ABSTRACT The subsurface of a tidal-flat sediment was analyzed down to 360 cm in depth by molecular and geochemical methods. A community structure analysis of all three domains of life was performed using domain-specific PCR followed by denaturing gradient gel electrophoresis analysis and sequencing of characteristic bands. The sediment column comprised horizons easily distinguishable by lithology that were deposited in intertidal and salt marsh environments. The pore water profile was characterized by a subsurface sulfate peak at a depth of about 250 cm. Methane and sulfate profiles were opposed, showing increased methane concentrations in the sulfate-free layers. The availability of organic carbon appeared to have the most pronounced effect on the bacterial community composition in deeper sediment layers. In general, the bacterial community was dominated by fermenters and syntrophic bacteria. The depth distribution of methanogenic archaea correlated with the sulfate profile and could be explained by electron donor competition with sulfate-reducing bacteria. Sequences affiliated with the typically hydrogenotrophic Methanomicrobiales were present in sulfate-free layers. Archaea belonging to the Methanosarcinales that utilize noncompetitive substrates were found along the entire anoxic-sediment column. Primers targeting the eukaryotic 18S rRNA gene revealed the presence of a subset of archaeal sequences in the deeper part of the sediment cores. The phylogenetic distance to other archaeal sequences indicates that these organisms represent a new phylogenetic group, proposed as “tidal-flat cluster 1.” Eukarya were still detectable at 360 cm, even though their diversity decreased with depth. Most of the eukaryotic sequences were distantly related to those of grazers and deposit feeders.

Monoalkylether phospholipids in the sulfate-reducing bacteria Desulfosarcina variabilis and Desulforhabdus amnigenus

Abstract. In this study, cellular lipid compositions of two mesophilic sulfate-reducing bacteria were analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS). In Desulfosarcina variabilis and Desulforhabdus amnigenus, alkylether-containing phospholipids were detected which had previously only been found in significant amounts in deeply branching hyperthermophilic bacteria and archaea. Combining information from HPLC-MS analysis and chemical degradation experiments, ether lipids were identified as 1-alkyl-2-acyl-phosphatidyl ethanolamines, glycerols and cholines. In Desulforhabdus amnigenus, n-penta-, n-hexa- and n-heptadecyl ethers were present (in order of decreasing abundance), whereas Desulfosarcina variabilis solely contained n-hexadecyl ether side chains.