Bernhard Schink

Syntrophism among Prokaryotes

The study of pure cultures in the laboratory has provided an amazingly diverse diorama of metabolic capacities among microorganisms, and has established the basis for our understanding of key transformation processes in nature. Pure culture studies are also prerequisites for research in microbial biochemistry and molecular biology. However, desire to understand how microorganisms act in natural systems requires the realization that microorganisms don’t usually occur as pure cultures out there, but that every single cell has to cooperate or compete with other microor macroorganisms. The pure culture is, with some exceptions such as certain microbes in direct cooperation with higher organisms, a laboratory artifact. Information gained from the study of pure cultures can be transferred only with great caution to an understanding of the behavior of microbes in natural communities. Rather, a detailed analysis of the abiotic and biotic life conditions at the microscale is needed for a correct assessment of the metabolic activities and requirements of a microbe in its natural habitat. In many cases, relationships of bacteria with other organisms may be relatively unimportant, as appears to be the case with most aerobes: they can usually degrade even fairly complex substrates to water and carbon dioxide without any significant cooperation with other organisms. Nutritional cooperation may exist, but may be re stricted to the transfer of minor growth factors, such as vitamins, from one organism to the other. However, we have to realize that this assumption is based on experience gained from pure cultures that were typically enriched and isolated in simple media, and the selection aimed at organisms

Clostridium ultunense sp. nov., a mesophilic bacterium oxidizing acetate in syntrophic association with a hydrogenotrophic methanogenic bacterium.

A syntrophic acetate-oxidizing bacterium, strain BST (T = type strain), was isolated from a previously described mesophilic triculture that was able to syntrophically oxidize acetate and form methane in stoichiometric amounts. Strain BST was isolated with substrates typically utilized by homoacetogenic bacteria. Strain BST was a spore-forming, gram-positive, rod-shaped organism which utilized formate, glucose, ethylene glycol, cysteine, betaine, and pyruvate. Acetate and sometimes formate were the main fermentation products. Small amounts of alanine were also produced from glucose, betaine, and cysteine. Strain BST grew optimally at 37 degrees C and pH 7. The G+C content of the DNA of strain BST was 32 mol%. A 16S rRNA sequence analysis revealed that strain BST was a member of a new species of the genus Clostridium. We propose the name Clostridium ultunense for this organism; strain BS is the type strain of C. ultunense.

Physiology, Ecology, Phylogeny, and Genomics of Microorganisms Capable of Syntrophic Metabolism

Syntrophic metabolism is diverse in two respects: phylogenetically with microorganisms capable of syntrophic metabolism found in the Deltaproteobacteria and in the low G+C gram‐positive bacteria, and metabolically given the wide variety of compounds that can be syntrophically metabolized. The latter includes saturated fatty acids, unsaturated fatty acids, alcohols, and hydrocarbons. Besides residing in freshwater and marine anoxic sediments and soils, microbes capable of syntrophic metabolism also have been observed in more extreme habitats, including acidic soils, alkaline soils, thermal springs, and permanently cold soils, demonstrating that syntrophy is a widely distributed metabolic process in nature. Recent ecological and physiological studies show that syntrophy plays a far larger role in carbon cycling than was previously thought. The availability of the first complete genome sequences for four model microorganisms capable of syntrophic metabolism provides the genetic framework to begin dissecting the biochemistry of the marginal energy economies and interspecies interactions that are characteristic of the syntrophic lifestyle.

Anaerobic and aerobic oxidation of ferrous iron at neutral pH by chemoheterotrophic nitrate-reducing bacteria

Nine out of ten anaerobic enrichment cultures inoculated with sediment samples from various freshwater, brackish-water, and marine sediments exhibited ferrous iron oxidation in mineral media with nitrate and an organic cosubstrate at pH 7.2 and 30° C. Anaerobic nitrate-dependent ferrous iron oxidation was a biological process. One strain isolated from brackish-water sediment (strain HidR2, a motile, nonsporeforming, gram-negative rod) was chosen for further investigation of ferrous iron oxidation in the presence of acetate as cosubstrate. Strain HidR2 oxidized between 0.7 and 4.9 mM ferrous iron aerobically and anaerobically at pH 7.2 and 30° C in the presence of small amounts of acetate (between 0.2 and 1.1 mM). The strain gained energy for growth from anaerobic ferrous iron oxidation with nitrate, and the ratio of iron oxidized to acetate provided was constant at limiting acetate supply. The ability to oxidize ferrous iron anaerobically with nitrate at approximately pH 7 appears to be a widespread capacity among mesophilic denitrifying bacteria. Since nitrate-dependent iron oxidation closes the iron cycle within the anoxic zone of sediments and aerobic iron oxidation enhances the reoxidation of ferrous to ferric iron in the oxic zone, both processes increase the importance of iron as a transient electron carrier in the turnover of organic matter in natural sediments.

Electron shuttling via humic acids in microbial iron(III) reduction in a freshwater sediment

The biological and chemical potential for electron shuttling via humic acids was evaluated by analyzing the depth distribution of humic-acid-reducing and iron-reducing bacteria in a freshwater sediment, and correlating it to the redox characteristics of humic acids and iron. Physicochemical analysis of profundal sediments of Lake Constance revealed a distinct stratification, with oxygen respiration, microbial iron and sulfate reduction, and methanogenesis allocatable to defined layers. Among the acid-extractable iron in the surface layer, ferric iron (Fe(III)) was dominant, whereas ferrous iron (Fe(II)) prevailed below 2 cm depth. Humic acids showed a higher electron-accepting (oxidizing) capacity in the surface layer and a higher reducing capacity in deeper layers. The more reduced redox state of humic acids in deeper layers was probably due to reduction by humic-acid-reducing microorganisms. Most-probable-number analysis revealed that the sediments contained populations of humic-acid-reducing bacteria that (i) were substantially larger than those of the iron-reducing bacteria in the respective sediment layers and (ii) were in the same range as those of the fermenting bacteria. Our results suggest that microbial reduction of humic acids and subsequent chemical reduction of poorly soluble iron(III) minerals by the reduced humic acids represents an important path of electron flow in anoxic natural environments such as freshwater sediments.

Anaerobic Naphthalene Degradation by a Sulfate-Reducing Enrichment Culture

ABSTRACT Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3,4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-13C]naphthalene or deuterated D8-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [13C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, 13C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.

Synergistic interactions in the microbial world

After several decades of microbiological research has focused on pure cultures, synergistic effects between different types of microorganisms find increasing interest. Interspecies interactions between prokaryotic cells have been studied into depth mainly with respect to syntrophic cooperations involved in methanogenic degradation of electron-rich substrates such as fatty acids, alcohols, and aromatics. Partners involved in these processes have to run their metabolism at minimal energy increments, with only fractions of an ATP unit synthesized per substrate molecule metabolized, and their cooperation is intensified by close proximity of the partner cells. New examples of such syntrophic activities are anaerobic methane oxidation by presumably methanogenic and sulfate-reducing prokaryotes, and microbially mediated pyrite formation. Syntrophic relationships have also been discovered to be involved in the anaerobic metabolization of amino acids and sugars where energetical restrictions do not necessarily force the partner organisms into strict interdependencies. The most highly developed cooperative systems among prokaryotic cells appear to be the structurally organized phototrophic consortia of the Chlorochromatium and Pelochromatium type in which phototrophic and chemotrophic bacteria not only exchange metabolites but also interact at the level of growth coordination and tactic behaviour.

Fermentation of Trihydroxybenzenes by Pelobacter acidigallici gen. nov. sp. nov., a New Strictly Anaerobic, Non-Sporeforming Bacterium

Five strains of rod-shaped, Gram-negative, non-sporing, strictly anaerobic bacteria were isolated from limnic and marine mud samples with gallic acid or phloroglucinol as sole substrate. All strains grew in defined mineral media without any growth factors; marine isolates required salt concentrations higher than 1% for growth, two freshwater strains only thrived in freshwater medium. Gallic acid, pyrogallol, 2,4,6-trihydroxybenzoic acid, and phloroglucinol were the only substrates utilized and were fermented stoichiometrically to 3 mol acetate (and 1 mol CO2) per mol with a growth yield of 10g cell dry weight per mol of substrate. Neither sulfate, sulfur, nor nitrate were reduced. The DNA base ratio was 51.8% guanine plus cytosine. A marine isolate, Ma Gal 2, is described as type strain of a new genus and species, Pelobacter acidigallici gen. nov. sp. nov., in the family Bacteroidaceae. In coculture with Acetobacterium woodii, the new isolates converted also syringic acid completely to acetate. Cocultures with Methanosarcina barkeri converted the respective substrates completely to methane and carbon dioxide.

Diversity of Ferrous Iron-Oxidizing, Nitrate-Reducing Bacteria and their Involvement in Oxygen-Independent Iron Cycling

In previous studies, three different strains (BrG1, BrG2, and BrG3) of ferrous iron-oxidizing, nitrate-reducing bacteria were obtained from freshwater sediments. All three strains were facultative anaerobes and utilized a variety of organic substrates and molecular hydrogen with nitrate as electron acceptor. In this study, analyses of 16S rDNA sequences showed that strain BrG1 was affiliated with the genus Acidovorax, strain BrG2 with the genus Aquabacterium, and strain BrG3 with the genus Thermomonas. Previously, bacteria similar to these three strains were detected with molecular techniques in MPN dilution series for ferrous iron-oxidizing, nitrate-reducing bacteria inoculated with different freshwater sediment samples. In the present study, further molecular analyses of these MPN cultures indicated that the ability to oxidize ferrous iron with nitrate is widespread amongst the Proteobacteria and may also be found among the Gram-positive bacteria with high GC content of DNA. Nitrate-reducing bacteria oxidized ferrous iron to poorly crystallized ferrihydrite that was suitable as an electron acceptor for ferric iron-reducing bacteria. Biologically produced ferrihydrite and synthetically produced ferrihydrite were both well suited as electron acceptors in MPN dilution cultures. Repeated anaerobic cycling of iron was shown in a coculture of ferrous iron-oxidizing bacteria and the ferric iron-reducing bacterium Geobacter bremensis. The results indicate that iron can be cycled between its oxidation states +II and +III by microbial activities in anoxic sediments.

Chlorobium ferrooxidans sp. nov., a phototrophic green sulfur bacterium that oxidizes ferrous iron in coculture with a "Geospirillum" sp. strain

Abstract A green phototrophic bacterium was enriched with ferrous iron as sole electron donor and was isolated in defined coculture with a spirilloid chemoheterotrophic bacterium. The coculture oxidized ferrous iron to ferric iron with stoichiometric formation of cell mass from carbon dioxide. Sulfide, thiosulfate, or elemental sulfur was not used as electron donor in the light. Hydrogen or acetate in the presence of ferrous iron increased the cell yield of the phototrophic partner, and hydrogen could also be used as sole electron source. Complexed ferric iron was slowly reduced to ferrous iron in the dark, with hydrogen as electron source. Similar to Chlorobium limicola, the phototrophic bacterium contained bacteriochlorophyll c and chlorobactene as photosynthetic pigments, and also resembled representatives of this species morphologically. On the basis of 16S rRNA sequence comparisons, this organism clusters with Chlorobium, Prosthecochloris, and Pelodictyon species within the green sulfur bacteria phylum. Since the phototrophic partner in the coculture KoFox is only moderately related to the other members of the cluster, it is proposed as a new species, Chlorobium ferrooxidans. The chemoheterotrophic partner bacterium, strain KoFum, was isolated in pure culture with fumarate as sole substrate. The strain was identified as a member of the ɛ-subclass of the Proteobacteria closely related to “Geospirillum arsenophilum” on the basis of physiological properties and 16S rRNA sequence comparison. The “Geospirillum” strain was present in the coculture only in low numbers. It fermented fumarate, aspartate, malate, or pyruvate to acetate, succinate, and carbon dioxide, and could reduce nitrate to dinitrogen gas. It was not involved in ferrous iron oxidation but possibly provided a thus far unidentified growth factor to the phototrophic partner.