Abundance and activity of methanotrophic bacteria in littoral and profundal sediments of lake constance (Germany).

The abundances and activities of aerobic methane-oxidizing bacteria (MOB) were compared in depth profiles of littoral and profundal sediments of Lake Constance, Germany. Abundances were determined by quantitative PCR (qPCR) targeting the pmoA gene and by fluorescence in situ hybridization (FISH), and data were compared to methane oxidation rates calculated from high-resolution concentration profiles. qPCR using type I MOB-specific pmoA primers indicated that type I MOB represented a major proportion in both sediments at all depths. FISH indicated that in both sediments, type I MOB outnumbered type II MOB at least fourfold. Results obtained with both techniques indicated that in the littoral sediment, the highest numbers of methanotrophs were found at a depth of 2 to 3 cm, corresponding to the zone of highest methane oxidation activity, although no oxygen could be detected in this zone. In the profundal sediment, highest methane oxidation activities were found at a depth of 1 to 2 cm, while MOB abundance decreased gradually with sediment depth. In both sediments, MOB were also present at high numbers in deeper sediment layers where no methane oxidation activity could be observed.

Energetics of syntrophic cooperation in methanogenic degradation.

Fatty acids and alcohols are key intermediates in the methanogenic degradation of organic matter, e.g., in anaerobic sewage sludge digestors or freshwater lake sediments. They are produced by classical fermenting bacteria for disposal of electrons derived in simultaneous substrate oxidations. Methanogenic bacteria can degrade primarily only one-carbon compounds. Therefore, acetate, propionate, ethanol, and their higher homologs have to be fermented further to one-carbon compounds. These fermentations are called secondary or syntrophic fermentations. They are endergonic processes under standard conditions and depend on intimate coupling with methanogenesis. The energetic situation of the prokaryotes cooperating in these processes is problematic: the free energy available in the reactions for total conversion of substrate to methane attributes to each partner amounts of energy in the range of the minimum biochemically convertible energy, i.e., 20 to 25 kJ per mol per reaction. This amount corresponds to one-third of an ATP unit and is equivalent to the energy required for a monovalent ion to cross the charged cytoplasmic membrane. Recent studies have revealed that syntrophically fermenting bacteria synthesize ATP by substrate-level phosphorylation and reinvest part of the ATP-bound energy into reversed electron transport processes, to release the electrons at a redox level accessible by the partner bacteria and to balance their energy budget. These findings allow us to understand the energy economy of these bacteria on the basis of concepts derived from the bioenergetics of other microorganisms.

The role of mycoplasmas in a conservation project of the lesser kestrel (Falco naumanni).

The lesser kestrel (Falco naumanni) is one of the most endangered bird species in Europe, and a captive breeding and reintroduction project was established. A breeding project is vulnerable to pathogens, e.g., mycoplasmas, reducing the reproductive success and carrying the risk to release pathogens with the birds to the wild. Therefore, 18 infertile eggs and 43 dead in shell embryos of the breeding project, as well as 27 nestlings and 34 adult birds of the captive and three different free-ranging populations were investigated for the occurrence of mycoplasmas by culture and a Mycoplasma genus-specific polymerase chain reaction. All eggs, embryos, and hand-reared nestlings from the captive group were negative. In contrast, all parent-reared nestlings and 88% of the adults were positive. Mycoplasma falconis and unidentifiable mycoplasmas were detected in all groups. Mycoplasma buteonis was found in the captive and only in two of the three free-ranging populations. Sequencing the 16S rRNA gene of six randomly selected unidentified isolates showed that five isolates were similar and most likely had been found previously in a falcon from Germany. The remaining isolate demonstrated a very high homology to unidentified Mycoplasma isolates obtained previously from semen samples of raptors. The results suggest that these isolates might represent two new species. Mycoplasmas seem not to play a major role as pathogens in the breeding project, and there is no evidence that releasing birds poses a risk to the free-ranging population with regard to mycoplasmas. The study seems to be the first to describe the occurrence and role of mycoplasmas in the lesser kestrel.

Microbial degradation of natural and of new synthetic polymers.

In landfills, deposited waste material is usually faced with strictly anoxic conditions. This means that the design of new biodegradable polymers must take into consideration that degradation should be possible especially in the absence of molecular oxygen. Poly-beta-hydroxybutyrate is depolymerized by the anaerobic fermenting bacterium Ilyobacter delafieldii through an extracellular hydrolase. Monomers are degraded inside the cells through classical beta-oxidation. Polyalkanoates containing odd-numbered or branched-chain acid monomers should he degraded in an analogous manner; in most cases the final mineralization of these residues requires special pathways. A comparison of the chemistry of natural polymer biodegradation leads to the conclusion that synthetic biodegradable polymers should be designed in the future to contain linkages which can be cleaved by extracellular hydrolytic enzymes. Recent findings on aerobic and anaerobic bacterial degradation of synthetic polyethers suggest that natural evolution of new depolymerizing enzymes, perhaps from existing hydrolases, could be possible in a reasonable amount of time, provided that the monomers are likely energy sources for a broad variety of microbes.

Towards the reaction mechanism of pyrogallol-phloroglucinol transhydroxylase of Pelobacter acidigallici.

Conversion of pyrogallol to phloroglucinol was studied with the molybdenum enzyme transhydroxylase of the strictly anaerobic fermenting bacterium Pelobacter acidigallici. Transhydroxylation experiments in H218O revealed that none of the hydroxyl groups of phloroglucinol was derived from water, confirming the concept that this enzyme transfers a hydroxyl group from the cosubstrate 1,2,3, 5-tetrahydroxybenzene (tetrahydroxybenzene) to the acceptor pyrogallol, and simultaneously regenerates the cosubstrate. This concept requires a reaction which synthesizes the cofactor de novo to maintain a sufficiently high intracellular pool during growth. Some sulfoxides and aromatic N-oxides were found to act as hydroxyl donors to convert pyrogallol to tetrahydroxybenzene. Again, water was not the source of the added hydroxyl groups; the oxides reacted as cosubstrates in a transhydroxylation reaction rather than as true oxidants in a net hydroxylation reaction. No oxidizing agent was found that supported a formation of tetrahydroxybenzene via a net hydroxylation of pyrogallol. However, conversion of pyrogallol to phloroglucinol in the absence of tetrahydroxybenzene was achieved if little pyrogallol and a high amount of enzyme preparation was used which had been pre-exposed to air. Obviously, the enzyme was oxidized by air to form sufficient amounts of tetrahydroxybenzene from pyrogallol to start the reaction. A reaction mechanism is proposed which combines an oxidative hydroxylation with a reductive dehydroxylation via the molybdenum cofactor, and allows the transfer of a hydroxyl group between tetrahydroxybenzene and pyrogallol without involvement of water. With this, the transhydroxylase differs basically from all other hydroxylating molybdenum enzymes which all use water as hydroxyl source.

The membrane-bound hydrogenase of Alcaligenes eutrophus. I. Solubilization, purification, and biochemical properties.

The membrane-bound hydrogenase of Alcaligenes eutrophus was solubilized from washed membranes of autotrophically grown cells. The enzyme consists of two types of subunits and is an iron-sulfur protein. A flavin compound was not detected. The enzyme reacts only with few artificial electron acceptors.

Transhydroxylase of Pelobacter acidigallici: a molybdoenzyme catalyzing the conversion of pyrogallol to phloroglucinol.

Trihydroxybenzenes are degraded anaerobically through the phloroglucinol pathway. In Pelobacter acidigallici as well as in Pelobacter massiliensis, pyrogallol is converted to phloroglucinol in the presence of 1,2,3,5-tetrahydroxybenzene by intermolecular hydroxyl transfer. The enzyme catalyzing this reaction was purified to chromatographic and electrophoretic homogeneity. Gel filtration and electrophoresis revealed a heterodimer structure with an apparent molecular mass of 127 kDa for the native enzyme and 86 kDa and 38 kDa, respectively, for the subunits. The enzyme was not sensitive to oxygen. HgCl2, p-chloromercuribenzoic acid, and CuCl2 inhibited strongly the reaction indicating an essential function of SH-groups. Transhydroxylase had a pH-optimum of 7.0 and a pI of 4.1. The apparent temperature optimum was in the range of 53 degrees C to 58 degrees C. The activation energy for the conversion of pyrogallol and 1,2,3,5-tetrahydroxybenzene to phloroglucinol and tetrahydroxybenzene was 31.4 kJ per mol. Purified enzyme exhibited a specific activity of 3.1 mol min-1 mg-1 protein and an apparent Km for pyrogallol and 1,2,3,5-tetrahydroxybenzene of 0.70 mM and 0.71 mM, respectively. The enzyme was found to contain per mol heterodimer 1.1 mol molybdenum, 12.1 mol iron and 14.5 mol acid-labile sulfur. Requirement for molybdenum for transhydroxylating enzyme activity was proven also by cultivation experiments. No hints for the presence of flavins were obtained. The results presented here support the hypothesis that a redox reaction is involved in this intermolecular hydroxyl transfer.

Hydrogen metabolism in aerobic hydrogen-oxidizing bacteria.

A survey on organisms able to use molecular hydrogen as electron donor in the energy-yielding process is presented. In the group of the aerobic hydrogen-oxidizing bacteria so far two types of hydrogenases have been encountered, a NAD-reducing, soluble enzyme (H2 : NAD oxidoreductase) and a membrane-bound enzyme unable to reduce pyridine nucleotides. With respect to the distribution of both types of hydrogenases three groups of hydrogen-oxidizing bacteria can be diffentiated containing (i) both types (Alcaligenes eutrophus), (ii) a soluble enzyme only (Nocardia opaca lb), and (iii) a membrane-bound hydrogenase only (majority of genera and species). The results of studies on the NAD-specific hydrogenase of A. eutrophus are summarized. Results on the solubilization and purification of the membrane-bound hydrogenase of A. eutrophus are presented in detail. The enzyme was solubilized from purified membranes by Triton X-100 and sodium desoxycholate or phospholipase D. The crude membrane extract was fractionated by ammonium sulfate precipitation and chromatography on carboxymethylcellulose at pH 5.5. The enzyme was stable in potassium phosphate buffer; it resembles the soluble enzyme with respect to stability under oxidizing conditions. Further biochemical and immunological data indicate, however, that both enzymes are different with respect to their native structure.

Humic acid reduction by propionibacterium freudenreichii and other fermenting bacteria

Iron-reducing bacteria have been reported to reduce humic acids and low-molecular-weight quinones with electrons from acetate or hydrogen oxidation. Due to the rapid chemical reaction of amorphous ferric iron with the reduced reaction products, humic acids and low-molecular-weight redox mediators may play an important role in biological iron reduction. Since many anaerobic bacteria that are not able to reduce amorphous ferric iron directly are known to transfer electrons to other external acceptors, such as ferricyanide, 2,6-anthraquinone disulfonate (AQDS), or molecular oxygen, we tested several physiologically different species of fermenting bacteria to determine their abilities to reduce humic acids. Propionibacterium freudenreichii, Lactococcus lactis, and Enterococcus cecorum all shifted their fermentation patterns towards more oxidized products when humic acids were present; P. freudenreichii even oxidized propionate to acetate under these conditions. When amorphous ferric iron was added to reoxidize the electron acceptor, humic acids were found to be equally effective when they were added in substoichiometric amounts. These findings indicate that in addition to iron-reducing bacteria, fermenting bacteria are also capable of channeling electrons from anaerobic oxidations via humic acids towards iron reduction. This information needs to be considered in future studies of electron flow in soils and sediments.

Growth of geobacter sulfurreducens with acetate in syntrophic cooperation with hydrogen-oxidizing anaerobic partners

Pure cultures of Geobacter sulfurreducens and other Fe(III)-reducing bacteria accumulated hydrogen to partial pressures of 5 to 70 Pa with acetate, butyrate, benzoate, ethanol, lactate, or glucose as the electron donor if electron release to an acceptor was limiting. G. sulfurreducens coupled acetate oxidation with electron transfer to an anaerobic partner bacterium in the absence of ferric iron or other electron acceptors. Cocultures of G. sulfurreducens and Wolinella succinogenes with nitrate as the electron acceptor degraded acetate efficiently and grew with doubling times of 6 to 8 h. The hydrogen partial pressures in these acetate-degrading cocultures were considerably lower, in the range of 0.02 to 0.04 Pa. From these values and the concentrations of the other reactants, it was calculated that in this cooperation the free energy change available to G. sulfurreducens should be about -53 kJ per mol of acetate oxidized, assuming complete conversion of acetate to CO2 and H2. However, growth yields (18.5 g of dry mass per mol of acetate for the coculture, about 14 g for G. sulfurreducens) indicated considerably higher energy gains. These yield data, measurement of hydrogen production rates, and calculation of the diffusive hydrogen flux indicated that electron transfer in these cocultures may not proceed exclusively via interspecies hydrogen transfer but may also proceed through an alternative carrier system with higher redox potential, e.g., a c-type cytochrome that was found to be excreted by G. sulfurreducens into the culture fluid. Syntrophic acetate degradation was also possible with G. sulfurreducens and Desulfovibrio desulfuricans CSN but only with nitrate as electron acceptor. These cultures produced cell yields of 4.5 g of dry mass per mol of acetate, to which both partners contributed at about equal rates. These results demonstrate that some Fe(III)-reducing bacteria can oxidize organic compounds under Fe(III) limitation with the production of hydrogen, and they provide the first example of rapid acetate oxidation via interspecies electron transfer at moderate temperature.

Energy conservation in fermentative glutarate degradation by the bacterial strain WoG13.

Dicarboxylic acids with 2-5 carbon atoms can be degraded fermentatively by pure cultures of various strictly anaerobic bacteria. The small amount of free energy released in these decarboxylations (about 20-25 kJ mol-1) is conserved as sole source of growth energy either through sodium-pumping decarboxylases or through electrogenic substrate/product transport devices. In the glutarate-fermenting bacterial strain WoG13 a glutaconyl-CoA-decarboxylating enzyme activity was detected. This enzyme was inhibited by avidin and was stimulated by sodium ions. The enzyme activity was partially associated with the cytoplasmic membrane, indicating that energy conservation is accomplished through a sodium-ion-pumping glutaconyl-CoA decarboxylase enzyme.

Anaerobic degradation of 3-hydroxybenzoate by a newly isolated nitrate-reducing bacterium.

A Gram-negative nitrate-reducing bacterium, strain Asl-3, was isolated from activated sludge with nitrate and 3-hydroxybenzoate as sole source of carbon and energy. The new isolate was facultatively anaerobic, catalase- and oxidase-positive and polarly monotrichously flagellated. In addition to nitrate, nitrite, N2O, and O2 served as electron acceptors. Growth with 3-hydroxybenzoate and nitrate was biphasic: nitrate was completely reduced to nitrite before nitrite reduction to N2 started. Benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, protocatechuate or phenyl-acetate served as electron and carbon source under aerobic and anaerobic conditions. During growth with excess carbon source, poly-beta-hydroxybutyrate was formed. These characteristics allow the affiliation of strain Asl-3 with the family Pseudomonadaceae. Analogous to the pathway of 4-hydroxybenzoate degradation in other bacteria, the initial step in anaerobic 3-hydroxybenzoate degradation by this organism was activation to 3-hydroxy-benzoyl-CoA in an ATP-consuming reaction. Cell extracts of 3-hydroxybenzoate-grown cells exhibited 3-hydroxybenzoyl-CoA synthetase activity of 190 nmol min-1 mg protein-1 as well as benzoyl-CoA synthetase activity of 86 nmol min-1 mg protein-1. A reductive dehydroxylation of 3-hydroxybenzoyl-CoA could not be demonstrated due to rapid hydrolysis of chemically synthesized 3-hydroxybenzoyl-CoA by cell extracts.

Lithotrophic growth and hydrogen metabolism by Clostridium magnum.

Clostridium magnum, originally described as a non-autotrophic homoacetogenic bacterium, was found to be able to grow with H2/CO2, formate, or methanol with stoichiometric acetate formation, provided that the growth medium contained at least 0.025% (w/v) yeast extract. Hydrogen was also formed as a byproduct of glucose fermentation, and was consumed again after glucose consumption. Hydrogen formation from glucose was independent of growth conditions and reached similar maximal concentrations in mineral media with or without ammonia added as well as in non-growing cultures or in the presence of carbon monoxide.

Fermentative degradation of acetone by an enrichment culture in membrane-separated culture devices and in cell suspensions.

A mixed culture, WoAct, growing on acetone, consisted of two dominant morphotypes: a rod-shaped acetone-fermenting bacterium producing acetate, and an acetate-utilizing Methanosaeta species. Dense cell suspensions, largely free of the aceticlastic methanogen and supplemented with bromoethanesulfonate, were able to degrade acetone and grow in small volumes in membrane-separated culture devices in which the acetate produced could diffuse into a large volume of medium. Acetone degradation and growth halted when the acetate concentration reached about 10 to 12 mM. Cell suspensions were able to degrade acetone in the absence of active methanogenesis, but the addition of 10 mM acetate inhibited acetone metabolism. Addition of an active culture of Methanosaeta sp. greatly stimulated the rate of acetone degradation. The results show that acetate removal in the mixed culture is not a prerequisite for growth and acetone degradation by the acetone-fermenting bacterium.

Acetylene degradation by new isolates of aerobic bacteria and comparison of acetylene hydratase enzymes.

Aerobic acetylene-degrading bacteria were isolated from soil samples. Two isolates were assigned to the species Rhodococcus opacus, two others to Rhodococcus ruber and Gordona sp. They were compared with known strains of aerobic acetylene-, cyanide-, or nitrile-utilizing bacteria. The acetylene hydratases of R opacus could be measured in cell-free extracts only in the presence of a strong reductant like titanium(III) citrate. Expression of these enzymes was molybdenum-dependent. Acetylene hydratases in cell-free extracts of R ruber and Gordona spp. did not require addition of reductants. No cross-reactivity could be found between cell-free extracts of any of these aerobic isolates and antibodies raised against the acetylene hydratase of the strictly anaerobic fermenting bacterium Pelobacter acetylenicus. These results show that acetylene hydratases are a biochemically heterogeneous group of enzymes.

Factors influencing the cultivability of lake water bacteria.

Counting bacteria in natural water samples by cultivation yields only low recovery efficiencies (ca. 1%), compared to total counts obtained after 4,6-diamidino-2-phenylindol (DAPI) staining. In order to optimize the cultivation of heterotrophic planktonic bacteria from Lake Constance (Germany), selected parameters of the medium composition were modified. The most important factor was the concentration of organic substrate (nutrient broth plus yeast extract), which significantly influenced the "most probable number" obtained in liquid growth medium. Reduced oxygen concentrations (3-12%) lowered the "most probable number". Addition of N-acyl homoserine lactones to the medium increased the cultivability slightly. Low substrate concentrations [0.03-0.06% (w/v)], an incubation atmosphere of 21% oxygen at 16 degrees C for 4 weeks were optimal and increased the cultivability ("most probable number" related to total bacterial counts) to an average cultivability of 18+/-11%, (n=8). The results indicate that cultivabilities of heterotrophic bacteria from lakewater samples can be significantly increased by modifying the cultivation methods.

The fermenting bacterium Malonomonas rubra is phylogenetically related to sulfur-reducing bacteria and contains a c-type cytochrome similar to those of sulfur and sulfate reducers.

Malonomonas rubra is a microaerotolerant fermenting bacterium which can maintain its energy metabolism for growth by decarboxylation of malonate to acetate. 16S rRNA sequence analysis revealed that M. rubra is closely related to the cluster of mesophilic sulfur-reducing bacteria within the delta subclass of the Proteobacteria, with the fermenting bacterium Pelobacter acidigallici and the sulfur reducers Desulfuromusa kysingii, D. bakii and D. succinoxidans as closest relatives. The cells contain high amounts (up to 12% of the total cell protein content) of a c-type cytochrome which is present mainly (> 60%) in the cytoplasm and to minor parts in the periplasm (> 20%) and associated with the membrane fraction (> 10%), independent of the growth substrate. This cytochrome is a tetraheme cytochrome of 13,700 Da molecular mass with a midpoint redox potential of -0.210 V.M. rubra does not reduce sulfur or ferric iron compounds. Since this cytochrome appears not to be involved in the energy metabolism it is concluded that it is a remnant of sulfur-reducing ancestors of this bacterium, without a conceivable physiological function in its present energy metabolism.

Prediction of biodegradability from structure: imidazoles.

A project for the development of Structure-Activity Relationship for Biodegradation is presented. The aim of the project is to assemble sets of structural rules governing the potential microbial degradability of (classes of) chemicals. These rules will provide tools to take into account the biodegradation aspects of a product--and all precursors in the production process--early in the product development. The modeling concept is to take all experimental biodegradation data available and combine structural trends in the data with mechanistical information from degradation pathways. The rules that are derived should give insight into the possibility of biodegradation for specific classes of chemicals, thereby revealing why a compound is biodegradable or not. For the class of imidazole derivatives such rules are derived, and a model degradation mechanism is proposed in analogy to the urocanate-hydratase mechanism from histidine metabolism. The model is validated using 12 imidazole-compounds, which are all predicted correctly to be poorly biodegradable. It is demonstrated that both data analysis and information on enzymatic reaction mechanisms are necessary to yield valid Structure-Biodegradation Relationship.

Anaerobic digestion: concepts, limits and perspectives.

Anaerobic degradation processes are faced with limitations with respect to reaction energetics and reaction kinetics. The small amount of energy available in methanogenic degradation of complex organic compounds allows in most cases only the conservation of minimum amounts of energy in the lowest range of energy exploitable by biochemical reactions for ATP-synthesis. This limit has to be defined in the range of 1/3-1/4 of an ATP unit, or 15-20 kJ per mol reaction. Such small amounts of energy are exploited efficiently by syntrophic microbial communities co-operating e.g. in fatty acid conversion to methane and CO2. Methanogens also set the stage for efficient conversion of sugars or amino acids, and channel electron fluxes to the utmost efficiency. Kinetic limitations are set by the inertness of certain compounds, e.g. hydrocarbons, to react in the absence of a strong oxidant. New reactions have been found recently which activate such compounds, e.g. aromatic hydrocarbons such as toluene, xylenes, naphthalene, methane, or ammonia. Refined techniques for analysis of microbial activities in ill defined natural environments such as digestive tracts of small invertebrates or polluted aquifers have shown an amazing capacity for anaerobic or oxygen-limited degradation processes that are still to be exploited. Thus, anaerobic digestion is still a matter of fast increasing knowledge, both on the side of basic research as well as on the side of application in treatment of soil, waste materials, or in understanding complex living communities.

Characterization and phylogeny of a novel methanotroph, Methyloglobulus morosus gen. nov., spec. nov.

A novel methanotrophic gammaproteobacterium, strain KoM1, was isolated from the profundal sediment of Lake Constance after initial enrichment in opposing gradients of methane and oxygen. Strain KoM1 grows on methane or methanol as its sole source of carbon and energy. It is a Gram-negative methanotroph, often expressing red pigmentation. Cells are short rods and occur sometimes in pairs or short chains. Strain KoM1 grows preferably at reduced oxygen concentrations (pO2=0.05-0.1bar). It can fix nitrogen, and grows at neutral pH and at temperatures between 4 and 30°C. Phylogenetically, the closest relatives are Methylovulum miyakonense and Methylosoma difficile showing 91% 16S rRNA gene sequence identity. The only respiratory quinone is ubiquinone Q8; the main polar lipids are phosphatidyl ethanolamine and phosphatidyl glycerol. The major cellular fatty acids are summed feature 3 (presumably C16:1ω7c) and C16:1ω5c, and the G+C content of the DNA is 47.7mol%. Strain KoM1 is described as the type strain of a novel species within a new genus, Methyloglobulus morosus gen. nov., sp. nov.