Corrinoids in anaerobic bacteria

Abstract C₁-metabolizing bacteria were analyzed for their corrinoids. The autotrophic phototrophe Chloroflexus aurantiacus contains predominantly the light-sensitive coenzyme B₁₂. The corrinoid could be teh prostethic group of a methylmalonyl-CoA mutase, which is involved in the CO₂ fixing reaction sequence from proplonyl-CoA to succinyl CoA. Methanobacterium thermoautotrophicum and Sporomusa ovata contain only traces of light-sensitive corrinoids, indicating that the demethylation reaction is favored, if these corrinoids are involved in methyl transfer reactions. The chemical structure of the unique p -cresolyl cobamide is specific for the acetogenic bacterium S. ovata , rather than the corrinoid 'factor III' for methanogenic bacteria.     

Responses of lactic acid bacteria to oxygen

Abstract A small number of flavoprotein oxidase enzymes are responsible for the direct interaction of lactic acid bacteria (LAB) with oxygen; hydrogen peroxide or water are produced in these reactions. In some cultures exposed to oxygen, hydrogen peroxide accumulates to inhibitory levels.
Through these oxidase enzymes and NADH peroxidase, O₂ and H₂O₂ can accept electrons from sugar metabolism, and thus have a sparing effect on the use of metabolic intermediates, such as pyruvate or acetaldehyde, as electron acceptors. Consequently, sugar metabolism in aerated cultures of LAB can be substantially different from that in unaerated cultures. Energy and biomass yields, end-products of sugar metabolism and the range of substrates which can be metabolised are affected.
Lactic acid bacteria exhibit an inducible oxidative stress response when exposed to sublethal levels of H₂O₂. This response protects them if they are subsequently exposed to lethal concentrations of H₂O₂. The effect appears to be related to other stress responses such as heat-shock and is similar, in some but not all respects, to that previously reported for enteric bacteria.     

Aerobic and anaerobic microbial consumption of hydrogen in geothermal spring water

Abstract Hydrogen consumption was measured in hot geothermal water from two ponds of the San Federigo solfatara, Tuscany, Italy, where emanation gases contained approx. 4% H₂. H₂ consumption was completely inhibited by NaOH and partially by HgCl₂ indicating microbial utilization. Aerobic and anaerobic H₂ consumption activities coexisted in the same water with aerobic activity being higher in one pond and anaerobic activity in the other. The kinetics of H₂ consumption were consistent with those of 'Knallgas', methanogenic or sulfidogenic bacteria.     

Methyl sulfides as intermediates in the anaerobic oxidation of methane

While it is clear that microbial consortia containing Archaea and sulfate-reducing bacteria (SRB) can mediate the anaerobic oxidation of methane (AOM), the interplay between these microorganisms remains unknown. The leading explanation of the AOM metabolism is 'reverse methanogenesis' by which a methanogenesis substrate is produced and transferred between species. Conceptually, the reversal of methanogenesis requires low H₂ concentrations for energetic favourability. We used ¹³C-labelled CH₄ as a tracer to test the effects of elevated H₂ pressures on incubations of active AOM sediments from both the Eel River basin and Hydrate Ridge. In the presence of H₂, we observed a minimal reduction in the rate of CH₄ oxidation, and conclude H₂ does not play an interspecies role in AOM. Based on these results, as well as previous work, we propose a new model for substrate transfer in AOM. In this model, methyl sulfides produced by the Archaea from both CH₄ oxidation and CO₂ reduction are transferred to the SRB. Metabolically, CH₄ oxidation provides electrons for the energy-yielding reduction of CO₂ to a methyl group ('methylogenesis'). Methylogenesis is a dominantly reductive pathway utilizing most methanogenesis enzymes in their forward direction. Incubations of seep sediments demonstrate, as would be expected from this model, that methanethiol inhibits AOM and that CO can be substituted for CH₄ as the electron donor for methylogenesis.     

Activity and composition of ammonia oxidizing bacterial communities and emission dynamics of NH3 and N2O in a compost reactor treating organic household waste

Aims:  To monitor emissions of NH₃ and N₂O during composting and link these to ammonia oxidation rates and the community structure of ammonia oxidizing bacteria (AOB).
Methods and Results:  A laboratory-scale compost reactor treating organic household waste was run for 2 months. NH₃ emissions peaked when pH started to increase. Small amounts of N₂O and CH₄ were also produced. In total, 16% and less than 1% of the initial N was lost as NH₃-N and N₂O-N respectively. The potential ammonia oxidation rate, determined by a chlorate inhibition assay, increased fourfold during the first 9 days and then remained high. Initially, both Nitrosospira and Nitrosomonas populations were detected using DGGE analysis of AOB specific 16S rRNA fragments. Only Nitrosomonas europaea was detected under thermophilic conditions, but Nitrosospira populations re-established during the cooling phase.
Conclusions:  Thermophilic conditions favoured high potential ammonia oxidation rates, suggesting that ammonia oxidation contributed to reduced NH₃ emissions. Small but significant amounts of N₂O were emitted during the thermophilic phase. The significance of different AOBs detected in the compost for ammonia oxidation is not clear.
Significance and Impact of Study:  This study shows that ammonia oxidation occurs at high temperature composting and therefore most likely reduces NH₃ emissions.     

Comparative study of enrichment methods for the isolation of autotrophic nitrifying bacteria from soil, estuarine and marine sediments

Abstract A comparative study has been undertaken to determine the efficiency of methods for the enrichment and isolation of autotrophic nitrifying bacteria from soils and estuarine and marine sediments. Chemostat enrichments proved to be the most efficient means of isolating autotrophic NH⁺₄ oxidisers whereas NO⁻₂ oxidising bacteria were never successfully enriched by this method. In contrast, gel enrichment and traditional batch culture enrichments of nitrifying bacteria were comparatively time consuming procedures and the degree of enrichment obtained for NH⁺₄ oxidising bacteria never approached that obtained with continuous culture enrichments. Gel enrichments, however, because they have continuous physicochemical gradients provide qualitative advantages in that morphologically distinct types of nitrifying bacteria can be isolated from the same gel.     

Measurement of mercaptoacetate levels in anaerobic batch culture of colonic bacteria

Abstract Mercaptoacetate levels were measured by HPLC utilizing precolumn derivitisation with o -phthalaldehyde in bacteria suspensions incubated anaerobically in batch culture. Reproducibility of measurement had a coefficient of variation of 4.8% and the recovery was 98%. Suspensions of faecal bacteria were incubated under H₂/CO₂ or N₂/CO₂ (4:1 v/v) in anaerobic dilution solution, reduced with ascorbate, with either glucose, starch, dextran or dextran sulphate. Production of the short chain fatty acids (acetate, propionate and butyrate) and utilization of H₂ showed continuing microbial fermentation. Under these conditions mercaptoacetate was produced at variable rates between 0.06–12.34 μmol/g (dry weight) over 24 h. Incubations from 24 to 48 h revealed that mercaptoacetate was both produced and utilized. Endogenous mercaptoacetate production in the colon would assist in maintaining anaerobiosis in an environment exposed to variable amounts of oxygen.     

Key role of teichoic acids on aflatoxin B1 binding by probiotic bacteria

Aims:  To assess the ability of five probiotic bacteria to bind aflatoxin B₁ and to determine the key role of teichoic acids in the binding mechanism.
Methods and Results:  The strains were incubated in aqueous solutions containing aflatoxin B₁ (AFB₁). The amount of free toxin was quantified by HPLC. Stability of the bacteria–aflatoxin complex was evaluated by repeated washes with buffer. In order to understand the binding process, protoplasts, spheroplasts and cell wall components of two strains were analysed to assess their capacity to bind AFB₁. Additionally, the role of teichoic acids in the AFB₁ binding process was assessed. Lactobacillus reuteri strain NRRL14171 and Lactobacillus casei strain Shirota were the most efficient strains for binding AFB₁. The stability of the AFB₁–bacteria complex appears to be related to the binding ability of a particular strain; AFB₁ binding was also pH-dependent. Our results suggest that teichoic acids could be responsible for this ability.
Conclusions:  Our results provide information concerning AFB₁ binding by previously untested strains, leading to enhanced understanding of the mechanism by which probiotic bacteria bind AFB₁.
Significance and Impact of the Study:  Our results support the suggestion that some probiotic bacteria could prevent absorption of aflatoxin from the gastrointestinal tract.     

Temporal change of gas metabolism by hydrogen-syntrophic methanogenic bacterial associations in anoxic paddy soil

Abstract Interspecies H₂ transfer within methanogenic bacterial associations (MBA) accounted for 95–97% of the conversion of ¹⁴CO₂ to ¹⁴CH₄ in anoxic paddy soil. Only 3–5% of the ¹⁴CH₄ were produced from the turnover of dissolved H₂. The H₂-syntrophic MBA developed within 5 days after the paddy soil had been submerged and placed under anoxic atmosphere. Afterwards, both the contribution of MBA to H₂-dependent methanogenesis and the turnover of dissolved H₂ did not change significantly for up to 7 months of incubation. However, while the rates of H₂-dependent methanogenesis stayed relatively constant, the rates of total methanogenesis decreased. The contribution of MBA to H₂-dependent methanogenesis was further enhanced to 99% when the temperature was shifted from 30°C to 17°C, or when the soil had been planted with rice. This enhancement was partially due to an increased utilization of dissolved H₂ by chloroform-insensitive non-methanogenic bacteria, most probably homoacetogens, so that CH₄ production was almost completely restricted to H₂-syntrophic MBA. The activity of MBA, as measured by the conversion of ¹⁴CO₂ to ¹⁴CH₄, was stimulated by glucose, lactate, and ethanol to a similar or greater extent than by exogenous H₂. Propionate and acetate had no effect.     

Characterization of populations of aerobic hydrogen-oxidizing soil bacteria

Abstract Freshly isolated soil bacteria were screened for different characteristics of the H₂ metabolism without prior selection for growth on H₂. The bacteria were isolated from different grain size fractions of a neutral meadow cambisol and an acidic forest cambisol, and then tested (1) for the ability to oxidize H₂, (2) for chemolithoautotrophic growth on H₂ as sole electron donor and energy source, (3) for DNA-DNA-hybridization with two hydrogenase gene fragments from Alcaligenes eutrophus and Rhizobium leguminosarum , and (4) for reduction of 2,3,5-triphenyl-2H-tetrazoliumchloride (TTC) in the presence of H₂. Many (65–90%) of the isolates were able to reduce TTC, but only 30–65% were actually able to oxidize H₂ indicating that the TTC test was not a specific characteristic for H₂ oxidation ability. The TTC test was only reliable in pure cultures of known bacteria with optimized test conditions, here shown for Alcaligenes eutrophus, Bradyrhizobium japonicum and Nocardia opaca , but not in mixed cultures of unknown bacteria. Still less (< 30%) of the isolates were able to grow chemolithoautotrophically indicating that culturable aerobic bacteria with the ability for H₂ oxidation are more abundant than bacteria with the ability for chemolithoautotrophic growth. The DNA-DNA-hybridization test failed to detect many of the bacteria with H₂ oxidation activity, probably since the hydrogenase genes present in the isolates were too diverse to be all detected by the DNA probes applied.     

CO2 reduction to acetate in anaerobic bacteria

Abstract The reduction of 2 CO₂ to acetate is catalyzed in the energy metabolism of homoacetogenic bacteria, which couple acetate formation to the synthesis of ATP. The carboxyl group of acetate is formed from CO₂ via reduction to a bound carbonyl ([CO]), a redution that requires the input of methaolic energy when hydrogen is used as the electron donor. The methyl group of acetate is formed via formate and tetrahydrofolate bound C₁ intermediates including methyl tetrahydrofolate as the intermediates. The methyl group is the 'condensed' with the carbonyl and CoA to acetyl-CoA, which is converted to acetate in the energy metabolism or to cell carbon in the anabolism of the bacteria. The mechanism of ATP synthesis coupled to CO₂ reduction to acetate is still unclear. The only reaction sufficiently exergonic is the reduction of methylene tetrahydrofolate to methyl tetrahydrofolate. Indirect evidence was presented that this reaction in homoacetogens might be coupled to the electrogenic transport of sodium across the cytoplasmic membrane. The sodium gradient formed via methylene-THF reduction could be transformed into a proton gradient via a sodium/proton antiporter. ATP would then be synthesized by a proton translocating ATP synthase.     

Biological oxidation of hydrogen in soils flushed with a mixture of H2, CO2, O2 and N2

Abstract A stainless steel cylinder filled with soil was flushed upstream with a H₂/CO₂/air mixture. The consequence was a strong enrichment of the aerobic, autotrophic hydrogen-oxidising microflora, which reached densities enabling them to oxidize 84.5 ml H₂· dm⁻²· h⁻¹ in the first 25-cm layer. H₂ concentration profiles, hydrogen uptake activity and cell numbers correlated well with each other. Most of the organisms isolated were dinitrogen fixers. Thus, soils containing hydrogen-oxidising bacteria may act as a biological shield between H₂-rich environments and air, and may be utilized as biofilters, e.g., in the waste-processing industry.     

Incorporation of [methyl-3H]thymidine by obligate and facultative anaerobic bacteria when grown under defined culture conditions

Abstract Incorporation of [ methyl -³H]thymidine into bacterial DNA was determined for a range of axenic anaerobic bacterial cultures: fermentative heterotrophs, sulphate-reducing bacteria, purple sulphur bacteria, acetogens and methanogens. Anaerobically growing Bacillus sp. and the obligate aerobe Thiobacillus ferrooxidans were also investigated. Actively growing cultures of sulphate-reducing bacteria belonging to the genera Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfobotulus and Desulfobulbus , purple sulphur bacteria ( Chromatium vinosum OP2 and Thiocapsa roseopersicina OP1), methanogens ( Methanococcus GS16 and Methanosarcina barkeri ) and an acetogen ( Acetobacterium woodii ) did not incorporate [ methyl -³H]thymidine into DNA. The only obligate anaerobes in which thymidine incorporation into DNA could be unequivocally demonstrated were members of the genus Clostridium . Anaerobically growing Bacillus sp. also incorporated thymidine. These data demonstrate that pure culture representatives of major groups of anaerobic bacteria involved in the terminal oxidation of organic carbon and anoxygenic phototrophs within sediments are unable to incorporate [ methyl -³H]thymidine into DNA, although some obligate and facultative anaerobes can. Variability in thymidine incorporation amongst pure culture isolates indicates that unless existing techniques can be calibrated to take this into consideration then productivity estimates in both aerobic and anaerobic environments may be greatly underestimated using the [ methyl -³H]thymidine technique.     

The acetyl-CoA pathway of autotrophic growth

Abstract The most direct conceivable route for synthesis of multicarbon compounds from CO₂ is to join two molecules of CO₂ together to make a 2-carbon compound and then polymerize the 2-carbon compound or add CO₂ successively to the 2-carbon compound to make multicarbon compounds. Recently, it has been demonstrated that the bacterium, Clostridium thermoaceticum , grows autotrophically by such a process. The mechanism involves the reduction of one molecule of CO₂ to a methyl group and then its combination with a second molecule of CO₂ and CoA to form acetyl-CoA. We have designated this autotrophic pathway the acetyl-CoA pathway [1]. Evidence is accumulating that this pathway is utilized by other bacteria that grow with CO₂ and H₂ as the source of carbon and energy. This group includes bacteria which, like C. thermoaceticum , produce acetate as a major end product and are called acetogens or acetogenic bacteria. It also includes the methane-producing bacteria and sulfate-reducing bacteria.
The purpose of this review is to examine critically the evidence that the acetyl-CoA pathway occurs in other bacteria by a mechanism that is the same or similar to that found in C. thermoaceticum . For this purpose, the mechanism of the acetyl-CoA pathway, as found in C. thermoaceticum , is described and hypothetical mechanisms for other organisms are presented based on the acetyl-CoA pathway of C. thermoaceticum . The available data have been reviewed to determine if the hypothetical schemes are in accord with presently known facts. We conclude that the formation of acetyl-CoA by other acetogens, the methanogens and sulphate-reducing bacteria occurs by a mechanism very similar to that of C. thermoaceticum .     

Time-resolved microfluorescence for measuring coenzyme F420 in Methanobacterium thermoautotrophicum

Abstract The time course of photobleaching and the nanosecond fluorescence decay have been measured from microscopic samples of methanogenic bacteria, to our knowledge the first application of these methods in this field. Decay times of about 1 ns and 3 ns were obtained for the specific coenzymes F₄₂₀ and 7-methylpterin, respectively. In contrast to methylpterin and other fluorescent compounds the intensity of F₄₂₀ fluorescence was reduced selectively due to photobleaching. This effect, as well as the different decay time constants could be used to discriminate F₄₂₀ from other fluorescent components. In addition, active and inactive bacterial cells could be differentiated following the course of photobleaching.     

Manganese in biogenic magnetite crystals from magnetotactic bacteria

Magnetotactic bacteria produce either magnetite (Fe₃O₄) or greigite (Fe₃S₄) crystals in cytoplasmic organelles called magnetosomes. Whereas greigite magnetosomes can contain up to 10 atom% copper, magnetite produced by magnetotactic bacteria was considered chemically pure for a long time and this characteristic was used to distinguish between biogenic and abiogenic crystals. Recently, it was shown that magnetosomes containing cobalt could be produced by three strains of Magnetospirillum . Here we show that magnetite crystals produced by uncultured magnetotactic bacteria can incorporate manganese up to 2.8 atom% of the total metal content (Fe+Mn) when manganese chloride is added to microcosms. Thus, chemical purity can no longer be taken as a strict prerequisite to consider magnetite crystals to be of biogenic origin.     

Isolation and characterization of thermophilic methanogenic bacteria from mushroom compost

Abstract During the first stage of the preparation of mushroom compost oxygen is believed to be readily available. However we measured methane in the evoking air above the compost piles and were able to isolate thermophilic methanogenic bacteria from this compost. The isolates grow only on H₂ and CO₂ as energy and carbon source and do not require complex factors for growth. On the basis of nutritional and morphological characteristics these methanogens were identified as strains of Methanobacterium thermoautotrophicum .     

Sodium dependent acetate formation from CO2 in Peptostreptococcus productus (strain Marburg)

Abstract Washed cells of Peptostreptococcus productus (strain Marburg), which were incubated in the presence of CO/CO₂/N₂ (50%/ 17%/ 33%; 200 kPa) catalyzed the synthesis of acetate from carbon monoxide. The rate of acetate formation from CO was stimulated more than threefold by the addition of sodium (10 mM); potassium did not effect acetate synthesis. The degree of stimulation was dependent on the sodium concentration; the dependence followed simple Michaelis-Menten kinetics. The apparent K _m for sodium was determined to be about 2 mmol/1. Sodium also stimulated acetate synthesis from H₂ plus CO₂. In the absence of added sodium the formation of formate as an intermediate in methyl group synthesis was stimulated. It is suggested that the sodium dependent reaction(s) is one (or more) of the reactions involved in methyl group synthesis from CO₂.     

Formation of vitamins by pure cultures of tempe moulds and bacteria during the tempe solid substrate fermentation

The formation of water soluble vitamins (vitamin B₁₂, vitamin B₆, riboflavin, thiamine, nicotinic acid and nicotinamide) during the tempe solid substrate fermentation was investigated. The role of several strains of Rhizopus oligosporus, R. arrhizus , and R. stolonifer and the role of several bacteria in the vitamin formation process were checked. All fungal and bacterial strains were isolated from Indonesian tempe and soaking water samples. The Rhizopus strains formed riboflavin, nicotinic acid, nicotinamide and vitamin B₆. The final concentrations of these substances depended on the different strains involved and on the fermentation time. Isolates of R. oligosporus were generally the best vitamin formers. The moulds did not produce physiologically active vitamin B₁₂. The thiamine content decreased during fermentation. The addition of bacteria, which had been selected in a screening for vitamin B₁₂ production, resulted in an increase of physiologically active vitamin B₁₂. Citrobacter freundii and Klebsiella pneumoniae showed the best formation capabilities. Furthermore, the bacteria produced riboflavin and vitamin B₆ in addition to the moulds. The influence of Rhizopus on the vitamin B₁₂ formation of Cit. freundii was also investigated. The vitamin content of tempe that was fermented with the mould and the bacterium was three times as high as a control fermentation with Cit. freundii only.     

Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway

Ladderane lipids are unusual membrane lipids of bacteria that anaerobically oxidize ammonium to dinitrogen gas (anammox). Ladderane lipids contain linearly concatenated cyclobutane rings for which the pathway of biosynthesis is currently unknown. To investigate the possible biosynthetic routes of these lipids, 2-¹³C-labelled acetate was added to a culture of the anammox bacterium Candidatus Brocadia fulgida. Labelling patterns obtained by high-field ¹³C nuclear magnetic resonance spectroscopy of isolated lipids indicated that C . Brocadia fulgida synthesizes C_16:0 and iso C_16:0 fatty acids according to the known pathway of type II fatty acid biosynthesis. The ¹³C-labelling pattern of the C₈ alkyl chain of the C₂₀ [3] ladderane monoether also indicated the use of this route. However, carbon atoms in the cyclobutane rings and the cyclohexane ring were nonspecifically labelled and did not correspond to known patterns of fatty acid synthesis. Taken together, our results indicate that it is unlikely that ladderane lipids are formed from the cyclization of polyunsaturated fatty acids as hypothesized previously and suggest an alternative, although as yet unknown, pathway of biosynthesis.