Hydrogen-oxidizing Methane Bacteria II. Electron Microscopy

Comparison of the fine structure of the methanogenic organism from the culture known as Methanobacillus omelianskii with that of Methanobacterium formicicum revealed a great similarity. Both organisms exhibited a large number of intracytoplasmic membranous elements when stained with phosphotungstic acid. In contrast, no such elements were observed in Methanobacterium ruminantium.     

Inhibition of Rumen Methanogenesis by Methane Analogues

Extremely low concentrations of chloroform and carbon tetrachloride and somewhat larger concentrations of methylene chloride inhibited the formation of methane by the rumen microbiota in the presence or absence of added substrate. The accumulation of hydrogen at these low concentrations indicates a selective inhibition of methanogenesis. Presumably, these inhibitors affect one or more of the reactions by which methane is formed from hydrogen and carbon dioxide.     

大豆根际土壤中氢氧化细菌促生效应研究

以大豆根际土壤样品为研究材料,采用一个气体循环培养体系(持续通氢气装置),在适当的H2、O2和CO2下以H2作为唯一能源分离氢氧化细菌,结果共分离出40余株细菌,对其进行耗氢能力测定,结果显示有20株菌具有氧化氢功能和自养生长能力。对20株氢氧化细菌进行小麦促生试验,筛选出11株具有促生效果的根际促生细菌。采用薄层层析法,对11株氢氧化细菌进行了ACC降解的分析,确定了菌株A06具有ACC脱氨酶活性,证实ACC脱氨酶是导致大豆根际土壤中氢氧化细菌促进小麦幼苗生长的原因之一。     

Effect of Gramicidin on Methanogenesis by Various Methanogenic Bacteria

Methanogenesis by Methanobacterium thermoautotrophicum strains was extremely sensitive to gramicidin, total inhibition being observed at 0.2 μg/ml. In contrast, methane synthesis by Methanococcus voltae, Methanogenium marisnigri, Methanosarcina mazei, and Methanospirillum hungatei were resistant to the highest concentrations of gramicidin tested (40 μg/ml), although spheroplasts of Methanospirillum hungatei were extremely sensitive. Other species tested showed intermediate sensitivity to gramicidin, methanogenesis inhibition occurring at 4 to 20 μg/ml.     

氢氧化细菌分离、筛选及促生机制研究进展

氢氧化细菌是一类以氢作为电子供体,通过氧化H2获得能量并同化CO2的无机化能自养菌。近年来,发现作为豆科作物根际主要生理类群的氢氧化细菌促生作用明显,因而受到各国学者的广泛关注。氢氧化细菌在农业、环境保护和人类健康等领域有巨大的应用潜力。目前,由于人们对氢氧化细菌的分类、分离和筛选技术的认识不足,严重影响了氢氧化细菌的研究进程和水平。概述氢氧化细菌的分离及筛选方法,并着重论述了氢氧化细菌促生作用及促生机理的最新研究进展,最后探讨其应用前景。随着研究的深入,氢氧化细菌将受到各国学者的广泛关注。     

Populations of Methane-Producing Bacteria and In Vitro Methanogenesis in Salt Marsh and Estuarine Sediments

Most probable numbers (MPNs) of methanogens in various salt marsh and estuarine sediments were determined with an anaerobic, habitat-simulating culture medium with 80% H₂ plus 20% CO₂ as substrate. Average MPNs for the short Spartina (SS) marsh sediments of Sapelo Island, Ga., were maximal at the 5- to 7-cm depth (1.2 × 10⁷/g of dry sediment). Populations decreased to approximately 880/g of dry sediment at the 34- to 36-cm depth. There was no significant difference between summer and winter populations. In tall Spartina (TS) marsh sediments, average populations were maximal (1.2 × 10⁶/g of dry sediment) in the upper 0- to 2-cm zone; populations from the 5- to 36-cm zones were similar (average of 9 × 10⁴/g of dry sediment). Methanogenic populations for TS sediments of James Island Creek marsh, Charleston, S.C., were similar (average of 3 × 10⁶/g of dry sediment) for all depths tested (0 to 22 cm), which was comparable to the trend observed for TS sediments at Sapelo Island, Ga. Sediment grab samples collected along a transect of James Island Creek and its adjacent Spartina marsh had MPNs that were approximately 20 times greater for the region of Spartina growth (average of 10⁶/g of dry sediment) compared with the channel (approximately 5 × 10⁴ methanogens per g of dry sediment). A similar trend was found at Pawley's Island marsh, S.C., but populations were approximately one order of magnitude lower. In vitro rates of methanogenesis with SS sediments incubated under 80% H₂-20% CO₂ showed that the 5- to 7-cm region exhibited maximal activity (51 nmol of CH₄ g⁻¹ h⁻¹), which was greater than rates for sediments above and below this depth. SS sediment samples (5 to 7 cm) incubated under 100% N₂ and supplemented with formate exhibited rates of methanogenesis similar to those generated by samples under 80% H₂-20% CO₂. Replacing the N₂ atmosphere with H₂ resulted in an eightfold decrease in the rate of methanogenesis. In vitro methanogenic activity by TS salt marsh sediments, incubated under 80% H₂-20% CO₂, was similar for all depths tested (0 to 22 cm). TS sediment samples (0 to 7 cm) supplemented with formate and incubated under 100% N₂ had greater rates of methanogenesis compared with unsupplemented samples.     

14C in Methane and DIC in the Deep Terrestrial Subsurface: Implications for Microbial Methanogenesis

A comparison between the ¹⁴C content of the methane and dissolved inorganic carbon (DIC) in deep, terrestrial subsurface systems was used to assess the timing of microbial methanogenesis contributing to gases in fracture water samples from three mines in the Witwatersrand Basin, South Africa. The results demonstrated that the majority of methane was produced over geologic timescales. In four of the samples, the methane contained no significant radiocarbon, indicating that the estimated 90% microbial methane in these samples was produced in the geologic past by indigenous microbial communities. In two samples from different mines, methane Δ¹⁴C levels indicated a primarily ancient origin for the microbial methane with the potential for more recent contributions from ongoing indigenous microbial activities constrained to between 0 and 40%, and 0 and 24%, respectively. Microbiological evidence for methanogenic archaea was observed in both of these samples. One sample had a Δ¹⁴C CH₄ that was higher than the corresponding DIC, indicating an extreme decoupling between these species and raising concerns over the representative quality of this sample. The variations in the Δ¹⁴C of DIC and CH₄ between and within mines demonstrate the need for a thorough assessment of each sample to obtain an accurate understanding of the role and timing of microbiological gas production in these complex, heterogeneous, terrestrial subsurface systems. The approach detailed here introduces timing as a new and widely applicable signature for the recognition of a major geochemical marker of indigenous life in the deep subsurface.     

盐胁迫对氢氧化细菌WMQ7浸种小麦萌发和生长的影响

从紫花苜蓿根际土壤分离得到的一株含有ACC脱氨酶以及铁载体的氢氧化细菌WMQ7,通过皿内试验,考查其在不同盐浓度下对小麦‘陕优225’的促生效应。结果表明,低盐胁迫(0.4%NaCl)下,用氢氧化细菌WMQ7菌悬液浸种的小麦发芽粒数、发芽率、发芽势、发芽指数、胚芽、胚根长有明显的增高;同时脯氨酸积累速度与对照组相比显著减缓,对照组脯氨酸含量于胁迫第11天时已达1 383μg.mL-1,高出接菌组3.8倍;丙二醛含量与对照组相比减少了69.28%,过氧化物酶活性显著升高,高出对照组44.44%。而高盐胁迫下(0.8%NaCl),与对照组相比,接菌组对小麦萌发指标有一定影响,丙二醛含量变化不显著,但脯氨酸含量及过氧化物酶活性变化显著。研究表明,氢氧化细菌WMQ7浸种小麦处理对低盐胁迫的伤害有一定的缓解作用,但在高盐胁迫下却无明显效果。     

Hydrogen-oxidizing electron transport components in nitrogen-fixing Azotobacter vinelandii.

Membranes from N2-fixing Azotobacter vinelandii were isolated to identify electron transport components involved in H2 oxidation. We found direct evidence for the involvement of cytochromes b, c, and d in H2 oxidation by the use of H2-reduced minus O2-oxidized absorption difference spectra. Carbon monoxide spectra showed that H2 reduced cytochrome d but not cytochrome o. Inhibition of H2 oxidation by cyanide was monophasic with a high Ki (135 microM); this was attributed to cytochrome d. Cyanide inhibition of malate oxidation showed the presence of an additional, low Ki (0.1 microM cyanide) component in the membranes; this was attributed to cytochrome o. However, H2 oxidation was not sensitive to this cyanide concentration. Chlorpromazine (at 160 microM) markedly inhibited malate oxidation, but it did not greatly inhibit H2 oxidation. Irradiation of membranes with UV light inhibited H2 oxidation. Adding A. vinelandii Q8 to the UV-damaged membranes partially restored H2 oxidation activity, whereas addition of UV-treated Q8 did not increase the activity. 2-n-Heptyl-4-hydroxyquinoline-N-oxide inhibited both H2 and malate oxidation.     

Hydrogen-oxidizing electron transport components in the hyperthermophilic archaebacterium Pyrodictium brockii.

The hyperthermophilic archaebacterium Pyrodictium brockii grows optimally at 105 degrees C by a form of metabolism known as hydrogen-sulfur autotrophy, which is characterized by the oxidation of H2 by S0 to produce ATP and H2S. UV-irradiated membranes were not able to carry out the hydrogen-dependent reduction of sulfur. However, the activity could be restored by the addition of ubiquinone Q10 or ubiquinone Q6 to the UV-damaged membranes. A quinone with thin-layer chromatography migration properties similar to those of Q6 was purified by thin-layer chromatography from membranes of P. brockii, but nuclear magnetic resonance analysis failed to confirm its identity as a ubiquinone. P. brockii quinone was capable of restoring hydrogen-dependent sulfur reduction to UV-irradiated membranes. Hydrogen-reduced-minus-air-oxidized absorption difference spectra on membranes revealed absorption peaks characteristic of c-type cytochromes. A c-type cytochrome with alpha, beta, and gamma peaks at 553, 522, and 421 nm, respectively, was solubilized from membranes with 0.5% Triton X-100. Pyridine ferrohemochrome spectra confirmed its identity as a c-type cytochrome, and heme staining of membranes loaded on sodium dodecyl sulfate gels revealed a single heme-containing component of 13 to 14 kDa. Studies with the ubiquinone analog 2-n-heptyl-4-hydroxyquinoline-N-oxide demonstrated that the P. brockii quinone is located on the substrate side of the electron transport chain with respect to the c-type cytochrome. These first characterizations of the strictly anaerobic, presumably primitive P. brockii electron transport chain suggest that the hydrogenase operates at a relatively high redox potential and that the H2-oxidizing chain more closely resembles those of aerobic eubacterial H2-oxidizing bacteria than those of the H2-metabolizing systems of anaerobes or the hyperthermophile Pyrococcus furiosus.     

Nitrous Oxide Production and Methane Oxidation by Different Ammonia-Oxidizing Bacteria

Ammonia-oxidizing bacteria (AOB) are thought to contribute significantly to N₂O production and methane oxidation in soils. Most of our knowledge derives from experiments with Nitrosomonas europaea, which appears to be of minor importance in most soils compared to Nitrosospira spp. We have conducted a comparative study of levels of aerobic N₂O production in six phylogenetically different Nitrosospira strains newly isolated from soils and in two N. europaea and Nitrosospira multiformis type strains. The fraction of oxidized ammonium released as N₂O during aerobic growth was remarkably constant (0.07 to 0.1%) for all the Nitrosospira strains, irrespective of the substrate supply (urea versus ammonium), the pH, or substrate limitation. N. europaea and Nitrosospira multiformis released similar fractions of N₂O when they were supplied with ample amounts of substrates, but the fractions rose sharply (to 1 to 5%) when they were restricted by a low pH or substrate limitation. Phosphate buffer (versus HEPES) doubled the N₂O release for all types of AOB. No detectable oxidation of atmospheric methane was detected. Calculations based on detection limits as well as data in the literature on CH₄ oxidation by AOB bacteria prove that none of the tested strains contribute significantly to the oxidation of atmospheric CH₄ in soils.     

Methanogenesis from acetate: enrichment studies.

An acetate enrichment culture was initiated by inoculating anaerobic sludge from a mesophilic methane digestor into a mineral salts medium with calcium acetate as the sole carbon and energy source. This enrichment was maintained indefinitely by weekly transfer into medium of the same composition. A study of this enrichment disclosed an unexpected age-dependent inhibition of methanogenesis by H2 and formate which apparently differed from the inhibition by chloroform and benzyl viologen. This age-dependent inhibition indicated that microbial interactions of the mixed enrichment population may play a regulatory role in methane formation. Futhermore, stimulation of methanogenesis in the acetate enrichment by addition of yeast extract showed a nutrient limitation which indicated that syntrophic interactions leading to formation of growth factors may also occur. A model is presented to illustrate the possible interrelationships between methanogenic and nonmethanogenic bacteria in their growth and formation of methane and carbon dioxide from acetate.     

Pelomyxa palustris Greeff. I. Cultivation and general observations

SUMMARY. A rich source of supply of Pelomyxa palustris has recently been found. The findings and views of previous investigators on the geographical and seasonal distribution, longevity in the laboratory and reproduction of this organism are reviewed and discussed. Clone and mass cultures were successfully obtained in Carrel flasks with Spirogyra as food. Feeding is an extremely slow process so that this Pelomyxa is incapable of capturing and ingesting actively motile organisms under ordinary circumstances. Multiplication is solely by simple or multiple plasmotomy.