从乙烷氧化富集物中分离到的乙醇氧化不动杆菌的生理生化特点

采用floating filter方法从乙烷氧化富集物中分离出杆状菌株Z1,其最适生长温度为37℃,最适p H为6. 0,最适盐浓度为0. 2 mol/L,过氧化氢酶阳性,氧化酶阴性。其全长16S r DNA序列与Acinetobacter pittii CIP 70. 29T完全一致,可判定其为不动杆菌属(Acinetobacter)细菌。此菌株可以壬烷、十三烷为碳源生长,具有甲醇、乙醇氧化活性,却不能利用乙烷。不动杆菌菌株Z1可应用于利用floating filter方法的乙烷氧化菌的分离培养以及污染石油烃的降解。     

Autotrophic growth and inorganic sulphur compound oxidation by Sulfolobus sp. in chemostat culture

Sulfolobus strain LM was grown in tetrathionate and thiosulphate-limited continuous culture. CO₂ limitation resulted in a decrease of the steady-state biomass and an increase in the specific rate of thiosulphate oxidation so that substrate did not accumulate in the medium. The initial step in thiosulphate utilization appeared to be its conversion to tetrathionate. The affinity for tetrathionate oxidation appeared to increase with prolonged continuous culture giving an apparent K _m of about 6 M tetrathionate, a higher affinity than for thiosulphate oxidation and in the same range as values observed with acidophilic, sulphur-oxidizing eubacteria.     

Kinetic studies on ammonia and methane oxidation by Nitrosococcus oceanus

The kinetics of ammonia oxidation and the ability of a marine ammonia-oxidizing bacterium, Nitrosococcus oceanus, to metabolize methane were investigated in semicontinuous batch culture. The effects of inhibitors (acetylene and nitrapyrin) and coreactants were determined in order to elucidate the behavior of the ammonia oxygenase enzyme in N. oceanus. Acetylene and nitrapyrin were potent inhibitors and their effects were not mitigated by increased ammonia concentrations. Oxygen concentration had the effect of a mixed-type inhibitor; reduced oxygen inhibited the rate or ammonia oxidation at high substrate concentration but may enhance the rate at low substrate concentrations. Substrate affinity in terms of NH ₄ ⁺ increased (K _m decreased) with increasing pH. Optimal pH was about 8. Methane inhibited ammonia oxidation; the interaction was not simple competitive inhibition and the presence of multiple active sites on the enzyme was indicated by the behaviour of the inhibited treatments. Half-saturation constants for methane (K _i=6.6 M) and ammonia (K _m=8.1 M) were similar. N. oceanus oxidized methanol and methane linearly over time, with CO₂ and cell material being produced at approximately equal rates.     

Ammonia oxidation by the methane oxidising bacterium Methylococcus capsulatus strain bath

Soluble extracts of Methylococcus capsulatus (Bath) that readily oxidise methane to methanol will also oxidise ammonia to nitrite via hydroxylamine. The ammonia oxidising activity requires O₂, NADH and is readily inhibited by methane and specific inhibitors of methane mono-oxygenase activity. Hydroxylamine is oxidised to nitrite via an enzyme system that uses phenazine methosulphate (PMS) as an electron acceptor. The estimated K _mvalue for the ammonia hydroxylase activity was 87 mM but the kinetics of the oxidation were complex and may involve negative cooperativity.Abbreviations PMS Phenazine methosulphate - NADH nicotinamide adenine dinucleotide, reduced form - K _m Michaelis constant - NO ₂ ^- nitrite - NH₂OH hydroxylamine     

Observations on the biology ofThiothrix

Thiothrix nivea grows profusely in tufts of greyish white material on pebbles in a local sulfide spring. The spring remained a constant 10.8° C during the course of the study. Chemical analyses indicated that the spring water contained 0.27 mg/l sulfide, 0.37 mg/l oxygen, and 0.77 mg/l dissolved organic carbon. Tufts ofThiothrix growth were removed from the pebbles and examined by phase and electron microscopy. The cell filaments contained numerous sulfur granules which disappeared upon incubation in the refrigerator. The average cell diameter was 1.5 m and the length 4.0 m. When cells were lysed by hyperplasmolysis a sheath was readily discernible. The presence of a sheath, 60 nm thick, was confirmed in thin sections. Fine structure analysis also revealed that the organism was a gram negative bacterium. Sulfur granules were bound by a unit membrane extending from the cytoplasmic membrane.Thiothrix nivea was grown for short periods of time in slide culture. In some cases the filaments fragmented into short segments ca 15m long. These may represent gonidia as initially reported by Winogradsky.     

Uranous ion oxidation and carbon dioxide fixation byThiobacillus ferrooxidans

The stoichiometric oxidation of uranous-to uranyl-uranium byThiobacllus ferrooxidans is demonstrated. Fixation of¹⁴CO₂ and the effect of inhibitors demonstrate that energy is conserved during the oxidation and used for energy-dependent reverse electron flow and carbon dioxide fixation.Abbreviations HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide - 8-HQ 8-hydroxyquinoline - TTFA thenoyltrifluoroacetone     

Microbiological resolution of chiral arylethyl carbinols by Nocardia corallina

Whole cells of Nocardia corallina B-276 oxidized enantioselectively racemates of arylethyl carbinols, at 0.5 mM, to give ketones in yields from 4 to 97% (w/w) and the unreacted alcohols showing enantiomeric excess ranging from 5 to > 99%. The configuration of the resulting alcohol is (R).     

Oxidation of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids by Acetobacter and Serratia strains

Conversion of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids was carried out using Acetobacter rancens IFO3297, A. pasteurianus IFO13753 and Serratia liquefaciens LF14. IFO3297 produced 110g 2-furoic acid l^-1 from furfural with a 95% molar yield. 5-Hydroxymethyl-2-furancarboxylic acid was produced from the corresponding aldehyde by using whole cells LF14. IFO13753 and LF14 both converted isophthalaldehyde, 2,5-furandicarbaldehyde, 2,5-thiophenedicarbaldehyde and 2,2 biphenyldicarbaldehyde to the corresponding formylcarboxylic acid with 86--91% molar yields.Revisions requested 21 July 2004; Revisions received 7 September 2004     

Ferric iron reduction by Thiobacillus ferrooxidans at extremely low pH-values

When ferrous iron and sulfur were supplied, cells of T. ferrooxidans in a well-aerated medium started growth by oxidizing ferrous iron. After ferrous iron depletion a lagphase followed before sulfur oxidation started. During sulfur oxidation at pH-values below 1.3 (±0,2) the ferrous iron concentration increased again, although the oxygen saturation of the medium amounted to more than 95%. The number of viable cells did not increase. Thus resting cells of T. ferrooxidans, which are oxidizing sulfur to maintain their proton balance, reduce ferric to ferrous iron. The ferrous iron-oxidizing system seemed to be inhibited at pH-values below 1.3. At a pH-value of 1.8 the ferrous iron was reoxidized at once. A scheme for the linkage of iron- and sulfur metabolism is discussed.     

Oxidation of ethylene by cotyledon extracts from Vicia faba L.

Improved rates of ethylene oxidation by cell-free preparations from cotyledons of Vicia faba L. have been obtained using cryogenic storage techniques and by developing a method for the hydrolysis of ethylene oxide. Gel permeation chromatography showed that a low-molecular-size fraction was required for activity; accordingly, the kinetics of ethylene oxidation in the presence of this fraction were studied. Reduced pyridine nucleotides could substitute for the low-molecular-size fraction. Activity under a nitrogen atmosphere was 60% lower than in air. The need for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen indicated that the enzyme might be a mixed-function oxidase. Using sufficient NADPH to approach saturation, the apparent Michaelis constant (K _m) for ethylene was 1.94±0.38 · 10^-8 M (aqueous phase), and when ethylene was saturating, the K _m for NADPH was 3.7 · 10^-5 M. Carbon monoxide was found to inhibit by competing with ethylene, and the inhibitor constant was 5.97 · 10^-7 M in solution. In the presence of excess ethylene and NADPH, activity was highest in phosphate-buffered medium pH 7.9. The bulk of the activity was found in a microsomal fraction.Abbreviations Epps N-2-hydroxyethylpiperazine-N-3-propane sulphinic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-porpanediol     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

Respiration-driven proton translocation in Thiobacillus versutus and the role of the periplasmic thiosulphate-oxidizing enzyme system

The periplasmic location of enzymes A and B of the thiosulphate-oxidizing multienzyme system of Thiobacillus versutus has been further confirmed by differential radiolabelling of periplasmic and cytoplasmic proteins. The stoichiometries of respiration-driven proton translocation in T. versutus were determined using the oxygen pulse and the initial rate methods. A value for the H⁺/O quotient (number of protons translocated per oxygen atom reduced) of about 2.8 was found for the oxidation of thiosulphate, and of about 2.5 for sulphite. The H⁺/O quotient for endogenous respiration was about 5.7. The data are shown to be in good agreement with the scheme proposed previously for thiosulphate oxidation by this organism. Proton generation during the oxidation of thiosulphate or sulphite is indicated to occur in the periplasm rather than by pumping across the cytoplasmic membrane. The results also suggest that a H⁺/O quotient of six occurs during NADH oxidation (from endogenous metabolism measurements) and that the terminal cytochrome oxidase, aa₃, does not function as a proton pump.Abbreviations DCCD dicyclohexyl carbodiimide - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - IEF isoelectric focusing - HIC hydrophobic interaction chromatography - EAI ethyl acetimidate hydrochloride - IAI isethionyl acetimidate     

Microdynamics and seasonal changes in manganese oxide epiprecipitation in Pinal Creek, Arizona

In Pinal Creek, Arizona, Mn oxyhydroxides (MnO_x) collect as thick precipitates on surface sediment, within the streambed, beneath algal mats, and on submerged and emergent plants and mosses. The proximate source of Mn is a thick, alluvial alkaline aquifer that was contaminated by past acid mine waste disposal practices associated with copper mines located upstream in the Globe–Miami area. Almost every organism in Pinal Creek is coated with MnO_x. Some are actively precipitating manganese, and others are doing it passively. The variety and seasonality of epilithic biological processes resulting in Mn oxidation (epiprecipitation) was studied for more than a year by analyzing artificial substrates placed in surface water having different flows and different vegetation types and densities. Most epiprecipitation took place on the holdfasts of the green alga, Ulothrix sp., and the iron bacterium, Leptothrix discophora. Extensive patches of MnO_x also coated extracellular polymeric substances of fungal hyphae and bacterial filaments. The dominant macroscopic precipitation was in the form of MnO_x clumps on mosses, green algae, and cyanobacterial mats, consistent with precipitation by pH elevation during photosynthesis. Most oxidation occurred in the spring and summer, in agreement with thermal, biological, and chemical activity models. More biological oxidation occurred in swifter water, consistent with oxygen elevation models. The efficiency of this naturally occurring, diverse ecosystem suggests that remediation efforts to remove metal contaminants such as Mn should focus on creation of habitats that raise biodiversity.US Geological Survey, retired     

Electron transport and respiration in Beggiatoa and Vitreoscilla

Seven strains of bacteria belonging to the Beggiatoa-Vitreoscilla group were studied for their respiratory activity and for the presence of electron transport conponents. All strains tested oxidized [1-¹⁴C] and [2-¹⁴C] acetate to ¹⁴CO₂ at relatively high rates. All strains tested were N,N,N,N-tetramethylphenylenediamine (TMPD)-oxidase positive and contained spectra representing a-type and carbon monoxide-binding cytochromes. Most of the strains also contained spectra representing c-type and b-type cytochromes. Beggiatoa alba B18LD contained b-type, a-type, c-type and CO-binding cytochromes, the latter two being located in the 144,000 x g soluble fraction. B. alba also contained ubiquinone-8 as its only detectable quinone.Non-standard abbreviations BSS basal salts solution - BH Beggiatoa heterotrophic medium - BSO Beggiatoa sulfide oxidation medium - TMPD N,N,N,N-tetramethylphenylenediamine - Q8 ubiquinone-8     

Morphology of bacterial leaching patterns by Thiobacillus ferrooxidans on synthetic pyrite

Thiobacillus ferrooxidans has been cultivated on synthetic pyrite (FeS₂) single crystals as the only energy source and the pyrite interface investigated with respect to characteristic morphological changes using scanning electron microscopy. Corrosion patterns of bacterial size were identified in different stages of development and correlated with bacterial activity. It appears that bacterial attack of the sulfide interface starts by secretion of organic substances around the contact area between the bacterial cell and the sulfide energy source. They might either be part of a pseudo capsule which shields the contact area or may form a sulfur absorbing and transporting organic film. Degradation of the sulfide occurs in the contact area below the bacterial cell leading to a corrosion pit which the bacterium may abandon after it has reached a depth of bacterial dimension. Electron spectroscopic (XPS) and X-ray fluorescence studies indicate a layer of organic substances covering the sulfide surface under bacterial leaching conditions, which is sufficiently thick for consideration in interfacial chemical mechanisms.     

Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

Utilization of formate as an additional energy source by glucose-limited chemostat cultures ofCandida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066

The oxidation of benzene to phenol by whole cells of Nitrosomonas europaea is catalysed by ammonia monooxygenase, and therefore requires a source of reducing power. Endogenous substrates, hydrazine, hydroxylamine and ammonium ions were compared as reductants. The highest rates of benzene oxidation were obtained with 4 mM benzene and hydrazine as reductant, and equalled 6 mol· h^-1·mg protein^-1. The specificity of ammonia monooxygenase for benzene as a substrate was determined by measuring k _cat/K _m for benzene relative to k _cat/K _m for uncharged ammonia, a value of 0.4 being obtained. Phenol was found to be further hydroxylated to yield hydroquinone. This reaction, like benzene oxidation, was sensitive to the ammonia monooxygenase inhibitor allylthiourea. Catechol and resorcinol were not detected as products of phenol oxidation, implying that at least 88% of the hydroxylation is para-directed.     

Transition of Thiobacillus ferrooxidans KG-4 from heterotrophic growth on glucose to autotrophic growth on ferrous-iron

The reputedly obligately organotrophic Thiobacillus ferrooxidans KG-4 cultured on glucose contained a small proportion of cells which grew autotrophically on ferrous-iron.     

典型草地土壤好氧甲烷氧化的微生物生态过程

【目的】针对我国甘肃三个典型生态区草地土壤(玛曲MQ、临泽LZ和环县HX),研究其甲烷氧化潜力、甲烷氧化菌(methane-oxidizingbacteria,MOB)丰度及可能存在的群落分异规律。【方法】通过原位分析、室内高浓度甲烷模拟培养三种典型土壤及实时荧光定量、高通量测序的方法研究甲烷氧化菌标靶基因pmoA序列的组成及其丰度变化规律。【结果】三种典型草地土壤的原位甲烷氧化菌的丰度存在显著差异,表现为MQ>HX>LZ,其数量范围为为0.18–6.86×10^7g/d.w.s.;甲烷氧化潜力也表现出类似规律,其通量为109–169mg/(m^2·h);甲烷氧化潜力与原位土壤中甲烷氧化菌丰度有正相关。三种草地土壤甲烷氧化菌存在明显的空间异质性,采用高通量测序的方法,发现三种草地原位土壤中的优势类群为USCγ(Upland Soil Cluster gamma,USCγ);然而,室内高浓度甲烷氧化过程中,传统的甲烷氧化菌均发生明显增加,MQ土壤中TypeⅡ的Methylocystis为优势类群,而LZ和HX土壤的优势类群均为TypeⅠ型Methylosarcina。【结论】这些研究结果表明,我国甘肃典型草地土壤中也存在难培养的大气甲烷氧化菌和经典的可培养甲烷氧化菌,这些微生物极可能氧化极低浓度的大气甲烷,也可能利用闭蓄于土壤中的高浓度甲烷生长。未来应采用先进技术原位观测大气甲烷氧化过程并分离相应微生物类群,研究草地土壤甲烷氧化菌地理分异规律及其环境驱动机制。     

Mixotrophic and autotrophic growth of Thiobacillus acidophilus on tetrathionate

Thiobacillus acidophilus can grow in batch and chemostat culture as a heterotroph on glucose, a chemolithoautotroph on tetrathionate and CO₂, or as a mixotroph. Mixotrophically it obtains energy from the simultaneous oxidation of tetrathionate and glucose, and carbon from both glucose and CO₂. Mixotrophic cultures contain lower activities of ribulose 1,5-bisphosphate carboxylase and exhibit lower specific rates of tetrathionate oxidation than do autotrophic cultures. Mixotrophic cultures with low concentrations of glucose have growth rates that are intermediate between slow autotrophic growth and fast heterotrophic growth. Slightly more glucose-carbon is assimilated by mixotrophic cultures than by heterotrophic ones provided with the same concentrations of glucose. Mixotrophic yield in the chemostat is also slightly greater than predicted from autotrophic and heterotrophic yields. These observations indicate that there is preferential assimilation of glucose, at the expense of energy from tetrathionate oxidation, during mixotrophy, resulting in an overall energy saving that produces enhanced growth yield. These observations are relevant to understanding the regulatory behaviour of T. acidophilus in its acidic, mineral-leaching habitats.