Nitrate reduction by Pseudomonas spp.: antagonism by fermentative bacteria

The reduction of nitrate by Pseudomonas denitrificans in a culture medium containing glycerol, yeast extract and 700 mg/1 NO₃–N, was antagonized by Aeromonas hydrophila, Escherichia coli and Enterobacter aerogenes. Nitrate reduction by Ps. denitrificans in mixed culture with a fermentative heterotroph was inhibited when 100–150 mg/1 NO₂–N had accumulated in the medium. The number of Ps. denitrificans declined concomitantly with the appearance of NO₂ in the culture medium, but there was only a slight increase in the numbers of fermentative hetero-trophs. The fermentative heterotrophs did not antagonize nitrate reduction by Ps. denitrificans , when the culture medium contained 140 mg/1 NO₃–N. When mixtures of equal parts of Ps. denitrificans and Esch. coli cultures were added to autoclaved river water relatively high concentrations of NO₂ were produced from the nitrate present in the water.     

Effect of different nitrogen nutrients on the viability, protein synthesis and tannin production of Scots pine callus

The effect of two different media on the growth, metabolism and viability of Scots pine ( Pinus sylvestris ) callus cultures was studied. Inorganic nitrogen in the culture media (modified MS) was in the form of either KNO₃ or NH₄NO₃. The cultures were started from buds of mature Scots pine. Growth was poor on the medium with KNO₃, but this compound had a noticeable effect on the metabolism of the callus, which was reflected in alterations in protein and polyphenol synthesis and the pH of the culture medium. Although the fresh mass, water content and viability of the callus decreased when KNO₃ was the exclusive inorganic nitrogen nutrient, protein synthesis was more abundant. Electrophoretic analyses indicated alterations in the patterns of soluble proteins and purified glycoproteins. Phenylalanine ammonia-lyase (EC 4.3.1.5) activities were high in all the calluses, and concentrations of condensed tannins and their precursors, catechins, were higher than in intact buds. The role of inorganic nitrogen nutrition in the deterioration of tissues is discussed on the basis of the effect of ammonium on the metabolism of pine callus.     

Mineral sulphide oxidation by enrichment cultures of novel thermoacidophilic bacteria

Abstract: Enrichment culture of hot spring water samples with pyrite as substrate has provided acidophiles with novel growth characteristics: with these bacteria, the range of conditions under which rapid microbial oxidation of mineral sulphides has been demonstrated has been extended. The upper temperature limit for bacterial mineral oxidation in reactors has been raised. The dissolution of pyrite occurred during growth of Sulfolobus -like thermophiles up to almost 90C. The most efficient extraction of copper from chalcopyrite occurred at 80–85C. With moderate thermophiles, rapid oxidation of minerals was obtained during autotrophic growth in the absence of supplemental CO₂: a mixed enrichment culture was active in pyrite dissolution at 45–50C in reactors gassed only with air which contrasted with poor growth by well-studied moderate thermophiles in the absence of enhanced CO₂ concentrations.     

Fixation of molecular nitrogen by Methanosarcina barkeri

Abstract Methanosarcina barkeri cells were observed in ammonia-free anaerobic acetate enrichments for sulfate-reducing bacteria. The capacity of Methanosarcina to grow diazotrophically was proved with a pure culture in mineral media with methanol. The cell yields with N₂ or NH₄⁺ ions as nitrogen source were 2.2 g and 6.1 g dry weight, respectively, per mol of methanol. Growth experiments with ¹⁵N₂ revealed that 84% of the cell nitrogen was derived from N₂. Acetylene was highly toxic to Methanosarcina and only reduced at concentrations lower than 100 μmol dissolved per 1 of medium. Assimilation of N₂ and reduction of acetylene were inhibited by NH₄⁺ ions. The experiments show that N₂ fixation occurs not only in eubacteria but also in archaebacteria. The ecological significance of diazotrophic growth of Methanosarcina is discussed.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest IV. Lifetime of the Metronidazole Radical Anion

Electrochemical studies on metronidazole using mixed aqueous/dimethylformamide (DMF) solvents have allowed us to generate the one-electron addition product, the nitro radical anion, RNO^-₂. Cyclic volt-ammetric techniques have been employed to study the tendency of RNO^-₂ to undergo further chemical reaction. The return-to-forward peak current ratio. ip/ip_f. was found to increase towards unity with increasing DMF content of the medium, indicating the extended lifetime of RNO^-₂. Second order kinetics for the decay of RNO^-₂ were established at all DMF concentrations examined. Extrapolation has allowed the rate constant and a first half-life of 8.4 × 10⁴dm²/mol-sec and 0.059 seconds respectively, to be determined for the decay of RNO^-₂ in a purely aqueous media. This is impossible by direct electrochemical measurement in water. due to a different reduction mechanism, giving the hydroxylamine derivative in a single 4-electron step. The application of the technique to other nitro-aromatic compounds is discussed.     

高矿浆浓度下黄铜矿浸出过程中微生物群落结构变化规律

目的：为阐明微生物群落演替及功能与浸出效率之间关系奠定基础,以及如何提高黄铜矿生物浸出效率和铜回收率提供理 论依据。方法：通过连续传代培养进行驯化,使得复合菌群的矿浆浓度耐受能力达到25 %（w/v）。采用该复合菌群在25 %矿浆浓 度下浸出黄铜矿,同时利用变性梯度凝胶电泳和克隆文库技术分析浸出过程中的微生物多样性。最后,采用实时荧光定量PCR 对 浸出过程中微生物群落结构进行定量解析。结果：28天内黄铜矿浸出率能够达到95.1 %,而驯化前的浸出率只有51.5%。该复合 菌群主要由Acidithiobacillus caldus, Sulfobacillus acidophilus,和Fereoplasma theroplasma thermophilum组成,其中Acidithbacillus caldus是浸出前期和后期的优势种群,而Sulfobacillus acidophilus在浸出中期均有竞争优势, Ferroplasma thermophilum在整个浸出过程中占 据整个群落的比例均较低。结论：本研究获得的复合菌群具有较强的浸出黄铜矿能力, Acidithiobacillus caldus和Sulfobacillus acidophilus在浸出过程中起着重要的作用,pH 值和铜浸出率与群落结构相关性较高。     

Microbial transformation of a fungicide-derived amine, dimethyl-p-phenylene diamine by Alcaligenes DM4

Abstract Microbial transformation of N , N -dimethyl- p -phenylene diamine (DMPDA), a microbial product formed from the fungicide fenaminosulf ( p -dimethylaminobenzenediazo sodium sulfonate) was studied by enriching microbes in soils treated with the amine. Microorganisms isolated from DMPDA-treated soil belonged to the genera of Micrococcus, Alcaligenes , and Corynebacterium . Of the various isolates, Alcaligenes DM₄ showed maximal growth on DMPDA utilizing it as sources of carbon and nitrogen. When grown in mineral salts basal medium containing 0.05% DMPDA to serve as carbon and nitrogen sources, Alcaligenes DM₄ grew exponentially up to 18 h. Even though the characterization of the complete pathway of microbial degradation of DMPDA could not be carried out due to the auto-oxidation of the compound, the initial transformation product of DMPDA by Alcaligenes DM₄ has been identified as a dimer. The dimer is generated into the culture medium presumably by the extra-cellular oxidase of Alcaligenes DM₄. It is suggested that the risk-benefit evaluation on the use of fenaminosulf is to be made taking into consideration the microbial transformations of the fungicide.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest: I. The Influence of Solvent

The electrochemical properties of three nitroimidazoles, a nitropyrazole, a nitrofuran and three nitroben-zenoid compounds have been extensively investigated in a range of solvents. The reduction pathway for the nitro group is independent of the cyclic function to which it is attached, but is strongly influenced by the nature of the solvent. In aqueous media, generally, a single, irreversible 4-electron reduction occurs to give the hydroxylamine. In aprotic media (dimethylformamide, methylene chloride or dimethylsulphoxide), a reversible one-electron reduction takes place to form a stable nitro radical anion. At more negative values, a further 3-electron reduction occurs, irreversibly to give the hydroxylamine. In mixed aqueous-organic systems, intermediate behaviour is found, with the reversibility of the RNO₂/RNO₂^- couple increasing with addition of organic medium. The control of the reduction pathway, by changing the electrolytic medium is discussed in relation to the biological activities of the drugs and identification of the short-lived reduction intermediate responsible for DNA damage.     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation

The kinetics of β-fructofuranosidase (Ffase) production by Aspergillus niger in submerged (SmF) and solid-state fermentation (SSF) systems was investigated. The maximum productivity of Ffase (81.8 U/l per h) was obtained in SSF for 72 h while it was 18.3 U/l per h in SmF for 120 h. The productivity of extra cellular Ffase produced in SSF was 5-fold higher than in SmF. Optimization of fermentation medium for Ffase production was carried out using De Meo's fractional factorial design with seven components such as (NH₄)₂SO₄, KH₂PO₄, FeSO₄, MgSO₄ · 7H₂O, sucrose, urea and yeast extract. The media designed for SmF after two steps of optimization supported the growth of A. niger and higher productivity of Ffase (58.3 U/l per h) than with the medium before optimization. The optimized medium of SmF when used in SSF, did not improve the Ffase productivity and therefore medium for SSF was optimized independent of SmF. After two optimization steps, the media was defined for SSF which supported the growth and high level of Ffase productivity (149.1 U/l per h) in SSF compared to the medium before optimization (81.8 U/l per h) and optimized medium for SmF (58.3 U/l per h). Our results suggested that the optimized media for SmF and SSF for the production of Ffase have to be different.     

The plant-growth-promoting rhizobacteria Bacillus pumilus and Bacillus licheniformis produce high amounts of physiologically active gibberellins

The plant-growth-promoting rhizobacteria (PGPR), Bacillus pumilus and Bacillus licheniformis, isolated from the rhizosphere of alder ( Alnus glutinosa [L.] Gaertn.) have a strong growth-promoting activity. Bioassay data showed that the dwarf phenotype induced in alder seedlings by paclobutrazol (an inhibitor of gibberellin [GA] biosynthesis) was effectively reversed by applications of extracts from media incubated with both bacteria and also by exogenous GA₃. Full-scan gas chromatography-mass spectrometry analyses on extracts of these media showed the presence of GA₁, GA₃, GA₄and GA₂₀, in addition to the isomers 3- epi -GA₁ and iso -GA₃. Isotope dilution analysis indicated that epi -GA₁ was an artefact. Likewise, iso -GA₃ is also probably an artifact spontaneously formed during extraction and/or analysis. In both culture media, GA₁ was present in higher concentrations (130–150 ng ml⁻¹) than GA₃ (50–60 ng ml⁻¹), GA₄ (8–12 ng ml⁻¹) and GA₂₀ (2–3 ng ml⁻¹). The data indicated that culture of both bacteria accumulate bioactive C19-gibberellins in relative high amounts and that these GAs appear to be physiologically active in the host plant. The evidence suggests that the promotion of stem elongation induced by the PGPR could be mediated by bacterial GAs.     

Transformation of vivianite by anaerobic nitrate-reducing iron-oxidizing bacteria

In phosphate-rich environments, vivianite (Fe^II₃(PO₄)₂, 8H₂O) is an important sink for dissolved Fe(II) and is considered as a very stable mineral due to its low solubility at neutral pH. In the present study, we report the mineralogical transformation of vivianite in cultures of the nitrate-reducing iron-oxidizing bacterial strain BoFeN1 in the presence of dissolved Fe(II). Vivianite was first transformed into a greenish phase consisting mostly of an amorphous mixed valence Fe-phosphate. This precipitate became progressively orange and the final product of iron oxidation consisted of an amorphous Fe(III)-phosphate. The sub-micrometer analysis by scanning transmission X-ray microscopy of the iron redox state in samples collected at different stages of the culture indicated that iron was progressively oxidized at the contact of the bacteria and at a distance from the cells in extracellular minerals. Iron oxidation in the extracellular minerals was delayed by a few days compared with cell-associated Fe-minerals. This led to strong differences of Fe redox in between these two types of minerals and finally to local heterogeneities of redox within the sample. In the absence of dissolved Fe(II), vivianite was not significantly transformed by BoFeN1. Whereas Fe(II) oxidation at the cell contact is most probably directly catalyzed by the bacteria, vivianite transformation at a distance from the cells might result from oxidation by nitrite. In addition, processes leading to the export of Fe(III) from bacterial oxidation sites to extracellular minerals are discussed including some involving colloids observed by cryo-transmission electron microscopy in the culture medium.     

高效杀蚊苏云金芽孢杆菌BRC-LLP29的发酵优化

苏云金芽孢杆菌BRC-LLP29为新型高效杀蚊菌株,应用快速有效的数学统计方法对其杀蚊毒力的发酵培养进行优化.通过单因素筛选确定最佳碳源为葡萄糖、麦芽糖、可溶性淀粉,氮源为typetone、大豆蛋白胨、干酪素;最佳金属离子为Mg²⁺、Al³⁺.采用二水平Placlkett-Burman设计对影响毒力的8因素进行显著性筛选,获得培养基成分中3个重要影响因子:葡萄糖、干酪素和Al₂（SO₄）₃;运用爬坡路径法对这3种因子进行试验,获得3种重要因子的最适浓度范围;通过响应面分析法得到3个重要因子的交互作用和最佳条件,确定BRC-LLP29菌株最佳毒力水平的发酵培养基为:葡萄糖19.8g/L、干酪素28.4g/L、Al₂（SO₄）₃1.2g/L、MgSO₄ 2g/L、K₂HPO₄ 3g/L、CaCO₃ 0.5g/L,优化后毒力水平达到致死率61.11%,与响应面数学模型的预测值只有5.91%的误差.发酵条件优化结果表明:发酵温度为31°,发酵初始pH为7.0,摇瓶装量为40mL/250mL三角瓶,每瓶的接种量为3.5%,发酵72h,对致倦库蚊最终致死率达到最高为83.33%.     

Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37°C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H₂SO₄ both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5·5 resulted in lower mycobacterial counts than egg media at pH 6·5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green–cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6·5.     

Bacterial Reduction of Hexavalent Chromium: Kinetic Aspects of Chromate Reduction by Enterobacter cloacae HO1

Kinetic aspects of the bacterial reduction of hexavalent chromium (chromate: CrO^2-₄) were investigated using Enterobacter cloacae strain HO1. E. cloacae strain HO1 could reduce hexavalent chromium to the trivalent form (Cr³⁺) anaerobically. High concentrations of CrO^2-₄ inhibited the reduction, and a substrate inhibition model gave a good fit to the observed data. The rate of chromate reduction was proportional to cell density. The effect of temperature on the reduction rate followed the Arrhenius equation. The rate of chromate reduction was also dependent on pH and the concentrations of carbon and energy sources in the culutre medium. Amino acids including asparagine, methionine, serine and threonine were utilized effectively as carbon and energy sources for chromate reduction.     

Increasing progesterone secretion and 3β-hydroxysteroid dehydrogenase activity of human cumulus cells and granulosa-lutein cells concurrent with successful fertilization of the corresponding oocyte

In many studies it has been documented that the induction of multiple follicular growth in humans results in an asynchrony between the degree of cumulus mucification, oocyte meiotic maturation, fertilizability, and follicular cell progesterone (P₄) secretion. The present study was carried out on oocytes enclosed in fully mucified cumulus. Thus, oocyte fertilizability was correlated to human cumulus cell (hCC) and human granulosa-lutein (G-L) cell competence for P₄ secretion in culture. In the G-L cells, P₄ secretion and percentage of cells manifesting 3β-hydroxysteroid dehydrogenase (3β-HSD) activity increased concurrently with the period of culture. In the hCC, however, P₄ secretion decreased concurrently with elongation of the culture period, whereas the percentage of 3β-HSD-positive cells increased. In hCC corresponding to the fertilized oocytes, P₄ accumulation in culture medium was 1.9-fold (P < 0.001) and 1.6-fold (P < 0.02) higher on days 0–3 and 3–5 of culture, respectively, as compared to P₄ accumulation in hCC of unfertilized oocytes. Also, in hCC corresponding to the fertilized oocytes, the degree of 3β-HSD activity was found to be significantly higher shortly after aspiration and after either 3 or 5 days, compared to hCC of unfertilized oocytes. In the G-L cells pooled from all follicles yielding mature cumulus-oocyte complexes, P₄ accumulation and percentage of 3β-HSD-positive cells increased concurrently with the increase in percentage of fertilized eggs of each individual woman. These results indicate that in stimulated cycles, follicles yielding mature cumulus-oocyte complex, oocyte fertilizability, and G-L cell or hCC competence for P₄ secretion are correlated and synchronous.     

宜昌百合胚性愈伤组织诱导及植株再生体系的研究

采用L₉（3⁴）正交试验等方法,以野生宜昌百合（Lilium leucanthum）鳞片为外植体,分别在光照和黑暗培养条件下,研究了TDZ、NAA、2,4-D不同浓度配比对宜昌百合的胚性愈伤组织诱导效果的影响,并采用石蜡切片技术对愈伤组织进行了的组织学观察。在此基础上,探讨了NAA对体胚苗壮苗生根的影响。结果表明：在光照条件下诱导胚性愈伤组织的最佳培养基为MS+TDZ 0.5 mg·L^-1+NAA 1.0 mg·L^-1+2,4-D 0.05 mg·L^-1,诱导率为62.690%;黑暗条件下诱导胚性愈伤组织的最佳培养基为MS+TDZ 0.5 mg·L^-1+NAA 1.5 mg·L^-1+2,4-D 0.1 mg·L^-1,诱导率为59.423%。胚性愈伤组织黄绿色或深绿色,颗粒状明显;非胚性愈伤组织松散易碎、呈水渍状,经石蜡切片观察可观察到胚性愈伤组织细胞核大、胞质浓,且细胞内含丰富淀粉粒,细胞直径为50~80 μm。非胚性愈伤组织细胞较大,未见明显细胞核,直径约为100~180 μm。体胚苗壮苗生根的最适宜培养基为1/2MS+NAA 0.2 mg·L^-1+AC 1 g·L^-1,培养60 d后,幼苗移至草炭：蛭石：珍珠岩=1：1：1基质中,成活率为90%。本试验成功建立了宜昌百合鳞片胚性愈伤组织的再生体系,为宜昌百合利用胚状体进行无性育种以及种质资源的创新奠定了基础。     

水杨酸诱发的丹参悬浮培养细胞内H2O2迸发与其培养基碱化的关系

水杨酸(salicylic acid,SA)处理可诱导丹参悬浮培养细胞内H2O2产生及其培养基碱化。利用NADPH氧化酶抑制剂咪唑(imidazole,IMD)、H2O2淬灭剂二甲基硫脲(dimethylthiourea,DMTU)、质膜H+-ATPase抑制剂钒酸钠(Na3VO4)及激活剂壳梭孢菌素(fusicoccin,FC)处理丹参悬浮培养细胞,探讨SA诱导的H2O2迸发与培养基碱化之间的关系。结果表明,H2O2可促发培养基碱化,IMD和DMTU抑制SA诱发的培养基碱化,说明H2O2参与SA诱发的培养基碱化过程;SA抑制质膜H+-ATPase活性,Na3VO4引发培养基碱化并使H2O2迸发时间提前,FC处理逆转了SA诱导的培养基碱化及H2O2迸发,说明质膜H+-ATPase调控培养基pH值变化,培养基碱化促进了H2O2产生。因此,丹参悬浮培养细胞内H2O2水平与其培养基碱化程度之间相互关联、共同作用,协同响应SA的诱导。     

Investigations on Myelination In Vitro: Regulation of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase by Thyroid Hormone in Cultures of Dissociated Brain Cells from Embryonic Mice

Abstract: The direct influence of l -3,3',5-triiodothyronine (T₃) on the development of 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37, CNPase) is demonstrated by using an in vitro culture system of dissociated embryonic mouse brain cells. Serum from a thyroidectomized calf, which contained low levels of T₃ (31 ng/100 ml), and thyroxine, T₄ (<1 μg/ml), was used in the culture medium in place of normal calf serum (T₃, 103 ng/100 ml; T₄, 5.7 μg/ml) to render the culture responsive to exogenously added T₃. The lower levels of enzyme activity observed in the presence of such a deficient medium could be restored to normal values by T₃ supplementation. Half-maximal effect was obtained with 2.5 ± 10⁻⁹ m -T₃. Three days of hormone treatment resulted in the maximal stimulation of CNPase. T₄ was less effective in inducing CNPase activity and the inactive analog of the hormone, reverse T₃ (3,3',5'-T₃) was ineffective. The morphological appearance of the cells was characterized by deformed (smaller size and less in number) reaggregates in the cultures, lacking hormone.