Enumeration of Autotrophic Ammonium-oxidizing Bacteria in Marine Waters by a Direct Method

A membrane filter method was developed for enumerating, in mixed populations, autotrophic nitrifying bacteria which transform ammonium to nitrite. It gave counts in good agreement with estimates obtained by using a most-probable-number procedure. Compared to the most-probable-number procedure, the membrane method is convenient and precise. A means of extending the sensitivity of the membrane method is presented.     

Oxidation and Reduction of Iron by Acidophilic Bacteria

Abstract

Redox reactions of iron in acidic environments are of economic and environmental significance, for example, for the leaching of metal ores and for the formation of acid mine drainage and acid sulfate soils. Until recently, research on microbial iron metabolism in acidic environments has mainly been focused on the role of aerobic, autotrophic ferrous iron‐oxidizing bacteria. In the present paper, recent new developments in the field of acidophilic iron metabolism are reviewed. In addition to the well‐known autotrophic ferrous iron‐oxidizing organisms, new heterotrophic isolates have been described that are capable of oxidizing ferrous iron. Microorganisms can also play an important role in the reductive part of the iron cycle. Both heterotrophic and autotrophic organisms may also be involved in this process. The contribution of heterotrophic organisms to acidophilic iron cycling can be twofold: In addition to their direct role as a catalyst, these organisms may scavenge organic compounds that inhibit their autotrophic counterparts. Detailed studies of acidophilic ecosystems are needed to assess the significance of the various types of microorganisms for the overall rate of iron cycling in these extreme environments.     

Mechanisms of Oxidation of Inorganic Electron Donors in Autotrophic Bacteria

The enzymatic reactions involved in the oxidation of sulfide,sulfite, thiosulfate, ferrous ions, ammonia, and nitrite arereviewed for Chlorobium limicola f. thiosulfato philum, Thiobacillusnovellus, Thiobacillus ferrooxidans, Nitrosomonas europaea,and Nitrobacter winogradskyi. The properties of the purifiedredox enzymes and of proteins that participate in the oxidationof the inorganic compounds in these autotrophic bacteria aresummarized, and the mechanisms of the oxidation of the inorganiccompounds are discussed on the basis of the interactions betweenthe redox enzymes and carriers. (Received December 21, 1995; Accepted May 29, 1996)     

Nitrate-Dependent Ferrous Iron Oxidation by Anaerobic Ammonium Oxidation (Anammox) Bacteria

We examined nitrate-dependent Fe²⁺ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe²⁺ and reduced NO₃⁻ to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein⁻¹ min⁻¹, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca. Brocadia sinica” (10 to 75 nmol NH₄⁺ mg protein⁻¹ min⁻¹). A ¹⁵N tracer experiment demonstrated that coupling of nitrate-dependent Fe²⁺ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO₃⁻ by “Ca. Brocadia sinica.” The activities of nitrate-dependent Fe²⁺ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO₃⁻ ± SD of “Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe²⁺ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe²⁺ oxidation and NO₃⁻ reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein⁻¹ min⁻¹, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe²⁺ oxidation and the anammox reaction decreased the molar ratios of consumed NO₂⁻ to consumed NH₄⁺ (ΔNO₂⁻/ΔNH₄⁺) and produced NO₃⁻ to consumed NH₄⁺ (ΔNO₃⁻/ΔNH₄⁺). These reactions are preferable to the application of anammox processes for wastewater treatment.     

Anaerobic Nitrate-Dependent Iron (II) Oxidation by a Novel Autotrophic Bacterium,Citrobacter freundii Strain PXL1

Anaerobic Fe(II) Oxidizing Denitrifiers (AFODN), a type of newly found Fe(II)-oxidizing bacteria, play an important role in iron and nitrogen cycling. In the present study, a novel AFODN strain PXL1 was isolated from anaerobic activated sludge. Phylogenetic analysis of 16S rRNA gene sequence revealed similarity between this strain and Citrobactor freundii. The strain reduced 30% of nitrate and oxidized 85% of Fe(II) over 72 h with an initial Fe(II) concentration of 3.4 mM and nitrate concentration of 9.5 mM. Oxidation of iron was dependent on the reduction of nitrate to nitrite in the absence of other electron donors or acceptors. Nitrate reduction and Fe(II) oxidation followed first-order reaction kinetics. Iron oxides accumulated in the culture were analyzed by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) spectroscopy and scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS). The strain recovered deposited oxidized Fe in the form of amorphous Fe oxides.     

A New Fluorescence Method for the Continuous Determination of Surface Lipid Oxidation in Lipoproteins and Plasma

We report on a new method for the determination of lipid oxidation in lipoproteins and plasma. The biological lipid system is preloaded with a fluorescent analog of phosphatidylcholine containing diphenylhexatriene (DPH) propionic acid covalently linked to the sn-2 position. When externally added, the respective phospholipid label (DPHPC) localizes to the surface monolayer of a lipoprotein. Under oxidative conditions (e.g. in the presence of Cu²⁺ ions) the fluorophore undergoes decomposition, resulting in a continuous decrease of fluorescence intensity which reflects the oxidation of a chemically defined phospholipid molecule with well defined localization. When incorporated into LDL particles, the kinetics of the decrease in DPHPC fluorescence intensity upon exposure to Cu²⁺ is very similar to that of conjugated diene accumulation. Furthermore, our assay can be applied to follow the oxidation of lipids in diluted serum and may also be developed into a suitable test system for clinical studies of susceptibility of plasma lipids to oxidation.     

A New Fluorescence Method for the Continuous Determination of Surface Lipid Oxidation in Lipoproteins and Plasma

We report on a new method for the determination of lipid oxidation in lipoproteins and plasma. The biological lipid system is preloaded with a fluorescent analog of phosphatidylcholine containing diphenylhexatriene (DPH) propionic acid covalently linked to the sn-2 position. When externally added, the respective phospholipid label (DPHPC) localizes to the surface monolayer of a lipoprotein. Under oxidative conditions (e.g. in the presence of Cu²⁺ ions) the fluorophore undergoes decomposition, resulting in a continuous decrease of fluorescence intensity which reflects the oxidation of a chemically defined phospholipid molecule with well defined localization. When incorporated into LDL particles, the kinetics of the decrease in DPHPC fluorescence intensity upon exposure to Cu²⁺ is very similar to that of conjugated diene accumulation. Furthermore, our assay can be applied to follow the oxidation of lipids in diluted serum and may also be developed into a suitable test system for clinical studies of susceptibility of plasma lipids to oxidation.     

Direct and Mn-Controlled Indirect Iron Oxidation by Leptothrix discophora SS-1 and Leptothrix cholodnii

Direct biotic and homogeneous abiotic Fe(II) oxidation rates as well as oxidation rates of Fe(II) with MnO_X were determined in laboratory experiments and compared with biotic Mn(II) oxidation rates. In groundwaters and thermal installation waters, parameters for both Fe(II) oxidation steps were studied and products of biotic and abiotic Fe(II) and Mn(II) oxidation were analyzed. Direct Fe(II) oxidation of active Leptothrix cholodnii cultures can reach first-order rates of up to 1.17 ± 0.90 h^?1. Second-order rates of Fe(II) oxidation with biogenic, Leptothrix-cholodnii- and Leptothrix-discophora-SS-1-originating MnO_X lead to rates comparable with those as obtained with abiotic H⁺-birnessite.     

Adsorption of Colloidal Iron by Bacteria

The adsorption of iron from a positive-iron sol by species of seven bacterial genera was examined by electron microscopy. All species precipitated the iron from the sol, and the bacterial cells became encrusted with iron. This was related to iron deposition in surface water supplies.     

Use of an Intelligent Control System To Evaluate Multiparametric Effects on Iron Oxidation by Thermophilic Bacteria

A learning-based intelligent control system, the BioExpert, was developed and applied to the evaluation of multiparametric effects on iron oxidation by enrichment cultures of moderately thermophilic, acidophilic mining bacteria. The control system acquired and analyzed the data and then selected and maintained the sets of conditions that were evaluated. Through multiple iterations, the BioExpert selected sets of conditions that resulted in improved iron oxidation rates. The results obtained with the BioExpert suggested that temperature and pH were coupled, or interactive, parameters. Elevated temperatures (51.5°C) in combination with a moderately high pH (pH 1.84) impaired the growth of and iron oxidation by the enrichment culture. Moderate-to-high oxidation rates were achieved with a relatively high pH in combination with a relatively low temperature or, conversely, with a relatively low pH in combination with a relatively high temperature. The interactive effect of pH and temperature was not apparent from the results obtained in an experiment in which temperature was the only parameter that was varied. When the BioExpert was applied to a mixed culture containing mesophilic and thermophilic bacteria, the computer “learned” that pH 1.8, 45°C, and an inlet iron concentration from 30 to 35 mM were most favorable for iron oxidation. In conclusion, this study demonstrated that the learning-based intelligent control system BioExpert was an effective experimental tool that can be used to examine multiparametric effects on the growth and metabolic activity of mining bacteria.     

Carbon Monoxide Oxidation by Methanogenic Bacteria

Different species of methanogenic bacteria growing on CO(2) and H(2) were shown to remove CO added to the gas phase. Rates up to 0.2 mumol of CO depleted/min per 10 ml of culture containing approximately 7 mg of cells (wet weight) were observed. Methanobacterium thermoautotrophicum was selected for further study based on its ability to grow rapidly on a completely mineral medium. This species used CO as the sole energy source by disproportionating CO to CO(2) and CH(4) according to the following equation: 4CO + 2H(2)O --> 1CH(4) + 3CO(2). However, growth was slight, and the growth rate on CO was only 1% of that observed on H(2)/CO(2). Growth only occurred with CO concentrations in the gas phase of lower than 50%. Growth on CO agrees with the finding that cell-free extracts of M. thermoautotrophicum contained both an active factor 420 (F(420))-dependent hydrogenase (7.7 mumol/min per mg of protein at 35 degrees C) and a CO-dehydrogenating enzyme (0.2 mumol/min per mg of protein at 35 degrees C) that catalyzed the reduction of F(420) with CO. The properties of the CO-dehydrogenating enzyme are described. In addition to F(420), viologen dyes were effective electron acceptors for the enzyme. The apparent K(m) for CO was higher than 1 mM. The reaction rate increased with increasing pH and displayed an inflection point at pH 6.7. The temperature dependence of the reaction rate followed the Arrhenius equation with an activation energy (DeltaHdouble dagger) of 14.1 kcal/mol (59.0 kJ/mol). The CO dehydrogenase activity was reversibly inactivated by low concentrations of cyanide (2 muM) and was very sensitive to inactivation by oxygen. Carbon monoxide dehydrogenase of M. thermoautotrophicum exhibited several characteristic properties found for the enzyme of Clostridium pasteurianum but differed mainly in that the clostridial enzyme did not utilize F(420) as the electron acceptor.     

Continuous Steady-State Method Using Tenax for Delivering Tetrachloroethene to Chloro-Respiring Bacteria

Tenax-TA, a solid-phase sorbent, was used as an alternative to hexadecane for continuous delivery of tetrachloroethene (PCE) to Desulfuromonas strain BB1, a chloro-respiring microorganism. In both batch and bioreactor configurations, Tenax not only maintained low, steady-state concentrations of PCE in an active culture for several months but also adsorbed the product of dechlorination, cis-1,2-dichloroethene, before it approached toxic levels.     

一种鉴定过氧化氢酶活性的铁染色法

采用聚丙烯酰胺凝胶电泳(PAGE)技术,建立了一种新的鉴定过氧化氢酶活性的方法──铁染色法．结果表明,细菌过氧化氢酶及牛肝过氧化氢酶显示单一的酶活性带．该方法操作简单、快速、灵敏度高、专一性强.是一种切实可行的鉴定过氧化氢酶活性染色法.     

Stimulation of Autotrophic Ammonium Oxidation in Rice Rhizosphere Soil by the Insecticide Carbofuran

The application of the insecticide carbofuran (technical or formulated) to rice rhizosphere soil suspensions at 10 and 100 ppm (μg/ml) of active ingredient distinctly stimulated the autotrophic oxidation of ammonium. Evidence suggested that Nitrosomonas sp. was enriched in the presence of carbofuran. Formulated carbofuran (Furadan 3G) exhibited a more pronounced stimulation of ammonium oxidation than that exhibited by technical-grade (99.5%) carbofuran, a result which was attributed to the CaCO₃ present in the formulation.     

《细菌生长曲线测定》实验方法的研究

为深入学习细菌生长曲线测定方法,并使其在教学、科研和生产中得到更好应用,以实验室分离菌株为研究对象,以大肠埃希菌生长曲线的测定为实验方法,并对该方法的应用进行了研究并提出改进方案。研究结果表明,采用上述方法仅测出2株葡萄球菌属菌株的生长曲线,采取增加溶氧量、单瓶培养及取样后立即测定,得到3株芽胞杆菌属菌株的生长曲线,而对产色素的考克氏菌属菌株宜采用平板菌落计数的方法进行生长曲线的绘制。不同菌属菌株生长曲线的测定要根据实际情况选择合适方法,测定过程要防止低温保存对菌液光密度的影响。     

Ecological Implications of an Improved Direct Viable Count Method for Aquatic Bacteria

The direct viable count method first described by Kogure et al. (Can. J. Microbiol. 25:415-420, 1979) was improved by using an antibiotic cocktail instead of nalidixic acid alone. We screened 100 marine isolates from two coastal areas for their sensitivities to five replication-inhibiting antibiotics, including four quinolones (nalidixic, piromidic, and pipemidic acids and ciprofloxacin) and one (beta)-lactam (cephalexin). It was shown that growth inhibition of all isolates cannot be readily achieved by using a single antibiotic. Inhibition was much more efficient when all the antibiotics were combined, making it possible to use this method with natural communities. In combination, the concentration of each antibiotic could be lowered and the incubation time could be increased without any growth. Under such conditions, it was shown that the fraction of substrate-responsive cells within natural marine communities is much greater (1 to 2 orders of magnitude) than those reported by traditional procedures. Furthermore, the new procedure made substrate-responsive cells more clearly distinguishable. These improvements resulted in an increased incubation time and were related to metabolic expression of slow-growing cells and/or to the recovery of starved cells. The increased fraction of viable cells within marine communities has ecological implications on the metabolic role of nonculturable cells.     

一种利用RT-HPLC分析乳酸菌产生物胺的方法

具有脱羧酶活性的乳酸菌可通过氨基酸的脱羧反应产生具有潜在安全风险的生物胺。本文利用脱羧酶培养基初步筛查61株乳酸菌产生物胺情况,再通过RT-HPLC法测定其在发酵液和发酵乳中的生物胺含量。用10%的三氯乙酸提取样品中的生物胺,采用苯甲酰氯衍生处理后,以甲醇/水为流动相,进行梯度洗脱,流速0.8mL/min,紫外检测器波长为254nm。结果显示,组胺和酪胺得到良好的分离,在给定的浓度范围内呈现良好的线性关系(R20.995)。在发酵液和发酵乳中添加生物胺混合标准溶液,平均回收率为97.92%-101.14%,相对偏差RSD5%。结果表明,发酵液与发酵乳中生物胺的RT-HPLC法,是一种快捷、稳定、灵敏度高的检测方法,其与脱羧酶培养基法结合可以准确地实现对乳酸菌产生物胺的评价。