Robotized incubation system for monitoring gases (O2, NO, N2O N2) in denitrifying cultures

As genomic data for bacteria are unraveled at an increasing speed, there is a need for more efficient and refined techniques to characterize metabolic traits. The regulatory apparatus for denitrification, for instance, has been explored extensively for type strains, but we lack refined observations of how these and wild type denitrifiers respond metabolically to changing environmental conditions. There is a need for new "phenomic" approaches, and the present paper describes one; an automated incubation system for the study of gas kinetics in 15 parallel bacterial cultures. An autosampler with a peristaltic pump takes samples from the headspace, and replaces the sampled gas with He by reversing the pump. The sample flows through the injector of a micro GC (for determination of N(2), O(2), CH(4), CO(2), N(2)O) to the inlet of a chemoluminescence NO analyzer. The linear range for NO is 0.5-10(4) ppmv (CV=2%, detection limit 0.2 ppmv). The gas leakage of N(2) into the system is low and reproducible, allowing the quantification of N(2) production (in flasks with He+O(2) atmosphere) with a detection limit of 150-200 nmol N(2) for a single time increment. The gas loss by each sampling is taken into account, securing mass balance for all gases, thus allowing accurate estimation of electron flows to the various terminal acceptors (O(2), NO(2)(-), NO, N(2)O) throughout the culture's depletion of O(2) and NO(x). We present some experimental results with Agrobacterium tumefaciens, Paracoccus denitrificans and denitrifying communities, demonstrating the system's potential for unraveling contrasting patterns of denitrification gene expression as a function of concentrations of O(2) and NO in the medium.     

土壤中反硝化酶活性变化与N2O排放的关系

研究施肥条件下,土壤反硝化酶活性硝酸还原酶(NR)活性、亚硝酸还原酶(NiR)活性及羟胺还原酶(HyR)活性在玉米生长季节中的变化及其与土壤含水量、硝态氮含量、N₂O排放之间的关系。结果表明,3种还原酶都有明显的季节变化规律并受土壤水分含量及施肥的影响。通过研究3种反硝化酶活性与土壤含水量及N₂O排放量之间的关系后指出,反硝化酶活性变化可作为一个区分旱田N₂O产生途径的指标.     

N2O reduction by Vibrio succinogenes.

Vibrio succinogenes grew anaerobically at the expense of formate oxidation, with nitrous oxide (N2O) serving a terminal oxidant. N2O was quantitatively reduced to dinitrogen (N2). In the presence of 5 x 10(-2) atm (ca. 5 kPa) of acetylene (C2H2), which inhibits the reduction of N2O, growth of V. succinogenes was completely inhibited. Nitrate was reduced to nitrite or to ammonia, depending on the extent of availability of formate, but N2 was not produced by reduction of nitrate. During the reduction of nitrate to ammonia, all eight electrons transported to a molecule of nitrate appeared to be coupled for energy-yielding reactions.     

Effect of carbon source and competition for electrons on nitrous oxide reduction in a mixed denitrifying microbial community

The competition for electrons has been recently demonstrated to affect the reduction rates of the nitrogen oxides in a methanol enriched denitrifying community. The aim of this study was to test if electron competition also occurred when other substrates were used for denitrification and if that could have an effect on the potential nitrous oxide (N₂O) production and subsequent consumption. A denitrifying culture was developed in a sequencing batch reactor using nitrate as electron acceptor and a combination of acetate, ethanol and methanol as carbon sources. Four sets of batch tests were conducted using acetate, ethanol, methanol and a combination of the three carbon sources respectively. For each set the effect of nitrate, nitrite and nitrous oxide on each other reduction rates when present individually or in combination was assessed. Results show that reduction rates are affected by the type of substrate added, probably due to different microbial populations specialized with consuming a particular substrate. Also, N₂O reduction rate is the most reduced under the different electron competition scenarios tested, which results in N₂O accumulation in some cases. The effect of substrate limitation on N₂O reduction was also assessed.     

Effect of anaerobic reaction time on denitrifying phosphorus removal and N2O production

Nitrous oxide (N₂O) is a highly potent greenhouse gas; however, the characteristics of N₂O production during denitrification using poly-β-hydroxyalkanoates (PHA) as a carbon source are not well understood. In this study, effects of anaerobic reaction time (AnRT) on PHA formation, denitrifying phosphorus removal and N₂O production were investigated using a laboratory-scale anaerobic/anoxic/oxic sequencing batch reactor (An/A/O SBR). The results showed that operation of the An/A/O SBR for 0.78 SRT (47 cycles) after the AnRT was shortened from 90 min to 60 min resulted in anaerobically synthesized PHA improving by 1.8 times. This improvement was accompanied by increased phosphorus removal efficiency and denitrification. Accordingly, the N₂O-N production was reduced by 6.7 times. Parallel batch experiments were also conducted with AnRTs of 60, 90 and 120 min. All results indicated that in addition to the amount of anaerobically synthesized PHA, the kinetics of PHA degradation also regulated denitrifying phosphorus removal and N₂O production.     

Decreased N2O reduction by low soil pH causes high N2O emissions in a riparian ecosystem

Quantification of harmful nitrous oxide (N(2)O) emissions from soils is essential for mitigation measures. An important N(2)O producing and reducing process in soils is denitrification, which shows deceased rates at low pH. No clear relationship between N(2)O emissions and soil pH has yet been established because also the relative contribution of N(2)O as the denitrification end product decreases with pH. Our aim was to show the net effect of soil pH on N(2)O production and emission. Therefore, experiments were designed to investigate the effects of pH on NO(3)(-) reduction, N(2)O production and reduction and N(2) production in incubations with pH values set between 4 and 7. Furthermore, field measurements of soil pH and N(2)O emissions were carried out. In incubations, NO(3)(-) reduction and N(2) production rates increased with pH and net N(2)O production rate was highest at pH 5. N(2)O reduction to N(2) was halted until NO(3)(-) was depleted at low pH values, resulting in a built up of N(2)O. As a consequence, N(2)O:N(2) production ratio decreased exponentially with pH. N(2)O reduction appeared therefore more important than N(2)O production in explaining net N(2)O production rates. In the field, a negative exponential relationship for soil pH against N(2)O emissions was observed. Soil pH could therefore be used as a predictive tool for average N(2)O emissions in the studied ecosystem. The occurrence of low pH spots may explain N(2)O emission hotspot occurrence. Future studies should focus on the mechanism behind small scale soil pH variability and the effect of manipulating the pH of soils.     

Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase.

A comparison of the effect of temperature on the reduction of N2 by purified molybdenum nitrogenase and vanadium nitrogenase of Azotobacter chroococcum showed differences in behaviour. As the assay temperature was lowered from 30 degrees C to 5 degrees C N2 remained an effective substrate for V nitrogenase, but not Mo nitrogenase, since the specific activity for N2 reduction by Mo nitrogenase decreased 10-fold more than that of V nitrogenase. Activity cross-reactions between nitrogenase components showed the enhanced low-temperature activity to be associated with the Fe protein of V nitrogenase. The lower activity of homologous Mo nitrogenase components, although dependent on the ratio of MoFe protein to Fe protein, did not equal that of V nitrogenase even under conditions of high electron flux obtained at a 12-fold molar excess of Fe protein.     

Competition for electrons between mono-oxygenations of pyridine and 2-hydroxypyridine

Pyridine and its heterocyclic derivatives are widely encountered in industrial wastewaters, and they are relatively recalcitrant to biodegradation. Pyridine biodegradation is initiated by two mono-oxygenation reactions that compete for intracellular electron donor (2H). In our experiments, UV photolysis of pyridine generated succinate, whose oxidation augmented the intracellular electron donor and accelerated pyridine biodegradation and mineralization. The first mono-oxygenation reaction always was faster than the second one, because electrons provided by intracellular electron donors were preferentially utilized by the first mono-oxygenase; this was true even when the concentration of 2HP was greater than the concentration of pyridine. In addition, the first mono-oxygenation had faster kinetics because it had higher affinity for its substrate (pyridine), along with less substrate self-inhibition.     

Symbiotic Bradyrhizobium japonicum reduces N2O surrounding the soybean root system via nitrous oxide reductase

N(2)O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N(2)O uptake and conversion of (15)N-N(2)O into (15)N-N(2). Free-living cells of USDA110 showed N(2)O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N(2)O reductase activity. When detached soybean nodules formed with USDA110 were fed with (15)N-N(2)O, they rapidly emitted (15)N-N(2) outside the nodules at a ratio of 98.5% of (15)N-N(2)O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N(2)O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N(2)O (0.34 ppm). These results indicate that the conversion of N(2)O to N(2) depends exclusively on the respiratory N(2)O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N(2)O.     

Symbiotic Bradyrhizobium japonicum Reduces N2O Surrounding the Soybean Root System via Nitrous Oxide Reductase

N₂O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N₂O uptake and conversion of ¹⁵N-N₂O into ¹⁵N-N₂. Free-living cells of USDA110 showed N₂O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N₂O reductase activity. When detached soybean nodules formed with USDA110 were fed with ¹⁵N-N₂O, they rapidly emitted ¹⁵N-N₂ outside the nodules at a ratio of 98.5% of ¹⁵N-N₂O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N₂O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N₂O (0.34 ppm). These results indicate that the conversion of N₂O to N₂ depends exclusively on the respiratory N₂O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N₂O.     

Divalent metal addition restores sulfide-inhibited N2O reduction in Pseudomonas aeruginosa

Hydrogen sulfide (H₂S) inhibits the last step of the denitrification process, i.e. the reduction of nitrous oxide (N₂O) to dinitrogen gas (N₂), both in natural environments (marine sediments) and industrial processes (activated sludge, methanogenic sludge, BioDeNOx process). In a previously published study, we showed that the inhibitory effect of sulfide to N₂O reduction in mixed microbial communities is reversible and can be counteracted by dosing trace amounts of copper. It remained, however, unclear if this was due to copper sulfide precipitation or a retrofitting of the copper containing N₂O-reductase (N₂OR). The present study aimed to elucidate the mechanism of the restoration of sulfide-inhibited N₂O reducing activity by metal addition to a pure Pseudomonas aeruginosa culture. This was done by using other metals (zinc, cobalt and iron) in comparison with copper. Zinc and cobalt clearly alleviated the sulfide inhibition of N₂OR to the same extent as copper and the activity restoration was extremely fast (within 15 min, Fig. 3) for zinc, cobalt and copper. This suggests that the alleviation of the inhibitory effect of sulfide is due to metal sulfide precipitation and thus not exclusively limited to Cu. This work also underlines the importance of metal speciation: supply of iron did not restore the N₂OR activity because it was precipitated by the phosphates present in the medium and thus could not precipitate the sulfide.     

Absolute scattering probabilities for subexcitation electrons in condensed H2O

Absolute elastic and inelastic collision probabilities per unit length for subexcitation electrons scattering in condensed water were determined by analysis of electron energy loss and transmission data for electron energies between the vacuum level (zero incident kinetic energy) and 3.2 eV. Highly disordered solid films were deposited on a metal substrate with thickness varying between 1 and 50 layers. Analysis by an N-channel model of hot electron transport provides values of 0.017 and 0.068 per layer for the elastic and total inelastic collision probabilities, respectively.     

Molecular characterization of nosRZDFYLX genes coding for denitrifying nitrous oxide reductase of Bradyrhizobium japonicum

The nosRZDFYLX gene cluster for the respiratory nitrous oxide reductase from Bradyrhizobium japonicum strain USDA110 has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ, the structural gene, nosD, nosF, nosY, nosL, and nosX were detected. The deduced amino acid sequence exhibited a high degree of similarity to other nitrous oxide reductases from various sources. The NosZ protein included a signal peptide for protein export. Mutant strains carrying either a nosZ or a nosR mutation accumulated nitrous oxide when cultured microaerobically in the presence of nitrate. Maximal expression of a P nosZ-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Microaerobic activation of the fusion required FixLJ and FixK(2).     

短期落干对水稻土反硝化微生物丰度和N2O释放的影响

为了探明水稻土落干过程对温室气体排放和反硝化微生物的影响,通过模拟水稻土淹水落干过程,系统监测了落干开始后24 h内N2O的释放和氧化还原电位(Eh)的变化,并利用实时PCR(qPCR)方法测定了反硝化功能基因narG和nosZ的丰度.结果表明：落干开始后4 h N₂O释放量就明显增加,在24 h时N₂O的释放量比淹水对照增加了5倍多;narG和nosZ基因丰度也随着落干过程的推移而快速增加;而且N₂O排放通量与narG基因呈极显著相关(P<0.01).表明水稻土短期淹水落干过程中,含narG基因反硝化微生物是驱动N₂O释放的主要功能微生物.     

硫化物抑制潮土反硝化过程中氧化亚氮还原的菌群机制

【背景】土壤中的反硝化作用形成气态产物N₂O和N₂,会导致氮素的气态损失,并造成温室效应。硫化物对土壤的N₂O还原具有抑制作用,但其对菌群和功能基因的影响机制还不清楚。【目的】研究有无外加碳源情况下,硫化物对反硝化作用中间产物(NO、N₂O)的积累、反硝化功能基因(narG、nirS、nirK和nosZ)表达量以及菌群结构的影响。【方法】分别设置不同量葡萄糖(0和1000mg-C/kg干重土壤)和硫化钠(0和150mg-S/kg干重土壤)添加的交叉处理,进行室内微宇宙培养实验,利用自动化培养与实时气体检测系统检测培养过程中NO、N₂O和N₂的积累量,通过反转录定量PCR测定反硝化功能基因表达量,利用MiSeq技术平台基于16S rRNA基因序列的高通量测序分析样品的菌群结构。【结果】硫化钠的添加显著抑制N₂O还原,但是其对于N₂O积累量没有显著影响,却显著降低了NO的积累量。硫化钠的添加短时间内在转录水平上显著抑...     

Competition by Bradyrhizobium Strains for Nodulation of the Nonlegume Parasponia andersonii

Bradyrhizobium strains isolated from the nonlegume Parasponia spp. formed a group of strains that were highly competitive for nodulation of P. andersonii when paired with strains isolated from legumes. Strains from legumes, including those of similar effectiveness to NGR231 and CP283, were not able to form nodules as single occupants on P. andersonii in the presence of Parasponia strains. However, NGR86, an isolate from Macroptilium lathyroides, jointly occupied one-third of the nodules formed with each of the three strains isolated from Parasponia spp. Time taken for nodules to appear may have influenced the outcome of competition, since CP283 and all isolates from legumes were slow to nodulate P. andersonii. Among the Parasponia strains, competitiveness for nodulation of P. andersonii was not associated with effectiveness of nitrogen fixation. The highly effective strain CP299 was a poor competitor when paired with the least effective strain NGR231. CP283 was the least competitive of the Parasponia strains but was still able to dominate nodules when paired with legume isolates. Dual occupancy was high, up to 67% when the inoculum contained CP299 and CP273. Both the Muc⁺ and Muc^- types of CP283 form a symbiosis of similar effectiveness and were similarly competitive at high inoculation densities, but the Muc^- form was more competitive at low inoculum densities. Both forms frequently occupied the same nodule. Bradyrhizobium strains isolated from Parasponia spp. may have specific genetic information that favor their ability to competitively and effectively infect plants in the genus Parasponia (Ulmaceae) outside the Leguminosae.     

Competition for nitrate between denitrifying Pseudomonas stutzeri and nitrate ammonifying enterobacteria

Abstract Competition for nitrate between nitrate ammonifying enterobacteria and a denitrifying pseudomonad was studied in electron acceptor-limited chenostats. In pure cultures, using different carbon and energy sources, the C/N-ratio needed for denitrification is far lower than that required for nitrate ammonification. In mixed cultures of Citrobacter freundii and Pseudomonas stutzeri , competing for nitrate with l -lactate as electron donor, the nitrate ammonifying organism dominated at dilution rates of D ≤ 0.14 h⁻¹. Competition for both nitrate and l -lactate at a dilution rate of D = 0.05 h⁻¹ always resulted in the coexistence of both species. Using glucose as additional carbon source, the final ratio of nitrate ammonifying and denitrifying organism depended on the C/N-ratio as well as on the dilution rate. The results of the study are discussed with respect to field data.