Enumeration of denitrifying microbial populations in turf

Summary Denitrifer populations of a silt and silt loam soil under a Kentucky bluegrass turf were enumerated using the most probable number (MPN) procedure. The influence of soil texture, soil depth, soil moisture, and additions of nitrate fertilizer on denitrifier populations were determined. Saturated soil conditions increased denitrifier populations 87-fold in the silt soil and 121-fold in the silt loam soil. Denitrifier populations did not differ significantly between soil depths and additions of fertilizer nitrate did not influence populations.     

An integrated network analysis reveals that nitric oxide reductase prevents metabolic cycling of nitric oxide by Pseudomonas aeruginosa

Nitric oxide (NO) is a chemical weapon within the arsenal of immune cells, but is also generated endogenously by different bacteria. Pseudomonas aeruginosa are pathogens that contain an NO-generating nitrite (NO₂⁻) reductase (NirS), and NO has been shown to influence their virulence. Interestingly, P. aeruginosa also contain NO dioxygenase (Fhp) and nitrate (NO₃⁻) reductases, which together with NirS provide the potential for NO to be metabolically cycled (NO→NO₃⁻→NO₂⁻→NO). Deeper understanding of NO metabolism in P. aeruginosa will increase knowledge of its pathogenesis, and computational models have proven to be useful tools for the quantitative dissection of NO biochemical networks. Here we developed such a model for P. aeruginosa and confirmed its predictive accuracy with measurements of NO, O₂, NO₂⁻, and NO₃⁻ in mutant cultures devoid of Fhp or NorCB (NO reductase) activity. Using the model, we assessed whether NO was metabolically cycled in aerobic P. aeruginosa cultures. Calculated fluxes indicated a bottleneck at NO₃⁻, which was relieved upon O₂ depletion. As cell growth depleted dissolved O₂ levels, NO₃⁻ was converted to NO₂⁻ at near-stoichiometric levels, whereas NO₂⁻ consumption did not coincide with NO or NO₃⁻ accumulation. Assimilatory NO₂⁻ reductase (NirBD) or NorCB activity could have prevented NO cycling, and experiments with ΔnirB, ΔnirS, and ΔnorC showed that NorCB was responsible for loss of flux from the cycle. Collectively, this work provides a computational tool to analyze NO metabolism in P. aeruginosa, and establishes that P. aeruginosa use NorCB to prevent metabolic cycling of NO.     

Denitrifying population dynamic and evolution of nitrogen gas during a composting process

Abstract Little information exists about nitrogen losses through microbial activity during treatment of solid urban waste (SUW) by processes such as composting. In the present study, in addition to evaluating the pattern of nitrogen losses by denitrification at different stages of the process, a comparison between the method of Pochon and Tardieux, and an improved gas chromatographic method for estimating denitrifying populations was undertaken, Though the MPN (Most Probable Number) enumerations were higher using the colorimetric method than the gas chromatographic one, the patterns of the two graphs showing numbers of denitrifiers during composing were the same. The highest numbers were revealed immediately after loading the reactor (10⁷–10⁸/g d.w.), lower numbers of denitrifiers were found in the second sampling corresponding to the thermophilic phase (10³–10⁴/g d.w.). These numbers increased gradually as the waste material stabilized (10th to 123rd day of composting) to again reach values of 10⁷–10⁸/g d.w.     

反硝化基因工程菌株YP4S的构建及对污水除氮特性研究

从养殖场污泥中筛选出菌株YP4,经16S rDNA分子发育树的同源序列比对,确定为克雷伯什菌属(Klebsiella sp.)。由NCBI数据库查编码亚硝酸还原酶(Nir)的基因nirS序列,设计引物,以铜绿假单胞菌PAOI基因组DNA为模板,应用PCR技术扩增目的片段nirS,经过双酶切、克隆和转化,得到重组质粒pYP4S,然后转化野生菌株YP4,构建反硝化基因工程菌YP4S。菌株生长曲线测定表明,工程菌株YP4S与YP4的生长特性基本一致。工程菌株YP4S对模拟污水COD、TN、NH_4^+-N和NO_3^--N具有较高的去除率,YP4S与YP4相比,对NO_2^--N积累的减少量为(32.44±3.96)%,明显减少了NO_2^--N的积累。通过正交试验获得工程菌株YP4S在C/N=10、T=30℃、r=200 r/min和pH=7.0的最佳组合条件下,对模拟污水TN去除率较高。应用工程菌株YP4S处理猪场沉淀池的实际污水,COD、TN、TP、NH_4^+-N和NO_3^--N去除率分别为(95.87±0.82)%、(76.38±3.84)%、(97.13±0.54)%和(75.35±2.57)%,NO_2^--N积累量为(3.31±1.24) mg/L,表明工程菌株YP4S具有较好反硝化作用,对含氮量高的实际污水修复具有潜在的应用前景。     

固氮施氏假单胞菌亚硝酸盐还原酶基因nirS转录特性及功能鉴定

【目的】研究固氮施氏假单胞菌(Pseudomonas stutzeri)A1501亚硝酸盐还原酶结构基因nir S的转录调控机制及其在反硝化过程中的功能。【方法】构建nir S-lac Z融合载体,利用三亲本结合法将其导入野生型A1501,通过β-半乳糖苷酶活性的测定,分析不同供氧状况、不同浓度的硝酸盐、亚硝酸盐对nir S基因表达的影响;同时将该载体导入rpo N突变株中,研究氮代谢调控因子Rpo N对nir S基因转录影响。通过同源重组方法构建nir S突变株,通过生化表型测定明确nir S在反硝化过程中的功能。【结果】启动子活性测定表明,nir S基因厌氧条件下高水平表达,是好氧条件下表达水平的4倍;nir S的表达受硝酸盐诱导,但不受亚硝酸盐的诱导;Rpo N突变株中,nir S的表达活性为野生型的1/4,nir S启动子未发现Rpo N的保守结合位点,表明nir S的表达受Rpo N间接调控。表型测定显示以硝酸盐为电子受体时Δnir S的反硝化能力降低了约20%;以亚硝酸盐为电子受体时Δnir S仅有微弱的反硝化能力,并且nir S的突变使得菌体在反硝化条件下利用亚硝酸盐的能力显著减弱。nir S突变提高了菌体在亚硝酸为电子受体的反硝化条件下的固氮酶活。【结论】A1501中nir S基因的转录受外界氧及硝酸盐的影响,同时受氮代谢Sigma因子Rpo N的调控。nir S在A1501菌反硝化过程中起关键作用,参与了亚硝酸盐的转化。     

Nitrogen removal from secondary effluent by using integrated constructed wetland system

The treatment capacity of an integrated constructed wetland system (CWS) that was designed to reduce nitrogen (N) from secondary effluent was explored. The integrated CWS consisted of vertical-flow constructed wetland, floating bed and sand filter. The vertical-flow wetland was filled with gravel, steel slag and peat from the bottom to the top. Vetiver zizanioides was selected to grow in the vertical-flow constructed wetland and Coix lacrymajobi L. was grown in the floating bed. The results showed that the integrated CWS displayed superior removal efficiency for nitrate nitrogen (NO₃⁻-N), ammonia nitrogen (NH₄⁺-N), nitrite nitrogen (NO₂⁻-N), and total nitrogen (TN). The average NO₃⁻-N, NO₂⁻-N, NH₄⁺-N and TN removal efficiencies of the integrated CWS were 98.83%, 95.60%, 98.05% and 92.41%, respectively, during the whole experimental operation. The integrated CWS may have a good potential for removing N from secondary effluent.     

Dynamics of gaseous nitrogen and carbon fluxes in riparian alder forests

We studied greenhouse gas (GHG) fluxes in two differently loaded riparian Alnus incana-dominated forests in agricultural landscapes of southern Estonia: a 33-year-old stand in Porijõgi, in which the uphill agricultural activities had been abandoned since the middle of the 1990s, and a 50-year-old stand in Viiratsi, which still receives polluted lateral flow from uphill fields fertilized with pig slurry. In Porijõgi, closed-chamber based sampling lasted from October 2001 to October 2009, whereas in Viiratsi the sampling period was from November 2003 to October 2009. Both temporal and spatial variations in all GHG gas fluxes were remarkable. Local differences in GHG fluxes between micro-sites (“Edge”, “Dry” and “Wet” in Porijõgi, and “Wet”, “Slope” and “Dry” in Viiratsi) were sometimes greater than those between sites. Median values of GHG fluxes from both sites over the whole study period and all microsites did not differ significantly, being 45 and 42 mg CO₂-C m⁻² h⁻¹, 8 and 0.5 μg CH₄-C m⁻² h⁻¹, 1.0 and 2.1 mg N₂-N m⁻² h⁻¹, and 5 and 9 μg N₂O-N m⁻² h⁻¹, in Porijõgi and Viiratsi, respectively. The N₂:N₂O ratio in Viiratsi (40-1200) was lower than in Porijõgi (10-7600). The median values-based estimation of the Global Warming Potential of CH₄ and N₂O was 19 and 185 kg CO₂ equivalents (eq) ha⁻¹ yr⁻¹ in Porijõgi and −14 and 336 kg CO₂ eq ha⁻¹ yr⁻¹ in Viiratsi, respectively. A significant Spearman rank correlation was found between the mean monthly air temperature and CO₂, CH₄ and N₂ fluxes in Porijõgi, and N₂O flux in Viiratsi, and between the monthly precipitation and CH₄ fluxes in both study sites. Higher groundwater level significantly increases CH₄ emission and decreases CO₂ and N₂O emission, whereas higher soil temperature significantly increases N₂O, CH₄ and N₂ emission values. In Porijõgi, GHG emissions did not display any discernable trend, whereas in Viiratsi a significant increase in CO₂, N₂, and N₂O emissions has been found. This may be a result of the age of the grey alder stand, but may also be caused by the long-term nutrient load of this riparian alder stand, which indicates a need for the management of similar heavily loaded riparian alder stands.     

Biofilm stratification during simultaneous nitrification and denitrification (SND) at a biocathode

The aeration of the cathode compartment of bioelectrochemical systems (BESs) was recently shown to promote simultaneous nitrification and denitrification (SND). This study investigates the cathodic metabolism under different operating conditions as well as the structural organization of the cathodic biofilm during SND. Results show that a maximal nitrogen removal efficiency of 86.9 ± 0.5%, and a removal rate of 3.39 ± 0.08 mg N L⁻¹ h⁻¹ could be achieved at a dissolved oxygen (DO) level of 5.73 ± 0.03 mg L⁻¹ in the catholyte. The DO levels used in this study are higher than the thresholds previously reported as detrimental for denitrification. Analysis of the cathodic half-cell potential during batch tests suggested the existence of an oxygen gradient within the biofilm while performing SND. FISH analysis corroborated this finding revealing that the structure of the biofilm included an outer layer occupied by putative nitrifying organisms, and an inner layer where putative denitrifying organisms were most dominant. To our best knowledge this is the first time that nitrifying and denitrifying microorganisms are simultaneously observed in a cathodic biofilm.     

Biological treatment of shrimp production wastewater

Over the last few decades, there has been an increase in consumer demand for shrimp, which has resulted in its worldwide aquaculture production. In the United States, the stringent enforcement of environmental regulations encourages shrimp farmers to develop new technologies, such as recirculating raceway systems. This is a zero-water exchange system capable of producing high-density shrimp yields. The system also produces wastewater characterized by high levels of ammonia, nitrate, nitrite, and organic carbon, which make waste management costs prohibitive. Shrimp farmers have a great need for a waste management method that is effective and economical. One such method is the sequencing batch reactor (SBR). A SBR is a variation of the activated sludge biological treatment process. This process uses multiple steps in the same reactor to take the place of multiple reactors in a conventional treatment system. The SBR accomplishes equalization, aeration, and clarification in a timed sequence in a single reactor system. This is achieved through reactor operation in sequences, which includes fill, react, settle, decant, and idle. A laboratory scale SBR was successfully operated using shrimp aquaculture wastewater. The wastewater contained high concentrations of carbon and nitrogen. By operating the reactors sequentially, namely, aerobic and anoxic modes, nitrification and denitrification were achieved as well as removal of carbon. Ammonia in the waste was nitrified within 4 days. The denitrification of nitrate was achieved by the anoxic process, and 100% removal of nitrate was observed within 15 days of reactor operation.     

Bioavailability of organic matter in a freshwater estuarine sediment: long<Emphasis Type="Bold">-</Emphasis>term degradation experiments with and without nitrate supply

Organic carbon degradation experiments were carried out using flow-through reactors with sediments collected from an intertidal freshwater marsh of an eutrophic estuary (The Scheldt, Belgium). Concentrations of nitrate, nitrite, dissolved inorganic carbon (DIC), dissolved organic carbon, methane, dissolved cations (Ca²⁺, Mg²⁺, Na⁺ and K⁺), total dissolved Fe, phosphate and alkalinity were measured in the outflow solutions from reactors that were supplied with or without the terminal electron acceptor nitrate. Organic carbon mineralization rates were computed from the release rates of DIC after correcting for the contribution of carbonate mineral dissolution. The experiments ran for several months until nitrate reducing activity could no longer be detected. In the reactors supplied with nitrate, 10–13% of the bulk sedimentary organic carbon (SOC) was mineralized by the end of the experiments. In reactors receiving no nitrate, only 3–9% of the initial SOC was mineralized. Organic matter utilization by nitrate reducers could be described as the simultaneous degradation of two carbon pools with different maximum oxidation rates and half-saturation constants. Even when nitrate was supplied in non-limiting concentrations about half of the carbon mineralization in the reactors was due to fermentative processes, rather than being coupled to nitrate respiration. Fermentation may thus be responsible for a large fraction of the DIC efflux from organic-rich, nearshore sediments.