三株异养硝化–好氧反硝化细菌对圆斑星鲽养殖水质的净化效果

为了考察3株异养硝化–好氧反硝化细菌对圆斑星鲽(Verasper variegates)养殖废水的净化效果,选择初始体重为(98±6)g的圆斑星鲽240尾,随机分为8组。分别接种花津滩芽孢杆菌(Bacillus hwajinpoensis)SLWX_2、嗜碱盐单胞菌(Halomonas alkaliphila)X_3和麦氏交替单胞菌(Alteromonas macleodii)SLNX2的不同组合。测定了不同组合中各项无机氮及有机物的变化情况。结果显示,在实验过程中,对照组氨氮、亚硝酸氮、硝酸氮、总氮和化学需氧量的浓度均呈持续上升趋势,分别从0.21 mg/L升至15.94mg/L,0.08 mg/L升至5.68 mg/L,1.10 mg/L升至7.05 mg/L,1.74 mg/L升至38.86 mg/L,1.19 mg/L升至22.87 mg/L。而加菌组的各指标浓度一直低于对照组,其中,SLWX_2+X_3+SLNX2组合对圆斑星鲽养殖废水净化效果最佳,氨氮、亚硝酸氮、硝酸氮、总氮、化学需氧量的浓度分别低于对照组68.55%、48.36%、58.38%、40.02%和27.47%,SLWX_2+X_3组合的净化效果次之。此外,第21天时,对照组出现大量死鱼现象,各实验组中仅有少量死鱼。研究表明,添加的异养硝化–好氧反硝化细菌可在不添加碳源的情况下实现脱氮功能,有效维护养殖水质,并且对圆斑星鲽无毒害及致病作用。     

Accumulation of Denitrifying Bacteria and Enhancement of Denitrification Activity in Deep Subsurface Soil in Relation to the Geological Profile

We investigated the denitrification activity and the distribution of the denitrifying bacteria of a boring survey site located on a volcanic plateau, where the geological profiles from surface to deep subsurface soil at the groundwater level had been examined. There were differences between the water quality in the Ito pyroclastic flow deposit (Shirasu) layer (44.2 to 54.5 m) and that in the Osumi pumice fall deposit (Pumice) layer (below 54.5 m) corresponding to the impermeable layer of unconfirmed groundwater: The nitrate concentration was less than 1 mg kg⁻¹ in the Shirasu layer and more than 10 mg kg⁻¹ in the Pumice layer (Kubota et al. 2005). Denitrification activity decreased from the surface to the loam layer and was enhanced in the Shirasu layer and the Haraigawa clay impermeable layer at a depth of 65 m. It was observed that the highest potential denitrification activity (10³ ng-N₂O d⁻¹ g⁻¹) in the impermeable layer was almost equal to that of a Kuroboku surface soil with slurry application. Viable counts of the sonic-samples, which indicated the presence of bacterial group with soil particles attached, increased in the impermeable layer. The ratios of viable or denitrifying bacterial counts in the sonic-samples to those in the wash-samples were significantly higher in the impermeable layer than those in the surface layer. These results suggest that the hydrogeological conditions enhanced the denitrification activity in the impermeable layer, the niches of which might be relatively anaerobic and have a sufficient supply of substrates to enable the denitrifying bacterial populations to multiply.     

Effect of the nitrification inhibitor nitrapyrin on the fate of nitrogen applied to a soil incubated under laboratory conditions

The aim of this study was to examine the effect of the nitrification inhibitor nitrapyrin on the fate and recovery of fertilizer nitrogen (N) and on N mineralization from soil organic sources. Intact soil cores were collected from a grassland field. Diammonium phosphate (DAP) and urea were applied as N sources. Cores were equilibrated at –5 kPa matric potential and incubated at 20 °C for 42 to 56 days. Changes in NH₄⁺‐N, accumulation of NO₃^–‐N, apparent recovery of applied N, and emission of N₂O (acetylene was used to block N₂O reductase) were examined during the study. A significant increase in NH₄⁺‐N released through mineralization was recorded when nitrapyrin was added to the control soil without N fertilizer application. In the soils to which N was added either as urea or DAP, 50–90 % of the applied N disappeared from the NH₄⁺‐N pool. Some of this N (8–16 %) accumulated as NO₃^–‐N, while a small proportion of N (1 %) escaped as N₂O. Addition of nitrapyrin resulted in a decrease and delay of NH₄⁺‐N disappearance, accumulation of much lower soil NO₃^–‐N contents, a substantial reduction in N₂O emissions, and a 30–40 % increase in the apparent recovery of added N. The study indicates that N recovery can be increased by using the nitrification inhibitor nitrapyrin in grassland soils at moisture condition close to field capacity.     

Nitrous oxide production of heavy metal contaminated soil

Arsenic (As), lead (Pb), copper (Cu) and zinc (Zn) can be found in large concentrations in mine spills of central and northern Mexico. Interest in these heavy metals has increased recently as they contaminate drinking water and aquifers in large parts of the world and severely affect human health, but little is known about how they affect biological functioning of soil. Soils were sampled in seven locations along a gradient of heavy metal contamination with distance from a mine in San Luis Potosí (Mexico), active since about 1800 AD. C mineralization and N₂O production were monitored in an aerobic incubation experiment. Concentrations of As in the top 0-10 cm soil layer ranged from 8 to 22,992 mg kg⁻¹, from 31 to 1845 mg kg⁻¹ for Pb, from 27 to 1620 mg kg⁻¹ for Cu and from 81 to 4218 mg kg⁻¹ for Zn. There was a significant negative correlation between production rates of CO₂ and concentrations of As, Pb, Cu and Zn, and there was a significant positive correlation with pH, water holding capacity (WHC), total N and soil organic C. There was a significant negative correlation (P<0.05) between production rate of nitrous oxide (N₂O) attributed to nitrification by the inhibition method in soil incubated at 50% WHC and total concentrations of Pb and Zn, and there was a significant positive correlation (P<0.05) with pH and total N content. There was a significant negative correlation (P<0.05) between the production rate of N₂O attributed to denitrification by the inhibition method in soil incubated at 100% WHC and total concentrations of Pb, Cu and Zn, and a significant positive correlation (P<0.01) with pH; there was a significant positive correlation (P<0.05) between the production of N₂O attributed to other processes by the inhibition method and WHC, inorganic C and clay content. A negative value for production rate of N₂O attributed to nitrifier denitrification by the inhibition method was obtained at 100% WHC. The large concentrations of heavy metals in soil inhibited microbial activity and the production rate of N₂O attributed to nitrification by the inhibition method when soil was incubated at 50% WHC and denitrification when soil was incubated at 100% WHC. The inhibitor/suppression technique used appeared to be flawed, as negative values for nitrifier denitrification were obtained and as the production rate of N₂O through denitrification increased when soil was incubated with C₂H₂.     

Effect of sodium chloride on denitrification in glucose amended soil treated with ammonium and nitrate nitrogen

Laboratory incubations were conducted to study the effect of sodium chloride (NaCl) on denitrification and respiratory gases (CO₂, O₂) from soil treated with ammonium or nitrate and incubated at 20 % moisture. The same samples were assayed for denitrifying enzyme activity (DEA) after incubation at 40 % moisture with glucose and NO₃^–. Under aerobic conditions (20 % water content), a flush of activity was observed at 6 hours after start of incubation and subsided to negligible levels at 12 hours. Sodium chloride significantly depressed N₂O and CO₂ emissions and O₂ consumption. Significantly more loss of N₂O occurred from NH₄⁺‐ than NO₃^–‐treated soil at all NaCl levels and was attributed to higher microbial activity. A highly significant positive correlation was obtained between N₂O emission and respiratory gases. The respiratory quotient (CO₂ evolved/O₂) was higher for NH₄⁺‐treated soil and decreased with the amount of NaCl. At 40 % moisture, N₂O emissions were higher than at 20 % and peaked at 37 hours followed by a sharp decrease. Short‐term incubations of soil with NH₄⁺ or NO₃^– did not have an effect on denitrifying enzyme activity (DEA) while NaCl had a positive effect, particularly in previously NO₃^–‐treated soil.     

Effect of increasing oxygen concentration on total denitrification and nitrous oxide release from soil by different bacteria

Summary The effect of increasing oxygen concentrations (0, 5, 10 and 20 Vol% O₂) on total denitrification and N₂0 release was studied in model experiments using a neutral pH loamy soil relatively rich in easily decomposable organic matter and supplied with nitrate (300 g nitrate N/g dry soil). The sterilized soil was inoculated with three different denitrifying bacteria (Bacillus licheniformis,Aeromonas denitrificans andAzospirillum lipoferum) and incubated (80% WHC, 30°C). The gas volume was analysed for O₂, CO₂, N₂O, NO and N₂ by gas chromatography and the soil investigated for changes in ammonium, nitrite, nitrate, pH, total N and C as well as water-extractable C. WithB. licheniformis andAeromonas denitrificans total denitrification increased remarkably with increasing pO₂ as the result of intensified mineralization.Azospirillum lipoferum, however, showed the highest activity at 5 vol% O₂. WithB. licheniformis N₂O was released only in anaerobic conditions and at 5 Vol% O₂ (maximum) or 10 Vol% 02, but not at 20 Vol%, whereasAeromonas denitrificans produced N₂O only in the presence of He gas (maximum) or at 5 Vol% O₂. In contrast to these bacteria, N₂O production withAzospirillum lipoferum was restricted to 10 Vol% O₂ (maximum) and to 20 Vol% 02, with some traces at 5 vol% O₂. With a certain set of conditions, total denitrification and N₂O formation seem to be governed by the mineralization rate of the organisms in question. The increased demand for electron acceptors by a high turnover rate rather than the presence of anaerobic conditions seems to have determined the rate of denitrification.     

Nitrous oxide production in grassland soils: assessing the contribution of nitrifier denitrification

Nitrifier denitrification is the reduction of NO₂⁻ to N₂ by nitrifiers. It leads to the production of the greenhouse gas nitrous oxide (N₂O) as an intermediate and possible end product. It is not known how important nitrifier denitrification is for the production of N₂O in soils. We explored N₂O production by nitrifier denitrification in relation to other N₂O producing processes such as nitrification and denitrification under different soil conditions. The influence of aeration of the soil, different N sources, and pH were tested in four experiments. To differentiate between sources of N₂O, an incubation method with inhibitors was used [Biol. Fertil. Soils 22 (1996) 331]. Sets of four incubations included controls without addition of inhibitors, incubations with addition of small concentrations of C₂H₂ (0.01-0.1 kPa), large concentrations of O₂ (100 kPa), or a combination of C₂H₂ and O₂. The results indicate that the availability of NO₂⁻ stimulated the apparent N₂O production by nitrifier denitrification. A decreasing O₂ content increased the total N₂O production, but decreased N₂O production by nitrifier denitrification. No significant effect of pH could be found. The study revealed problems concerning the use of the inhibitors C₂H₂ and O₂. Almost one-third of all incubations with inhibitors produced more N₂O than the controls. Possible reasons for the problems are discussed. The inhibitors C₂H₂ and O₂ need to be tested thoroughly for their effects on different N₂O producing processes before further application.     

异养硝化在水产养殖中的应用

比较了异养硝化作用与传统自养硝化作用,结果表明:异养硝化作用不仅客观存在,而且某些特殊的异养细菌能同步进行异养硝化和好氧反硝化,在养殖水体水质改善方面具有广泛的应用前景。     

不同固体碳源释碳特征及其对反硝化脱氮效果研究

碳源是低碳氮比废水反硝化过程的限制性因素之一,外加固体碳源可以强化微生物反硝化脱氮效果。为筛选出合适的外加碳源,本研究选用廉价的农业废弃物(稻草和锯木屑)和水生植物(绿狐尾藻和梭鱼草)作为固体碳源材料,分析不同固体碳源材料的释碳特征,比较其对反硝化过程的脱氮效果。结果表明,4种材料的释碳过程均符合二级动力学方程,其释碳能力大小为:稻草(25.64 mg/(g·L))梭鱼草(23.64 mg/(g·L))锯木屑(22.37 mg/(g·L))绿狐尾藻(20.45 mg/(g·L)),其中,绿狐尾藻的释放速率最快,其COD释放浓度达饱和浓度一半时所用时间仅为3.56 h。4种材料作为外加固体碳源可显著提高反硝化脱氮效率,其对水体硝态氮的去除率均达80%以上。由于梭鱼草在试验后期出现氨氮的大量积累,会造成水体二次污染。因此,稻草、锯木屑和绿狐尾藻适合作为外加碳源材料利用。     

Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment

Fungal denitrification in soils is receiving considerable attention as one of the dominant N₂O production processes. However, because of the lack of a methodology to detect fungal denitrification-related genes, the diversity and ecological behavior of denitrifying fungi in soil remains unknown. Thus, we designed a primer set to detect the fungal nitrite reductase gene (nirK) and validated its sensitivity and specificity. Through clone library analyses, we identified congruence between phylogenies of the 18S rRNA gene and nirK of denitrifying fungal isolates obtained from the surface-fertilized cropland soil and showed that fungi belonging to Eurotiales, Hypocreales, and Sordariales were primarily responsible for N₂O emissions in the soil.