Identification of novel denitrifying bacteria Stenotrophomonas sp. ZZ15 and Oceanimonas sp. YC13 and application for removal of nitrate from industrial wastewater

Two novel denitrifying bacteria were successfully isolated from industrial wastewater and soil samples. Using morphological, biochemical/biophysical and 16S rRNA gene analyses, these two bacteria were identified as Stenotrophomonas sp. ZZ15 and Oceanimonas sp. YC13, respectively. Both of these two bacteria showed efficient NO₃ ⁻-N removing abilities under a semi-anaerobic condition without obvious accumulation of NO₂ ⁻-N, N₂O-N and NH₄ ⁺-N. NO₃ ⁻-N removal from paper mill wastewater was also successful by treatments with either a denitrifier or an immobilization method. Therefore, this study provides valuable denitrifying bacteria in biotreatment of industrial wastewater and other environmental pollution caused by NO₃ ⁻/NO₂ ⁻.     

Nitrate removal with bacterial cells attached to quartz sand and zeolite from salty wastewaters

A mixed bacterial culture was acclimated to the removal of high nitrate-N concentrations (100–750 mg NO₃ ⁻-N L⁻¹) from salty wastewaters. The experiments were carried out under anoxic conditions in the presence of 0.5, 1.5 and 3% (w/v) NaCl at different temperatures. The acclimated mixed bacterial culture was attached to quartz sand and zeolite. Denitrification was monitored in a continuous-flow bioreactor at different hydraulic retention times (HRT). Nitrate removal with cells attached to quartz sand and zeolite was completed at HRT of 167 h and 25 h respectively. Then brine denitrification with bacterial cells attached to zeolite was monitored for 85 days. Under the increased nitrate loading rate, nitrate removal was above 90%. Furthermore, during denitrification, not more than 0.5 mg NO₂ ⁻-N L⁻¹ could be produced. It can be concluded that nitrate removal with the cells attached to zeolite is economically and operationally a promising solution to denitrification of brine wastewaters.     

Screening and characterization of an aerobic nitrifying-denitrifying bacterium from activated sludge

Taking advantage of the good biocompatibility and high efficiency of nitrogen removal with microbes, nitrifying and denitrifying bacteria, are becoming increasingly more widely used for wastewater treatment and prevention of eutrophication. In this research, an aerobic nitrifying-denitrifying bacterium was successfully screened from activated sludge and identified as Pseudomonas sp. (CCTCC No M2010209) by the 16S rDNA sequence. The activity verification confirmed its nitrifying-denitrifying capability of removing ammonium, nitrate and nitrite nitrogen. The types of carbon sources and carbon-nitrogen ratio greatly influenced the removal efficiency of NH₄ ⁺-N and NO₃ ⁻-N. When the initial concentrations of NH₄ ⁺-N and NO₃ ⁻-N in synthetic wastewater were less than 70 and 50 mg/L, the nitrogen removal rates reached 94 and 90% in 9 h, respectively. Preliminary comparisons of nitrogen removal capacity between this isolate and other commercial preparations in the treatment of synthetic wastewater revealed its promising potential to be used in the actual wastewater treatment.     

Removal of nitrogen by heterotrophic nitrification–aerobic denitrification of a novel halotolerant bacterium Pseudomonas mendocina TJPU04

Excess inorganic nitrogen in water poses a severe threat to enviroment. Removal of inorganic nitrogen by heterotrophic nitrifying–aerobic denitrifying microorganism is supposed to be a promising and applicable technology only if the removal rate can be maintained sufficiently high in real wastewater under various conditions, such as high concentration of salt and wide range of different nitrogen concentrations. Here, a new heterotrophic nitrifying–aerobic denitrifying bacterium was isolated and named as Pseudomonas mendocina TJPU04, which removes NH₄⁺-N, NO₃⁻-N and NO₂⁻-N with average rate of 4.69, 5.60, 4.99 mg/L/h, respectively. It also maintains high nitrogen removal efficiency over a wide range of nitrogen concentrations. When concentration of NH₄⁺-N, NO₃⁻-N and NO₂⁻-N was up to 150, 150 and 50 mg/L, 98%, 93%, and 100% removal efficiency could be obtained, respectively, after 30-h incubation under sterile condition. When it was applied under non-sterile condition, the ammonia removal efficiency was slightly lower than that under sterile condition. However, the nitrate and nitrite removal efficiencies under non-sterile condition were significantly higher than those under sterile condition. Strain TJPU04 also showed efficient nitrogen removal performance in the presence of high concentration of salt and nitrogen. In addition, the removal efficiencies of NH₄⁺-N, NO₃⁻-N and TN in real wastewater were 91%, 52%, and 75%, respectively. These results suggest that strain TJPU04 is a promising candidate for efficient removal of inorganic nitrogen in wastewater treatment.

     

Rapid Nitrate Loss and Denitrification in a Temperate River Floodplain

Nitrogen (N) pollution is a problem in many large temperate zone rivers, and N retention in river channels is often small in these systems. To determine the potential for floodplains to act as N sinks during overbank flooding, we combined monitoring, denitrification assays, and experimental nitrate (NO₃⁻ -N) additions to determine how the amount and form of N changed during flooding and the processes responsible for these changes in the Wisconsin River floodplain (USA). Spring flooding increased N concentrations in the floodplain to levels equal to the river. As discharge declined and connectivity between the river and floodplain was disrupted, total dissolved N decreased over 75% from 1.41 mg l⁻¹, equivalent to source water in the Wisconsin River on 14 April 2001, to 0.34 mg l⁻¹ on 22 April 2001. Simultaneously NO₃⁻ -N was attenuated almost 100% from 1.09 to <0.002 mg l⁻¹. Unamended sediment denitrification rates were moderate (0–483 μg m⁻² h⁻¹) and seasonally variable, and activity was limited by the availability of NO ₃⁻ -N on all dates. Two experimental NO₃⁻ -N pulse additions to floodplain water bodies confirmed rapid NO₃⁻ -N depletion. Over 80% of the observed NO ₃⁻ -N decline was caused by hydrologic export for addition #1 but only 22% in addition #2. During the second addition, a significant fraction (>60%) of NO₃⁻ -N mass loss was not attributable to hydrologic losses or conversion to other forms of N, suggesting that denitrification was likely responsible for most of the NO₃⁻ -N disappearance. Floodplain capacity to decrease the dominant fraction of river borne N within days of inundation demonstrates that the Wisconsin River floodplain was an active N sink, that denitrification often drives N losses, and that enhancing connections between rivers and their floodplains may enhance overall retention and reduce N exports from large basins.     

Spatial and Temporal Variability in Sediment Denitrification Within an Agriculturally Influenced Reservoir

Reservoirs are intrinsically linked to the rivers that feed them, creating a river–reservoir continuum in which water and sediment inputs are a function of the surrounding watershed land use. We examined the spatial and temporal variability of sediment denitrification rates by sampling longitudinally along an agriculturally influenced river–reservoir continuum monthly for 13 months. Sediment denitrification rates ranged from 0 to 63 μg N₂O g ash free dry mass of sediments (AFDM)⁻¹ h⁻¹ or 0–2.7 μg N₂O g dry mass of sediments (DM)⁻¹ h⁻¹ at reservoir sites, vs. 0–12 μg N₂O gAFDM⁻¹ h⁻¹ or 0–0.27 μg N₂O gDM⁻¹ h⁻¹ at riverine sites. Temporally, highest denitrification activity traveled through the reservoir from upper reservoir sites to the dam, following the load of high nitrate (NO₃⁻-N) water associated with spring runoff. Annual mean sediment denitrification rates at different reservoir sites were consistently higher than at riverine sites, yet significant relationships among theses sites differed when denitrification rates were expressed per gDM vs. per gAFDM. There was a significant positive relationship between sediment denitrification rates and NO₃⁻-N concentration up to a threshold of 0.88 mg NO₃⁻ -N l⁻¹, above which it appeared NO₃⁻-N was no longer limiting. Denitrification assays were amended seasonally with NO₃⁻-N and an organic carbon source (glucose) to determine nutrient limitation of sediment denitrification. While organic carbon never limited sediment denitrification, all sites were significantly limited by NO₃⁻-N during fall and winter when ambient NO ₃⁻-N was low.     

Enhancing biological nitrogen removal from tannery effluent by using the efficient Brachymonas denitrificans in pilot plant operations

Summary Laboratory scale and pilot plant reactors were inoculated with an efficient denitrifier, Brachymonas denitrificans(CCUG 45880), in order to evaluate whether a bio-augmentation approach can be used to enhance biological nitrogen removal from tannery effluents. To determine the effectiveness of the introduced strain, denitrifying activity in the activated sludge was monitored by nitrate uptake rate (NUR) measurement of NO₃-N. Fluorescent in situ hybridization (FISH) technique was used to monitor the growth of the augmented species. The laboratory scale nitrate removal efficiency with the introduced B. denitrificans (3.7±0.6 mg NO₃-N gVSS ⁻¹ h ⁻¹) was higher than that of the activated sludge without the addition of the bacteria (3.5±0.7 mg NO₃-N gVSS ⁻¹ h ⁻¹); the NUR in the pilot plant after and before the introduction of the strain was also of the magnitude of 12.0±1.4 and 10.6±1.4 mg NO₃-N gVSS ⁻¹ day ⁻¹ , respectively. In situ hybridization results revealed that the introduced denitrifying bacteria significantly facilitated the development of a dense denitrifying bacterial population in the activated sludge, which enhanced in situ denitrification activity. FISH data indicated that once introduced, B. denitrificans remained abundant throughout the experimental period. The ability to seed a bioreactor with bacterial strain capable of removing target pollutants from tannery effluents in a mixed microbial community suggests that this approach could have commercial applications.     

Continuous removal of aromatic hydrocarbons by an AF reactor under denitrifying conditions

Removal of three typical aromatic hydrocarbons, benzene, biphenyl and naphthalene by an anaerobic filter (AF) reactor under continuous mode and denitrifying conditions was studied. Results showed that the AF reactor could degrade these aromatic hydrocarbons effectively under above-mentioned conditions. When influent wastewater contained 900 mg COD/l and about 60 mg (total aromatic hydrocarbons)/l, 90% and 84% removal efficiency could be achieved for them respectively. When COD/NO₃ ⁻-N ratio (C/N) was in the range 5–30, the removal of benzene was slightly influenced by C/N and it remained stable at about 90%. However, degradation of naphthalene, biphenyl and total COD was greatly influenced by C/N, and highest removal was achieved at C/N = 15, it was 90%, 85% and 82% for COD, naphthalene and biphenyl, respectively. Degradation of these three aromatic hydrocarbons followed the order: benzene > naphthalene > biphenyl.     

Abiotic reaction of nitrite with dissolved organic carbon? Testing the Ferrous Wheel Hypothesis

The Ferrous Wheel Hypothesis (Davidson et al. 2003) postulates the abiotic formation of dissolved organic N (DON) in forest floors, by the fast reaction of NO₂ ⁻ with dissolved organic C (DOC). We investigated the abiotic reaction of NO₂ ⁻ with dissolved organic matter extracted from six different forest floors under oxic conditions. Solutions differed in DOC concentrations (15–60 mg L⁻¹), NO₂ ⁻ concentrations (0, 2, 20 mg NO₂ ⁻-N L⁻¹) and DOC/DON ratio (13.4–25.4). Concentrations of added NO₂ ⁻ never decreased within 60 min, therefore, no DON formation from added NO₂ ⁻ took place in any of the samples. Our results suggest that the reaction of NO₂ ⁻ with natural DOC in forest floors is rather unlikely.     

Bioaugmented sulfur-oxidizing denitrification system with <Emphasis Type="Italic">Alcaligenes defragrans</Emphasis> B21 for high nitrate containing wastewater treatment

The performance of enriched sludge augmented with the B21 strain of Alcaligenes defragrans was compared with that of enriched sludge, as well as with pure Alcaligenes defragrans B21, in the context of a sulfur-oxidizing denitrification (SOD) process. In synthetic wastewater treatment containing 100–1,000 mg NO₃⁻-N/L, the single strain-seeded system exhibited superior performance, featuring higher efficiency and a shorter startup period, provided nitrate loading rate was less than 0.2 kg NO₃⁻-N/m³ per day. At nitrate loading rate of more than 0.5 kg NO₃⁻-N/m³ per day, the bioaugmented sludge system showed higher resistance to shock loading than two other systems. However, no advantage of the bioaugmented system over the enriched sludge system without B21 strain was observed in overall efficiency of denitrification. Both the bioaugmented sludge and enriched sludge systems obtained stable denitrification performance of more than 80% at nitrate loading rate of up to 2 kg NO₃⁻-N/m³ per day.     

N-removal performance and underlying bacterial taxa of upflow filter bioreactor system under different dissolved oxygen and internal recycle conditions

Biological N-removal treatment of piggery wastewater in the upflow anaerobic–anoxic–aerobic floating filter (UA³FF) bioreactor based on the concept of nitritation–denitritation was studied along with the changes in internal recycle ratio and dissolved oxygen concentration (DO). Consecutive changes in the recirculation ratio between the anoxic and aerobic reactors has resulted in abundance and composition shifts of N-cycling bacteria as well as other bacterial groups, reflecting different survival strategies across (bio/physico)chemical milieu. The DO concentration was optimized to achieve nitritation in the aerobic reactor and denitritation in the anoxic reactor. Optimal nitritation–denitritation (270 and 130 g NO₂ ⁻–N produced or reduced/m³ filter media/day) was obtained at DO of 1.0–1.5 mg/l, inter-reactor recirculation ratio of 1:1–2:1, HRT of 24 h, pH of 7.6 ± 0.3, and temperature of 28 ± 4 °C. Since only well known nitrifying and denitrifying taxa were found, nitritation–denitritation was likely carried out by these bacteria rather than the yet unidentified novel taxa. Archaeal nitrifiers recently discovered to be important in the global N-cycle were not detected.     

Selection and dominance mechanisms of denitrifying phosphate-accumulating organisms in biological phosphate removal process

A sequencing batch reactor under different electron acceptor conditions was operated serially to investigate the selection and dominance mechanisms of denitrifying phosphate-accumulating organisms (DNPAOs) in a biological nutrient removal process. The presence of a small amount of NO^– ₃ at the start of the anaerobic phase stimulated the selection of DNPAOs in an anaerobic/aerobic system, and switching O₂ to NO^– ₃ as an electron acceptor enhanced the activity of anoxic phosphate uptake.     

Removal of a high load of ammonia by a marine bacterium,Vibrio alginolyticus in biofilter

A newly isolated heterotrophic marine bacterium,Vibrio alginolyticus, was used to remove a high load of ammonia gas under non-sterile condition. The cells were inoculated onto an inorganic packing material in a fixed-bed reactor (biofilter), and a high bad of ammonia, in the range of ammonia gas concentration of 170 ppm to 880 ppm, was introduced continuously. Sucrose solution and 3% NaCl was supplied intermittently to supplement the carbon source and water to the biofilter. The average percentage of gas removed exceeded 85% for 107-day operation. The maximum removal capacity and the complete removal capacity were 19 g-N kg⁻¹ dry packing material day⁻¹ and 16 g-N kg⁻¹ dry packing material day⁻¹, respectively, which were about three times greater than those obtained in nitrifying sludge inoculated onto the same packing material. On day 82, the enhanced pressure drop was restored to the normal one by NaOH treatment, and efficient removal characteristics were later observed. During this operation, the non-sterile condition had no significantly adverse effect on the removability of ammonia byV. alginolyticus.     

Use of an industrial effluent as a carbon source for denitrification of a high-strength wastewater

Denitrification of a high-strength synthetic wastewater (150 g NO^- ₃ l^-1) was carried out using a wine distillery effluent as an example of an industrial carbon source (22.7 g chemical oxygen demand l^-1). Two configurations were tested: one consisted of an acidogenesis reactor followed by a denitrifying reactor and the other was a single reactor directly fed with the raw effluents. In both cases, denitrification was achieved at a nitrate load of 9.54 g NO^- ₃ l^-1 day^-1 (2.19 g N as NO^- ₃ l^-1 day^-1) with good specific reduction rates: 32.6 mg and 35.2 mg N as NO _x  g volatile suspended solids h^-1, calculated on a single day, for the two-step and the one-step process respectively. Dissimilatory nitrate reduction to ammonium did not occur, even in the one-step process. Received: 26 October 1995/Received revision: 15 February 1996/Accepted: 20 February 1996     

The effects of drying on sediment nitrogen content in a Mediterranean intermittent stream: a microcosms study

Mediterranean climates predispose aquatic systems to both flood and drought periods, therefore, stream sediments may be exposed to desiccation periods. Changes in oxygen concentrations and sediment water content influence the biotic processes implicated in nitrogen dynamics. The objectives of this study were to identify (1) the changes of inorganic nitrogen in stream sediments during the transition from wet to dry conditions, and (2) the underlying processes in N dynamics and its regulation. Extractable sediment NO₃ ⁻-N and NH₄ ⁺-N, organic matter and extractable organic carbon content were assessed during natural desiccation in microcosms with sediments from an intermittent Mediterranean stream. In agreement with our initial hypothesis, our results showed how the NO₃ ⁻-N content of the sediment was enhanced during the first 10 days of sediment drying, whereas NH₄ ⁺-N was lost by 14 days post-drying. During the first 10 days, sediment desiccation seemed to stimulate the net N-mineralization and net nitrification from sediments. Afterwards, the extractable NO₃ ⁻-N concentration sharply dropped, which may be attributed to lower ammonium-oxidation rates as ammonium and organic matter are depleted, and to an increase in NO₃ ⁻-N consumption by microbial populations. Denitrification was inhibited, with a significant decrease as % water-filled pore space lowered. We hypothesize that the sediment inorganic N content enhanced during sediment desiccation could be released as part of the N pulse observed after sediment rewetting. However, the stream N availability after rewetting dried sediments would differ depending on desiccation period duration.     

Microbial Succession in a Compost-packed Biofilter Treating Benzene-contaminated Air

Air artificially contaminated with increasing concentrations of benzene was treated in a laboratory scale compost-packed biofilter for 240 days with a removal efficiency of 81–100%. The bacterial community in the packing material (PM) at different heights of the biofilter was analysed every 60 days. Bacterial plate counts and ribosomal intergenic spacer analysis (RISA) of the isolated strains showed that the number of cultivable aerobic heterotrophic bacteria and the species diversity increased with benzene availability. Identification of the isolated species and the main bands in denaturing gradient gel electrophoresis (DGGE) profiles from total compost DNA during the treatment revealed that, at a relatively low volumetric benzene load (1.2≤VBL≤6.4 g m⁻³ _PM h⁻¹), besides low G+C Gram positive bacteria, originally present in the packing compost, bacteroidetes and β- and γ-proteobacteria became detectable in the colonising population. At the VBL value (24.8 g m⁻³ _PM h⁻¹) ensuring the maximum elimination capacity of the biofilter (20.1 g m⁻³ _PM h⁻¹), strains affiliated to the genus Rhodococcus dominated the microflora, followed by β-proteobacteria comprising the genera Bordetella and Neisseria. Under these conditions, more than 35% of the isolated strains were able to grow on benzene as the sole carbon source. Comparison of DGGE and automated RISA profiles of the total community and isolated strains showed that a complex bacterial succession occurred in the reactor in response to the increasing concentrations of the pollutant and that cultivable bacteria played a major role in benzene degradation under the adopted conditions.     

Hillslope Nutrient Dynamics Following Upland Riparian Vegetation Disturbance

We investigated the effects of removing near-stream Rhododendron and of the natural blowdown of canopy trees on nutrient export to streams in the southern Appalachians. Transects were instrumented on adjacent hillslopes in a first-order watershed at the Coweeta Hydrologic Laboratory (35°03′N, 83°25′W). Dissolved organic carbon (DOC), K⁺, Na⁺, Ca²⁺, Mg²⁺, NO₃ ⁻-N, NH₄ ⁺-N, PO₄ ³⁻-P, and SO₄ ²⁻ were measured for 2 years prior to disturbance. In August 1995, riparian Rhododendron on one hillslope was cut, removing 30% of total woody biomass. In October 1995, Hurricane Opal uprooted nine canopy trees on the other hillslope, downing 81% of the total woody biomass. Over the 3 years following the disturbance, soilwater concentrations of NO₃ ⁻-N tripled on the cut hillslope. There were also small changes in soilwater DOC, SO₄ ²⁻, Ca²⁺, and Mg²⁺. However, no significant changes occurred in groundwater nutrient concentrations following Rhododendron removal. In contrast, soilwater NO₃ ⁻-N on the storm-affected hillslope showed persistent 500-fold increases, groundwater NO₃ ⁻-N increased four fold, and streamwater NO₃ ⁻-N doubled. Significant changes also occurred in soilwater pH, DOC, SO₄ ²⁻, Ca²⁺, and Mg²⁺. There were no significant changes in microbial immobilization of soil nutrients or water outflow on the storm-affected hillslope. Our results suggest that Rhododendron thickets play a relatively minor role in controlling nutrient export to headwater streams. They further suggest that nutrient uptake by canopy trees is a key control on NO₃ ⁻-N export in upland riparian zones, and that disruption of the root–soil connection in canopy trees via uprooting promotes significant nutrient loss to streams. Received 30 January 2001; accepted 25 July 2002.     

Effect of light/dark cycles on wastewater treatments by microalgae

Chlorella kessleri was cultivated in artificial wastewater using diurnal illumination of 12 h light/12 h dark (L/D) cycles. The inoculum density was 10⁵ cells/mL and the irradiance in light cycle was 45 μmol m² s⁻¹ at the culture surface. As a control culture, another set of flasks was cultivated under continuous illumination. Regardless of the illumination scheme, the total organic carbon (TOC) and chemical oxygen demand (COD) was reduced below 20% of the initial concentration within a day. However, cell concentration under the L/D lighting scheme was lower than that under the continuous illuminating scheme. Thus the specific removal rate of organic carbon under L/D cycles was higher than that under continuous illumination. This result suggested thatC. kessleri grew chemoorganotrophically in the dark periods. After 3 days, nitrate was reduced to 136.5 and 154.1 mg NO₃ ⁻-N/L from 168.1 mg NO₃ ⁻-N/L under continuous illumination and under diurnal cycles, respectively. These results indicate nitrate removal efficiency under continuous light was better than that under diurnal cycles. High-density algal cultures using optimized photobioreactors with diurnal cycles will save energy and improve organic carbon sources removal.     

反硝化除磷戈登氏菌属的富集及其菌剂制备

【背景】投加微生物菌剂是强化生物处理效能的重要手段,反硝化是污水脱氮除磷的关键步骤,但目前对于反硝化微生物菌剂相关的研究报道较少。【目的】驯化高效反硝化聚磷菌菌剂,并对系统进行生物强化。【方法】采用两阶段法快速富集反硝化聚磷菌,筛选高效脱氮除磷功能菌株NC1-1并进行鉴定,以NC1-1为菌种来源制备干粉菌剂,研究菌剂强化A²SBR系统污水处理效果。【结果】历经36 d后反硝化聚磷菌富集成功,菌株NC1-1经鉴定属于戈登氏菌属,其脱氮除磷率分别为89.46%和91.68%。麦麸、玉米粉配比为85%:15%、NC1-1投菌量为20 mL、发酵液用量20 mL的条件下成功制得干粉菌剂,干粉菌剂最佳投加量为10%的A²SBR系统总磷（total phosphorus,TP）和NO₃^--N去除率比未投加菌剂的A²SBR系统提高12.06%和11.52%。【结论】菌剂NC1-1的投加使A²SBR系统的污染物去除效能进一步提高,研究结果为进一步研究反硝化聚磷菌菌剂提供了...     

Suppression of nitrate formation within an exotic conifer plantation

Summary Nitrate-N losses to stream waters and soil inorganic N pools, nitrifying potentials and NO₃-N production rates were measured in 2 adjacent watersheds, one used as pasture and the other planted in exotic conifer forest (Pinus radiata D. Don). Estimated NO₃-N loss to stream waters draining the pine and pasture watersheds were 0.6kg ha⁻¹ y⁻¹ and 7.6 kg ha⁻¹ y⁻¹ respectively. Ammonium-N pool sizes were not significantly different between soils in the two watersheds but NO₃−N pools and nitrifying potentials were always lower in the pine watershed soil samples. Laboratory incubation experiments indicated that suppression of NO₃−N formation in pine watershed soils required the presence of live tree roots and was not due to the direct action of allelopathic chemicals on nitrifiers.