Response of denitrification rate associated with wetting and drying cycles in a littoral wetland area of Lake Biwa,Japan

To clarify the relationship between denitrification activity and dry–wet levels in the littoral wetland sediments of Lake Biwa, Japan, denitrification rates and their regulating parameters (degree of dryness, redox potential, nitrate concentration) were measured on different moisture sediments. Redox potential in sediments was higher in the exposed region in contact with atmosphere than the flooded region covered with water. The nitrate concentration in interstitial waters was undetectable in the flooded region. On the other hand, concentration in the exposed region increased with increase in the degree of sediment dryness. The denitrification rate ranged from <0.001 to 0.88 μg N cm⁻³ h⁻¹ in the exposed region and increased with the increase in the degree of dryness. In the flooded region, on the other hand, no detectable rate (<0.001 μg N cm⁻³ h⁻¹) was observed. This indicates that the rates in the exposed region were mainly influenced by nitrate concentration in the interstitial waters accumulated by desiccation of sediments, whereas rates in the flooded region were strongly limited by no accumulation of nitrate in the anaerobic conditions. The potential denitrification rate, under the application condition of nitrate, ranged from 0.13 to 0.26 μg N cm⁻³ h⁻¹ in the flooded region and from 0.77 to 1.5 μg N cm⁻³ h⁻¹ in the exposed region. The potential rates in the flooded region had a tendency to be lower than those in the exposed region, implying that the number of denitrifying bacteria in the flooded region was low due to inactivation of aerobic respiration and denitrification in the denitrifying bacteria community. Kinetic parameters, maximum rate (V _max) and half-saturation constant (K _s) for denitrification were calculated on the experimental procedures of the wetting–drying cycles of sediments. Both parameters decreased by the wetting treatment and increased by the drying treatment. The fluctuation of V _max values with wetting–drying cycles indicated that the number of denitrifying bacteria was influenced by aerobic respiration and denitrification in the denitrifying bacteria community similar to the potential rates, and denitrifying enzyme was induced by the nitrate supplied by nitrification accelerated through the drying process. On the other hand, the fluctuation of K _s values implied that members of denitrifying bacteria were shifted to members of high nitrate affinity by wetting treatment and of low nitrate affinity by drying treatment.     

洱海沉积物中反硝化细菌的分离与反硝化作用研究

自洱海十个点位的沉积物中富集筛选出101株反硝化细菌并从中筛选出1株较强反硝化能力的细菌,命名为EH314。该细菌接触酶(过氧化氢酶)试验、产硫化氢试验和淀粉水解均为阳性,葡萄糖氧化发酵实验结果为氧化菌,产脂酶(Tween 80)试验结果为阴性;初步鉴定该菌为产碱杆菌属细菌;对细菌反硝化能力进行测定发现,菌株EH314能有效地降解水体中的硝酸盐且反硝化可在有氧条件下进行。     

Urban Stormwater Runoff Drives Denitrifying Community Composition Through Changes in Sediment Texture and Carbon Content

The export of nitrogen from urban catchments is a global problem, and denitrifying bacteria in stream ecosystems are critical for reducing in-stream N. However, the environmental factors that control the composition of denitrifying communities in streams are not well understood. We determined whether denitrifying community composition in sediments of nine streams on the eastern fringe of Melbourne, Australia was correlated with two measures of catchment urban impact: effective imperviousness (EI, the proportion of a catchment covered by impervious surfaces with direct connection to streams) or septic tank density (which affects stream water chemistry, particularly stream N concentrations). Denitrifying community structure was examined by comparing terminal restriction fragment length polymorphisms of nosZ genes in the sediments, as the nosZ gene codes for nitrous oxide reductase, the last step in the denitrification pathway. We also determined the chemical and physical characteristics of the streams that were best correlated with denitrifying community composition. EI was strongly correlated with community composition and sediment physical and chemical properties, while septic tank density was not. Sites with high EI were sandier, with less fine sediment and lower organic carbon content, higher sediment cations (calcium, sodium and magnesium) and water filterable reactive phosphorus concentrations. These were also the best small-scale environmental variables that explained denitrifying community composition. Among our study streams, which differed in the degree of urban stormwater impact, sediment grain size and carbon content are the most likely drivers of change in community composition. Denitrifying community composition is another in a long list of ecological indicators that suggest the profound degradation of streams is caused by urban stormwater runoff. While the relationships between denitrifying community composition and denitrification rates are yet to be unequivocally established, landscape-scale indices of environmental impact such as EI may prove to be useful indicators of change in microbial communities.     

Denitrification kinetics and denitrifier abundances in sediments of lakes receiving atmospheric nitrogen deposition (Colorado, USA)

The transport and deposition of anthropogenic nitrogen (N) to downwind ecosystems is significant and can be a dominant source of new N to many watersheds. Bacterially mediated denitrification in lake sediments may ameliorate the effects of N loading by permanently removing such inputs. We measured denitrification in sediments collected from lakes in the Colorado Rocky Mountains (USA) receiving elevated (5–8?kg?N?ha^?1?y^?1) or low (<2?kg?N?ha^?1?y^?1) inputs of atmospheric N deposition. The nitrate (NO₃ ^?) concentration was significantly greater in high-deposition lakes (11.3?μmol?l^?1) compared to low-deposition lakes (3.3?μmol?l^?1). Background denitrification was positively related to NO₃ ^? concentrations and we estimate that the sampled lakes are capable of removing a significant portion of N inputs via sediment denitrification. We also conducted a dose–response experiment to determine whether chronic N loading has altered sediment denitrification capacity. Under Michaelis–Menten kinetics, the maximum denitrification rate and half-saturation NO₃ ^? concentration did not differ between deposition regions and were 765?μmol?N?m^?2?h^?1 and 293?μmol?l^?1?NO₃ ^?, respectively, for all lakes. We enumerated the abundances of nitrate- and nitrite-reducing bacteria and found no difference between high- and low-deposition lakes. The abundance of these bacteria was related to available light and bulk sediment resources. Our findings support a growing body of evidence that lakes play an important role in N removal and, furthermore, suggest that current levels of N deposition have not altered the abundance of denitrifying bacteria or saturated the capacity for sediment denitrification.     

Denitrification in deep subsurface sediments

Dissimilatory nitrate reduction (denitrification) in subsurface sediments by indigenous microflora was investigated in samples obtained over a range of depths from 0 to 289 m. Denitrifying activity in sediment samples retrieved from similar stratigraphic horizons at four different sites was determined by measuring the accumulation of N₂O using the acetylene blockage technique. Denitrification was detected in unamended samples which received only prereduced deionized water at almost all depths in all sediments sampled. The surface sediments showed the highest denitrification activity. In the deeper sediments, denitrifying activity was much higher in saturated sandy samples and lower or absent in drier clay samples. Addition of nitrate enhanced denitrification activity in all samples from below the water table down to the maximum depth sampled (289 m), while addition of a carbon (succinate) source in general had no stimulatory effect. These results show that denitrifying microorganisms were present in all of the deep subsurface sediments tested in this study. Furthermore, these results suggest that adequate supplies of metabolizable organic carbon were available to support denitrifying activity. However, denitrification may be limited by inadequate supplies of nitrate in the sediments.     

Effect of crude oil on denitrification and sulfate reduction in marine sediments

The denitrifying activity was measured in different types of sediment from the Mediterranean coast of France before, and after, a massive contamination (30–100 g kg^-1 sediment) of hydrocarbons. A closed system was used in order to maintain anoxic conditions and to control substrates and gaseous products concentrations. We have demonstrated that the respiratory metabolism was inhibited in all cases following an incubation time of 20 to 50 days. At this time, the addition of lactate restore the denitrifying activity. The inhibitory effect of crude oil was not related to an alteration of bacterial cells, but to changes in environmental conditions allowing denitrification. The presence of hydrocarbons in the sediments causes a decrease in the redox potential and a concomitant stimulation of the sulfate reduction.     

Short-term influence of recolonisation by the polycheate worm [2pt] Nereis succinea on oxygen and nitrogen fluxes and [2pt] denitrification: a microcosm simulation

The short-term effects of sediment recolonisation by Nereis succinea on sediment-water column fluxes of oxygen and dissolved inorganic nitrogen, and rates of denitrification, were studied in microcosms of homogenised, sieved sediments. The added worms enhanced oxygen uptake by the sediments, due to the increased surface area provided by the burrow walls and the degree of stimulation was stable with time. Similarly, ammonium fluxes to the water column were stimulated by N. succinea, but declined over the 3 day incubation in all microcosms including the controls. Nitrate fluxes were generally greater in the faunated microcosms, but highly variable with time. Denitrification rates were positively stimulated by N. succinea populations, denitrification of water column nitrate was stimulated 10-fold in comparison to denitrification coupled to nitrification in the sediments. Rates of denitrification of water column nitrate were not significantly different from rates in undisturbed sediment cores with similar densities of N. succinea, whereas rates of coupled nitrification–denitrification were 3-fold lower in the experimental set-up. These results may reflect the relative growth rates of nitrifying and denitrifying bacteria, which allow more rapid colonisation of new burrow surfaces by denitrifier compared to nitrifier populations. The data indicate that recolonisation by burrowing macrofauna of the highly reduced sediments of the Sacca di Goro, Lagoon, Italy, following the annual dystrophic crisis, may play a significant role in the reoxidation and detoxification of the sediments. The increased rates of denitrification associated with the worm burrows, may promote nitrogen losses, but due to the low capacity of nitrifying bacteria to colonise the new burrow structures, these losses would be highly dependent upon water column nitrate concentrations.     

Denitrification with methane as electron donor in oxygen-limited bioreactors

The microbial population from a reactor using methane as electron donor for denitrification under microaerophilic conditions was analyzed. High numbers of aerobic methanotrophic bacteria (3 10⁷ cells/ml) and high numbers of acetate-utilizing denitrifying bacteria (2 10⁷ cells/ml) were detected, but only very low numbers of methanol-degrading denitrifying bacteria (4 10⁴ cells/ml) were counted. Two abundant acetate-degrading denitrifiers were isolated which, based on 16S rRNA analysis, were closely related to Mesorhizobium plurifarium (98.4% sequence similarity) and a Stenotrophomonas sp. (99.1% sequence similarity). A methanol-degrading denitrifying bacterium isolated from the bioreactor morphologically resembled Hyphomicrobium sp. and was moderately related to H. vulgare (93.5% sequence similarity). The initial characterization of the most abundant methanotrophic bacterium indicated that it belongs to class II of the methanotrophs. “In vivo”¹³C-NMR with concentrated cell suspensions showed that this methanotroph produced acetate under oxygen limitation. The microbial composition of reactor material together with the NMR experiments suggest that in the reactor methanotrophs excrete acetate, which serves as the direct electron donor for denitrification. Received: 19 October 1999 / Received revision: 11 January 2000 / Accepted: 14 January 2000     

Mathematical estimations of hyper-ammonia producing ruminal bacteria and evidence for bacterial antagonism that decreases ruminal ammonia production(1)

Bacteria capable of denitrification are spread among phylogenetically diverse groups. In the present investigation, molecular methods (amplified ribosomal DNA restriction analysis (ARDRA) and partial 16S rDNA gene sequencing) were used to determine the genetic diversity of culturable denitrifying soil bacteria. The purpose of this work was to study the microbial density and diversity of denitrifying communities isolated from two luvisols and a rendosol. The denitrifying bacterial density was significantly higher in the two luvisols (3x10(6) and 4x10(6) bacteria g(-1) dry soil) than in the rendosol (4x10(5) bacteria g(-1) dry soil). Denitrifying isolates from soils were grouped according to the similarity of their restriction patterns into 26 ARDRA types. Interestingly ARDRA analysis suggests that some denitrifying isolates are specific to a soil type while others seem to be geographically widespread. The number of individual isolates found in each ARDRA type appeared to be highly variable between the two sampling dates but some denitrifying types were capable of persisting in soil. The tree obtained from the partial sequences revealed five major branches exhibiting highest identity to the following genera: (i) Burkholderia-Ralstonia, (ii) Pseudomonas, (iii) Xanthomonas-Frateuria, (iv) Bacillus and (v) Streptomyces. Our 16S rDNA-based analysis clearly reveals broad diversity exceeding that previously described in the literature.     

Watershed urbanization alters the composition and function of stream bacterial communities

Watershed urbanization leads to dramatic changes in draining streams, with urban streams receiving a high frequency of scouring flows, together with the nutrient, contaminant, and thermal pollution associated with urbanization. These changes are known to cause significant losses of sensitive insect and fish species from urban streams, yet little is known about how these changes affect the composition and function of stream microbial communities. Over the course of two years, we repeatedly sampled sediments from eight central North Carolina streams affected to varying degrees by watershed urbanization. For each stream and sampling date, we characterized both overall and denitrifying bacterial communities and measured denitrification potentials. Denitrification is an ecologically important process, mediated by denitrifying bacteria that use nitrate and organic carbon as substrates. Differences in overall and denitrifying bacterial community composition were strongly associated with the gradient in urbanization. Denitrification potentials, which varied widely, were not significantly associated with substrate supply. By incorporating information on the community composition of denitrifying bacteria together with substrate supply in a linear mixed-effects model, we explained 45% of the variation in denitrification potential (p-value<0.001). Our results suggest that (1) the composition of stream bacterial communities change in response to watershed urbanization and (2) such changes may have important consequences for critical ecosystem functions such as denitrification.     

Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater

This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)⁻¹day⁻¹] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible. Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997     

Potential rates of nitrification and denitrification in an oligotrophic freshwater sediment system

Potential rates of nitrification and denitrification were measured in an oligotrophic sediment system. Nitrification potential was estimated using the CO oxidation technique, and potential denitrification was measured by the acetylene blockage technique. The sediments demonstrated both nitrifying and denitrifying activity. E_h, O₂, and organic C profiles showed two distinct types of sediment. One type was low in organic C, had high O₂ and E_h, and had rates of denitrification 1,000 times lower than the other which had high organic C, low O₂, and low E_h. Potential nitrification and denitrification rates were negatively correlated with E_h. This suggests that environmental heterogeneity in denitrifier and nitrifier populations in oligotrophic sediment systems may be assessed using E_h before sampling protocols for nitrification or denitrification rates are established. There was no correlation between denitrification and nitrification rates or between either of these processes and NH₄ ⁺ or NO₃ ^– concentrations. The maximum rate of denitrification was 0.969 nmole N cm^–3 hour^–1, and the maximum rate of nitrification was 23.6 nmole cm^–3 hour^–1, suggesting nitrification does not limit denitrification in these oligotrophic sediments. Some sediment cores had mean concentrations of 6.0 mg O₂/liter and still showed both nitrification and denitrification activity.     

人工遮光和营养添加对河流反硝化活性和反硝化细菌群落结构的影响

河流生态系统受到人类活动例如河岸带森林植被毁损和农业活动施肥等的干扰日益加剧,而这些活动使河流接收的光照增多、河流的氮磷营养盐浓度增加。微生物的反硝化作用是河流去除氮的有效途径。在汉江的一级支流金水河上游核心保护区内选取6条溪流开展野外控制实验,利用营养添加模拟河流中营养的增加,遮盖河面来模拟源头溪流的隐蔽状态,来研究河流沉积物中微生物的反硝化作用对光照和营养改变的响应,并利用高通量测序(Mi Seq)技术研究在两种处理下河流沉积物中nir S型反硝化细菌的群落结构变化。结果显示:营养元素添加促进了沉积物的反硝化活性,河面遮盖抑制了沉积物的反硝化活性。营养添加和遮盖两种处理均降低了控制实验区域内脱氯单胞菌属(优势菌属)的相对丰度,同时也降低了该区域nir S型反硝化菌群落的Chao多样性。本研究初步证实了光照增加和河流的营养增加提高了河流沉积物反硝化活性,并为提高河流的脱氮能力提供科学依据。     

Potential roles of anaerobic ammonium and methane oxidation in the nitrogen cycle of wetland ecosystems

Anaerobic ammonium oxidation (anammox) and anaerobic methane oxidation (ANME coupled to denitrification) with nitrite as electron acceptor are two of the most recent discoveries in the microbial nitrogen cycle. Currently the anammox process has been relatively well investigated in a number of natural and man-made ecosystems, while ANME coupled to denitrification has only been observed in a limited number of freshwater ecosystems. The ubiquitous presence of anammox bacteria in marine ecosystems has changed our knowledge of the global nitrogen cycle. Up to 50% of N₂ production in marine sediments and oxygen-depleted zones may be attributed to anammox bacteria. However, there are only few indications of anammox in natural and constructed freshwater wetlands. In this paper, the potential role of anammox and denitrifying methanotrophic bacteria in natural and artificial wetlands is discussed in relation to global warming. The focus of the review is to explore and analyze if suitable environmental conditions exist for anammox and denitrifying methanotrophic bacteria in nitrogen-rich freshwater wetlands.     

Denitrification in salt marsh sediments: Evidence for seasonal temperature selection among populations of denitrifiers

Direct measurements of bacterial denitrification in salt marsh sediments near Woods Hole, Massachusetts were made over a 10-month period using a simple and precise gas-chromatographic technique. Based on laboratory experiments at 5°, 10°, and 20°C, it is shown that seasonal temperature variations select for at least two distinct populations of denitrifiers.In situ incubations suggest that resident populations of denitrifying bacteria are cold-sensitive. Salt marsh denitrifying bacteria are not optimally adapted to their thermal environment, but to temperatures 5°–10°C higher. In these water-logged muds, rates of bacterial denitrification (0.3–1.5g N₂/gm sediment-hr) are up to three orders of magnitude greater than maximum potential rates of insitu bacterial and algal nitrogen fixation.Supported by the Victoria Foundation and NSF grants GA 28365, GA 28272, and GA 41506. This is Contribution No. 3730 from the Woods Hole Oceanographic Institution.     

Differential metabolism of dimethylsulfoniopropionate and acrylate in saline and brackish intertidal sediments

In anoxic Spartina altemiflora—dominated sediments along a naturally occuring salinity gradient (the Cooper River estuary, South Carolina, U.S.A.), dimethylsulfoniopropionate (DMSP) was metabolized to dimethyl sulfide (DMS) and acrylate by sediment microbes. The rate of DMSP degradation and acrylate mineralization by sediment microbes was similar at all sites along this 25-km transect. However, sediments amended with acrylate (or DMSP) showed significantly higher rates of N₂ fixation (measured as acetylene reduction activity) (ARA) in the saline sediments downstream than brackish sediments. These results are consistent with the fact that acrylate stimulated the rates of both denitrification and CO₂ production in the saline sediments at the mouth of the river more than tenfold over rates in brackish sediments. Enrichment experiments indicate that microbes capable of using DMSP or acrylate were not present in upstream sediments despite the fact that microbial biomass, percent organic matter, and both glucose-stimulated ARA and denitrification were highest upstream. It appears that acrylate utilizing, N₂ fixing, and denitrifying populations are insignificant in the lower salinity sediments of the estuary. These results may reflect the availability of DMSP, which averaged 10.3 nmol g wet wt^–1 of saline sediments and levels less than our detection limit (1 m) in brackish sediments. Correspondence to: D.C. Yoch.     

Community structures and activities of nitrifying and denitrifying bacteria in industrial wastewater-treating biofilms

The bacterial community structure, in situ spatial distributions and activities of nitrifying and denitrifying bacteria in biofilms treating industrial wastewater were investigated by combination of the 16S rRNA gene clone analysis, fluorescence in situ hybridization (FISH) and microelectrodes. These results were compared with the nitrogen removal capacity of the industrial wastewater treatment plant (IWTP). Both nitrification and denitrification occurred in the primary denitrification (PD) tank and denitrification occurred in the secondary denitrification (SD) tank. In contrast, nitrification and denitrification rates were very low in the nitrification (N) tank. 16S rRNA gene clone sequence analysis revealed that the bacteria affiliated with Alphaproteobacteria, followed by Betaproteobacteria, were numerically important microbial groups in three tanks. The many clones affiliated with Alphaproteobacteria were closely related to the denitrifying bacteria (e.g., Hyphomicrobium spp., Rhodopseudomonas palustris, and Rhodobacter spp.). In addition, Methylophilus leisingeri affiliated with Betaproteobacteria, which favorably utilized methanol, was detected only in the SD-tank to which methanol was added. Nitrosomonas europaea and Nitrosomonas marina were detected as the ammonia-oxidizing bacteria affiliated with Betaproteobacteria throughout this plant, although the dominant species of them was different among three tanks. Nitrifying bacteria were mainly detected in the upper parts of the PD-biofilm whereas their populations were low in the upper parts of the N-biofilm. The presence of denitrifying bacteria affiliated with Hyphomicrobium spp. in SD- and N-biofilms was verified by FISH analysis. Microelectrode measurements showed that the nitrifying bacteria present in the N- and PD-biofilms were active and the bacteria present in the SD-biofilm could denitrify.     

Niche construction by the invasive Asian knotweeds (species complex <Emphasis Type="Italic">Fallopia</Emphasis>): impact on activity,abundance and community structure of denitrifiers and nitrifiers

Big Asian knotweeds (Fallopia spp.) are among the most invasive plant species in north-western Europe. We suggest that their success is partially explained by biological and chemical niche construction. In this paper, we explored the microbial mechanisms by which the plant modifies the nitrogen cycle. We found that Fallopia spp. decreased potential denitrification enzyme activity (DEA) by reducing soil moisture and reducing denitrifying bacteria density in the soil. The plant also reduced potential ammonia and nitrite oxydizing bacteria enzyme activities (respectively, AOEA and NOEA) in sites with high AOEA and NOEA in uninvaded situation. Modification of AOEA and NOEA were not correlated to modifications of the density of implicated bacteria. AOB and Nitrobacter-like NOB community genetic structures were significantly different in respectively two and three of the four tested sites while the genetic structure of denitrifying bacteria was not affected by invasion in none of the tested sites. Modification of nitrification and denitrification functioning in invaded soils could lead to reduced nitrogen loss from the ecosystem through nitrate leaching or volatilization of nitrous oxides and dinitrogen and could be considered as a niche construction mechanism of Fallopia.     

Vertical distribution of nitrite reductase genes (nir S) in continental margin sediments of the Gulf of Mexico

Marine sediments account for up to 66% of the loss of nitrogen load to coastal areas. Sedimentary denitrification is the main sink for fixed nitrogen in the global nitrogen budget, and thus it is important to understand the structure and composition of denitrifying communities. To understand the structure and composition of denitrifying communities, the diversity of nitrite reductase (nirS) genes from sediments along the Gulf of Mexico was examined using a PCR-based cloning approach. Sediments were collected at three different depths (0-0.5, 4-5 and 19-21 cm). Geochemical analysis revealed decreasing nitrate and oxygen concentrations with increasing sediment depth. This trend coincided with the decrease in diversity of denitrifying bacteria. LIBSHUFF analysis indicated that the clone library in the shallowest sediment (depth, 0-0.5 cm) was significantly different from that in the deepest sediment (depth, 19-21 cm), and that the deeper sediments (depths of 4-5 and 19-21 cm) were significantly similar. Community structural shifts were evident between the shallowest (oxic zone) and deepest (anoxic zone) sediments. Community changes within the deepest sediments were more subtle, with the presence of different nirS clone sequences gradually becoming dominant or, alternatively, decreasing with depth. The changes in community structure at this depth are possibly driven by nutrient availability, with lower quality sources of carbon and energy leading to the disappearance of nirS sequences common in the top layer. The majority of recovered nirS sequences were phylogenetically divergent relative to known denitrifying bacteria in the database.