Response of sediment microbial community structure in a freshwater reservoir to manipulations in oxygen availability

Hypolimnetic oxygenation systems (HOx) are being increasingly used in freshwater reservoirs to elevate dissolved oxygen levels in the hypolimnion and suppress sediment-water fluxes of soluble metals (e.g. Fe and Mn) which are often microbially mediated. We assessed changes in sediment microbial community structure and corresponding biogeochemical cycling on a reservoir-wide scale as a function of HOx operations. Sediment microbial biomass as quantified by DNA concentration was increased in regions most influenced by the HOx. Following an initial decrease in biomass in the upper sediment while oxygen concentrations were low, biomass typically increased at all depths as the 4-month-long oxygenation season progressed. A distinct shift in microbial community structure was only observed at the end of the season in the upper sediment near the HOx. While this shift was correlated to HOx-enhanced oxygen availability, increased TOC levels and precipitation of Fe- and Mn-oxides, abiotic controls on Fe and Mn cycling, and/or the adaptability of many bacteria to variations in prevailing electron acceptors may explain the delayed response and the comparatively limited changes at other locations. While the sediment microbial community proved remarkably resistant to relatively short-term changes in HOx operations, HOx-induced variation in microbial structure, biomass, and activity was observed after a full season of oxygenation.     

Temporal variation of magnetotactic bacterial communities in two freshwater sediment microcosms

Magnetotactic bacteria (MTB), which can mineralize nanosized magnetite or greigite crystals within cells, play important roles in biogeochemical processes, for example iron and sulfur cycling, and depositional remanent magnetization acquisitions. Despite decades of research, the knowledge of MTB distribution and ecology is still limited. In the present study, we investigated the temporal variation of MTB communities in freshwater sediment microcosms based on 16S rRNA genes and unifrac analyses. Two microcosms (MY8 and MY11) collected from two separate sites in Lake Miyun (Beijing, China) were analyzed. The majority of retrieved sequences belonged to alphaproteobacterial magnetotactic cocci in both microcosms (representing 64.29% of clones from MY8 and 100% of clones from MY11), whereas so-called ' Magnetobacterium bavaricum '-like MTB affiliated within Nitrospira phylum were exclusively found in microcosm MY8. Over a 3-month period, the temporal variation of MTB communities was evident in both microcosms. In addition, the phylogenetic discrepancy of MTB communities between two microcosms is more prominent than that of the same microcosm at different times, implying adaptation of MTB phylogenetic lineages to specific microenvironments. Among the physical–chemical parameters measured, a strong correlation was shown between nitrate and the main genetic variability of MTB communities, indicating that nitrate may influence the occurrence of MTB phylogenetic lineages in natural environments.     

The effect of nitrate and nitrite on oxygen evolution and carbon-dioxide assimilation and the reduction of nitrate and nitrite by intact chloroplasts

Summary Intact chloroplasts isolated from spinach reduced NO₃ ^- and NO₂ ^- in the light without the addition of either co-factors or added enzymes. The maximum rate observed, however, for the reduction of NO₃ ^- was approximately 3Moles hr^-1 mg^-1 (chlorophyll) and for NO₂ ^- 6 Moles hr^-1 mg^-1 (chlorophyll). These rates were consistent with the enzyme content of whole chloroplasts, but much lower than those found in whole leaf extracts.The addition of both NO₃ ^- and NO₂ ^- in low concentrations resulted in transient increases in both O₂ evolution and CO₂ fixation. The increases in oxygen evolution were not consistent in amount and bore no relation to the amount of substrate reduced. Similar transients were observed in a number of experiments when NaCl or NH₄Cl were added.The addition of NO₂ ^- at concentrations of 10^-4 M and above resulted in marked inhibition of both O₂ evolution and CO₂ fixation. NO₂ ^- appears to inhibit by blocking the reduction of NADP. NO₃ ^- at similar concentrations had no such effect.An increase in the soluble amino nitrogen content of the chloroplasts was observed when NO₃ ^- or NO₂ ^- was reduced. There was, however, no increase in the incorporation of ¹⁴C from ¹⁴CO₂ into amino acids under these conditions. Even with the addition of ammonia the amount of ¹⁴C incorporated into the amino acids was not changed from less than 5% of the total ¹⁴C fixed. We conclude that while intact chloroplasts do have the ability to reduce both NO₃ ^- and NO₂ ^- at low rates, they do not synthesize appreciable amounts of amino acid directly, and this fact must be considered when formulating any pathways for nitrogen metabolism during photosynthesis.Supported in part by the National Research Council of Canada.     

Diversity and vertical distribution of magnetotactic bacteria along chemical gradients in freshwater microcosms

The vertical distribution of magnetotactic bacteria along various physico-chemical gradients in freshwater microcosms was analyzed by a combined approach of viable cell counts, 16S rRNA gene analysis, microsensor profiling and biogeochemical methods. The occurrence of magnetotactic bacteria was restricted to a narrow sediment layer overlapping or closely below the maximum oxygen and nitrate penetration depth. Different species showed different preferences within vertical gradients, but the largest proportion (63-98%) of magnetotactic bacteria was detected within the suboxic zone. In one microcosm the community of magnetotactic bacteria was dominated by one species of a coccoid "Alphaproteobacterium", as detected by denaturing gradient gel electrophoresis in sediment horizons from 1 to 10 mm depth. Maximum numbers of magnetotactic bacteria were up to 1.5 x 10(7) cells/cm3, which corresponded to 1% of the total cell number in the upper sediment layer. The occurrence of magnetotactic bacteria coincided with the availability of significant amounts (6-60 microM) of soluble Fe(II), and in one sample with hydrogen sulfide (up to 40 microM). Although various trends were clearly observed, a strict correlation between the distribution of magnetotactic bacteria and individual geochemical parameters was absent. This is discussed in terms of metabolic adaptation of various strains of magnetotactic bacteria to stratified sediments and diversity of the magnetotactic bacterial communities.     

Biphasic behavior of anammox regulated by nitrite and nitrate in an estuarine sediment

The production of N2 gas via anammox was investigated in sediment slurries at in situ NO2- concentrations in the presence and absence of NO3-. With single enrichment above 10 microM 14NO2- or 14NO3- and 15NH4+, anammox activity was always linear (P < 0.05), in agreement with previous findings. In contrast, anammox exhibited a range of activity below 10 microM NO2- or NO3-, including an elevated response at lower concentrations. With 100 microM NO3-, no significant transient accumulation of NO2- could be measured, and the starting concentration of NO2- could therefore be regulated. With dual enrichment (1 to 20 microM NO2- plus 100 microM NO3-), there was a pronounced nonlinear response in anammox activity. Maximal activity occurred between 2 and 5 microM NO2-, but the amplitude of this peak varied across the study (November 2003 to June 2004). Anammox accounted for as much as 82% of the NO2- added at 1 microM in November 2003 but only for 15% in May 2004 and for 26 and 5% of the NO2- added at 5 microM for these two months, respectively. Decreasing the concentration of NO3- but holding NO2- at 5 microM decreased the significance of anammox as a sink for NO2-. The behavior of anammox was explored by use of a simple anammox-denitrification model, and the concept of a biphasic system for anammox in estuarine sediments is proposed. Overall, anammox is likely to be regulated by the availability of NO3- and NO2- and the relative size or activity of the anammox population.     

Solute dynamics in a North Brazilian mangrove: the influence of sediment permeability and freshwater input

Although water in mangrove sediments influences nutrient cycling in both, mangrove forest and estuary, little information exists on seasonal and vertical distribution of dissolved organic and inorganic compounds in the sediment column. We studied the influence of sediment texture and chemistry, permeability (K), tides, and rainfall on dissolved organic carbon (DOC) and nitrogen (DON), dissolved inorganic phosphate (DIP) and salinity in creek and sediment waters of a mangrove in Pará, Brazil. Water samples were taken from boreholes and piezometers in the mangrove forest and from an adjacent tidal creek at neap and spring tides, during the dry and rainy season. Forest sediment was analysed for carbon (C), nitrogen (N), salinity and permeability. Clay, C and N decreased with depth. Sediment permeability (K) was lowest (<0.1 m day⁻¹) in the upper, clay-rich and crab-burrow-free mud layer. In the deeper, fine sand strata, K ranged from 0.7 to 1.8 m day⁻¹. Tidal range in the creek was 3.5 and 5.5 m for neap and spring tides, respectively. Salinity, DOC, DON and DIP in creek water were inversely related to tidal height. Piezometer data revealed significant water level changes in deeper, sandy sediment layer, which followed, time-lagged, the tidal fluctuations. In contrast, tide did not affect the water level in the upper sediment due to low permeability. Compared with creek water, sediment water was enriched in DOC, DON and DIP because of organic matter input and mineralization. In deeper layers, solute concentration was most likely affected by sorption processes (DOC and DIP) and reduction reactions (DIP). During the rainy season, DOC and DON in creek and sediment water were higher than in the dry season. DIP appeared invariant to seasonal changes. In the rainy season, salt flushing from surface sediments resulted in higher salinities at intermediate sediment depths, while in the deeper layers salinity was lower due to exchange with water from the tidal creek.     

The effect of sulfate and nitrate on methane formation in a freshwater sediment

A freshwater sediment from a ditch of a peat grassland near Zegveld (Province of Utrecht, The Netherlands) was investigated for its potential methanogenic and syntrophic activity and the influence of sulfate and nitrate on these potential activities. Methanogenesis started after a 10 days lagphase. After 35–40 days aceticlastic methanogens were sufficiently enriched to cause a net decrease of acetate. In the presence of sulfate methane formation was only slightly affected. The addition of nitrate led to an outcompetion of aceticlastic methanogens by nitrate reducers. When inorganic electron acceptors were absent, substrates like propionate and butyrate were converted by syntrophic methanogenic consortia. Addition of inorganic electron acceptors resulted in an outcompetition of the syntrophic propionate and butyrate degrading consortia by the sulfate and nitrate reducers.     

Experimental investigation of nitrogen and oxygen isotope fractionation in nitrate and nitrite during denitrification

In batch experiments, we studied the isotope fractionation of nitrogen and oxygen during denitrification of two bacterial strains (Azoarcus sp. strain DSM 9056 and Pseudomonas pseudoalcaligenes strain F10). Denitrification experiments were conducted with succinate and toluene as electron donor in three waters with a distinct oxygen isotope composition. Nitrate consumption was observed in all batch experiments. Reaction rates for succinate experiments were more than six times higher than those for toluene experiments. Nitrogen and oxygen isotopes became progressively enriched in the remaining nitrate pool in the course of the experiments; the nitrogen and oxygen isotope fractionation varied between 8.6–16.2 and 4.0–7.3‰, respectively. Within this range, neither electron donors nor the oxygen isotope composition of the medium affected the isotope fractionation process. The experimental results provide evidence that the oxygen isotope fractionation during nitrate reduction is controlled by a kinetic isotope effect which can be quantified using the Rayleigh model. The isotopic examination of nitrite released upon denitrification revealed that nitrogen isotope fractionation largely follows the fractionation of the nitrate pool. However, the oxygen isotope values of nitrite are clearly influenced by a rapid isotope equilibration with the oxygen of the ambient water. Even though this equilibration may in part be due to storage, it shows that under certain natural conditions (re-oxidation of nitrite) the nitrate pool may also be indirectly affected by an isotope equilibration.     

In situ distribution and activity of nitrifying bacteria in freshwater sediment

Nitrification was investigated in a model freshwater sediment by the combined use of microsensors and fluorescence in situ hybridization with rRNA-targeted oligonucleotide probes. In situ nitrification activity was restricted mainly to the upper 2 mm of the sediment and coincided with the maximum abundance of nitrifying bacteria, i.e. 1.5 x 107 cells cm-3 for ammonia-oxidizing Beta-proteobacteria (AOB) and 8.6 x 107 cells cm-3 for Nitrospira-like nitrite-oxidizing bacteria (NOB). Cell numbers of AOB decreased more rapidly with depth than numbers of NOB. For the first time, Nitrospira-like bacteria could be quantified and correlated with in situ nitrite oxidation rates in a sediment. Estimated cell-specific nitrite oxidation rates were 1.2-2.7 fmol NO2- cell-1 h-1.     

Spatial distribution of bacterial communities in high-altitude freshwater wetland sediment

Sediment microorganisms play a crucial role in a variety of biogeochemical processes in freshwater ecosystems. The objective of the current study was to investigate the spatial distribution of sediment bacterial community structure in Luoshijiang Wetland, located in Yunnan–Kweichow Plateau (China). Wetland sediments at different sites and depths were collected. Clone library analysis indicates bacterial communities varied with both sampling site and sediment depth. A total of fourteen bacterial phyla were identified in sediment samples, including Proteobacteria, Acidobacteria, Actinobacteria, Armatimonadetes, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Spirochaetes, and Verrucomicrobia. Proteobacteria (mainly Betaproteobacteria and Deltaproteobacteria) predominated in wetland sediments. Moreover, the proportions of Alphaproteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadete, and Planctomycetes were significantly correlated with chemical properties.     

A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake

Two related polytopic membrane proteins of the major facilitator family, NarK and NarU, catalyse nitrate uptake, nitrite export and nitrite uptake across the Escherichia coli cytoplasmic membrane by an unknown mechanism. A 12-helix model of NarU was constructed based upon six alkaline phosphatase and beta-galactosidase fusions to NarK and the predicted hydropathy for the NarK family. Fifteen residues conserved in the NarK-NarU protein family were substituted by site-directed mutagenesis, including four residues that are essential for nitrate uptake by Aspergillus nidulans: arginines Arg(87) and Arg(303) in helices 2 and 8, and two glycines in a nitrate signature motif. Despite the wide range of substitutions studied, in no case did mutation result in loss of one biochemical function without simultaneous loss of all other functions. A NarU+ NirC+ strain grew more rapidly and accumulated nitrite more rapidly than the isogenic NarU+ NirC(-) strain. Only the NirC+ strain consumed nitrite rapidly during the later stages of growth. Under conditions in which the rate of nitrite reduction was limited by the rate of nitrite uptake, NirC+ strains reduced nitrite up to 10 times more rapidly than isogenic NarU+ strains, indicating that both nitrite efflux and nitrite uptake are largely dependent on NirC. Isotope tracer experiments with [15N]nitrate and [14N]nitrite revealed that [15N]nitrite accumulated in the extracellular medium even when there was a net rate of nitrite uptake and reduction. We propose that NarU functions as a single channel for nitrate uptake and nitrite expulsion, either as a nitrate-nitrite antiporter, or more likely as a nitrate/H+ or nitrite/H+ channel.     

Mineralization budgets in sediment microcosms: Effect of the infauna and anoxic conditions

Abstract A number of sediment incubations were set up to reproduce some of the conditions used by Kristensen and Blackburn [1] and to make a comparison with their results. There were three types of microcosm: aerobic (OX), anaerobic (AN) and aerobic with Nephtys (NOX). In addition to other measurements, dissolved organic nitrogen (DON) pools and fluxes, were measured. The sediment in this experiment contained more particulate organic matter (POM). Nephtys (NOX) had the same effect as Nereis in increasing the rate of mineralization of POC and PON, compared with the OX-cores (2.1 and 2.6 times, respectively). Again, the AN-cores had a higher mineralization rate (loss of POM) than that of the OX-cores, but in addition, mineralization in NOX-cores was not significantly different from AN-cores. It was thus confirmed that anoxic mineralization could be as high, or higher, than the oxic process. Both the temporal patterns of O₂-and and CO₂-fluxes and their magnitudes were very similar to those reported earlier. This contrasts with the higher loss of POM in the present experiment. However, the loss of C in DOC (associated with the measured DON) can account for the extra POM loss. The pore-water profiles of σCO₂ and NH₄⁺ were similar to those in the earlier report, and the fluxes of σCO₂, O₂, NH₄⁺ and NO₃⁻ followed the same temporal pattern.     

Effects of nitrite, chlorate, and chlorite on nitrate uptake and nitrate reductase activity

Effects of NO₂⁻, ClO₃⁻, and ClO₂⁻ on the induction of nitrate transport and nitrate reductase activity (NRA) as well as their effects on NO₃⁻ influx into roots of intact barley (Hordeum vulgare cv Klondike) seedlings were investigated. A 24-h pretreatment with 0.1 mol m⁻³ NO₂⁻ fully induced NO₃⁻ transport but failed to induce NRA. Similar pretreatments with ClO₃⁻ and ClO₂⁻ induced neither NO₃⁻ transport nor NRA. Net ClO₃⁻ uptake was induced by NO₃⁻ but not by ClO₃⁻ itself, indicating that NO₃⁻ and ClO₃⁻ transport occur via the NO₃⁻ carrier. At the uptake step, NO₂⁻ and ClO₂⁻ strongly inhibited NO₃⁻ influx; the former exhibited classical competitive kinetics, whereas the latter exhibited complex mixed-type kinetics. ClO₃⁻ proved to be a weak inhibitor of NO₃⁻ influx (K_i = 16 mol m⁻³) in a noncompetitive manner. The implications of these findings are discussed in the context of the suitability of these NO₃⁻ analogs as screening agents for the isolation of mutants defective in NO₃⁻ transport.     

Diverse effects of formate on the dissimilatory metabolism of nitrate in Pseudomonas denitrificans ATCC 13867: Growth,nitrite accumulation in culture,cellular activities of nitrate and nitrite reductases

The growth of Pseudomonas denitrificans ATCC 13867 under denitrifying conditions was significantly stimulated by adding an appropriate amount of formate (2.5 mM or above) to the growth medium. The accumulation of nitrite in the culture was markedly depressed so long as formate remained in the culture above a certain level. Cellular activities of enzymes participating in denitrification also changed. The cells grown in the presence of formate exhibited a lower nitrate reductase activity and, in contrast, a higher nitrite reductase activity than the cells grown without added formate.