Anniet M. Laverman

Spatiotemporal stability of an ammonia oxidizing community in a nitrogen-saturated forest soil

Elevated levels of nitrogen input into various terrestrial environments in recent decades have led to increases in soil nitrate production and leaching. However, nitrifying potential and nitrifying activity tend to be highly variable over space and time, making broad-scale estimates of nitrate production difficult. This study investigates whether the high spatiotemporal variation in nitrate production might be explained by differences in the structure of ammonia-oxidizing bacterial communities in nitrogen-saturated coniferous forest soils. The diversity of ammonia-oxidizing bacteria of the β-subgroup Proteobacteria was therefore investigated using two different PCR-based approaches. The first targeted the 16S rRNA gene and involved temporal temperature gradient electrophoresis (TTGE) of specifically amplified PCR products, with subsequent band excision and nucleotide sequence determination. The second approach involved the cloning and sequencing of PCR-amplified amoA gene fragments. All recovered 16S rDNA sequences were closely related to the culture strain Nitrosospira sp. AHB1, which was isolated from an acid soil and is affiliated with Nitrosospira cluster 2, a sequence group previously shown to be associated with acid environments. All amoA-like sequences also showed a close affinity with this acid-tolerant Nitrosospira strain, although greater sequence variation could be detected in the amoA analysis. The ammonia-oxidizing bacterial community in the nitrogen-saturated coniferous forest soil was determined to be very stable, showing little variation between different organic layers and throughout the year, despite large differences in the total Bacterial community structure as determined by 16S rDNA DGGE community fingerprinting. These results suggest that environmental heterogeneity affecting ammonia oxidizer numbers and activity, and not ammonia oxidizer community structure, is chiefly responsible for spatial and temporal variation in nitrate production in these acid forest soils.

Temporal and spatial variation of nitrogen transformations in a coniferous forest soil

Forest soils show a great degree of temporal and spatial variation of nitrogen mineralization. The aim of the present study was to explain temporal variation in nitrate leaching from a nitrogen-saturated coniferous forest soil by potential nitrification, mineralization rates and nitrate uptake by roots. Variation in nitrate production in time and space, between the different organic horizons, has been related to temperature, moisture content, substrate availability and pH. Temporal variation in concentrations of nitrate and ammonium in the forest floor was significant during a one-year cycle, when randomly taken samples were pooled. Nitrogen concentrations differed between the different organic horizons with highest concentrations found in the litter layer, decreasing with increasing depth. Ammonium concentrations always exceeded nitrate concentrations by a factor ten, indicating that ammonium was not limiting nitrification. Nitrification potential, the nitrate production at field moisture at 25°C, was highest in the litter layer, lower in the fragmentation layer and hardly measurable in the mineral soil. Uptake of nitrate by roots and changes in mineralization rates turned out to be unimportant to explain variation in time, as seasonal fluctuations seem to be less important than spatial variation. We found that horizontal spatial variation in potential nitrate production, leaching of nitrate and nitrogen concentrations from non-pooled field samples was higher than variation in time. All this reflects the actual spatial variation in the field, which is not explained by differences in moisture content or temperature. Overall neither pH nor substrate availability could explain this observed variation, however, local variation in microsites may be responsible for small-scale spatial variation. Allelopathic compounds and/or the composition of the microbial community are suggested as factors possibly affecting nitrate production.

Cleavage of dimethylsulfoniopropionate and reduction of acrylate by Desulfovibrio acrylicus sp. nov.

Abstract From anoxic intertidal sediment, a dimethylsulfoniopropionate (DMSP)-cleaving anaerobe (strain W218) was isolated that reduced the acrylate formed to propionate. The bacterium was vibrio- to rod-shaped and motile by means of multiple polar flagella. It reduced sulfate, thiosulfate, and acrylate, and used lactate, fumarate, succinate, malate, pyruvate, ethanol, propanol, glycerol, glycine, serine, alanine, cysteine, hydrogen, and formate as electron donors. Sulfate and acrylate were reduced simultaneously; growth with sulfate was faster than with acrylate. Extracts of cells grown in the presence of DMSP contained high DMSP lyase activities (9.8 U/mg protein). The DNA mol% G+C was 45.1. On the basis of its characteristics and the 16S rRNA gene sequence, strain W218 was assigned to a new Desulfovibrio species for which the name Desulfovibrio acrylicus is proposed. A variety of other sulfate-reducing bacteria (eight of them originating from a marine or saline environment and five from other environments) did not reduce acrylate.

Comparison of deep‐sea sediment microbial communities in the Eastern Mediterranean

Bacterial and archaeal communities in sediments obtained from three geographically-distant mud volcanoes, a control site and a microbial mat in the Eastern Mediterranean deep-sea were characterized using direct 16S rRNA gene analyses. The data were thus in relation to the chemical characteristics of the (stratified) habitats to infer community structure-habitat relationships. The bacterial sequences in the different habitats were related to those of Actinobacteria, Bacilli, Chloroflexi, Alpha-, Beta-, Gamma-, Delta- and Epsilonproteobacteria and unclassified bacteria, including the JS1 group. The archaeal sequences found were affiliated with those of the Methanosarcinales, Thermoplasmales, Halobacteriales and Crenarchaea belonging to marine benthic group I and B, as well as MCG group archaea. In each sample, the communities were diverse and unique at the phylotype level. However, at higher taxonomic levels, similar groups were found in different sediments, and similar depth layers tended to contain similar communities. The sequences that dominated in all top layers (as well as in the mat) probably represented organisms involved in aerobic heterotrophy, sulfide-based chemoautotrophy and methanotrophy and/or methylotrophy. Sequences of organisms most likely involved in anaerobic methane oxidation, sulfate reduction and anaerobic heterotrophy were predominantly found in deeper layers. The data supported the notion of (1) uniqueness of each habitat at fine taxonomic levels, (2) stratification in depth and (3) conservation of function in the sediments.

Nitrous oxide production kinetics during nitrate reduction in river sediments

A significant amount of nitrogen entering river basins is denitrified in riparian zones. The aim of this study was to evaluate the influence of nitrate and carbon concentrations on the kinetic parameters of nitrate reduction as well as nitrous oxide emissions in river sediments in a tributary of the Marne (the Seine basin, France). In order to determine these rates, we used flow-through reactors (FTRs) and slurry incubations; flow-through reactors allow determination of rates on intact sediment slices under controlled conditions compared to sediment homogenization in the often used slurry technique. Maximum nitrate reduction rates (R(m)) ranged between 3.0 and 7.1microg Ng(-1)h(-1), and affinity constant (K(m)) ranged from 7.4 to 30.7mg N-NO(3)(-)L(-1). These values were higher in slurry incubations with an R(m) of 37.9microg Ng(-1)h(-1) and a K(m) of 104mg N-NO(3)(-)L(-1). Nitrous oxide production rates did not follow Michaelis-Menten kinetics, and we deduced a rate constant with an average of 0.7 and 5.4ng Ng(-1)h(-1) for FTR and slurry experiments respectively. The addition of carbon (as acetate) showed that carbon was not limiting nitrate reduction rates in these sediments. Similar rates were obtained for FTR and slurries with carbon addition, confirming the hypothesis that homogenization increases rates due to release of and increasing access to carbon in slurries. Nitrous oxide production rates in FTR with carbon additions were low and represented less than 0.01% of the nitrate reduction rates and were even negligible in slurries. Maximum nitrate reduction rates revealed seasonality with high potential rates in fall and winter and low rates in late spring and summer. Under optimal conditions (anoxia, non-limiting nitrate and carbon), nitrous oxide emission rates were low, but significant (0.01% of the nitrate reduction rates).

The effect of environmental and therapeutic concentrations of antibiotics on nitrate reduction rates in river sediment

The use of antibiotics in both human and veterinary medicine has led to increased presence of these compounds and antibiotic resistance in the environment. In this study, the effect of low, environmentally relevant (mg L(-1)) concentrations of vancomycin (VA), flumequine (FLU), and sulfamethoxazole (SMX) on nitrate reduction rates was studied in river sediments. Nitrate reduction rates were determined by supplying intact sediments for several weeks with both nitrate and antibiotics (ng L(-1), μg L(-1), and mg L(-1) concentrations), including a non-amended control. Furthermore the concentrations of the three investigated antibiotics were measured in the initial (natural) sediments and the sediments supplied with the antibiotics. The antibiotic concentrations in the sediments decreased (on average 62% for FLU and 93% for SMX) during the experiments, indicating loss of antibiotics due to sorption or (bio) degradation. Nitrate reduction rates were not affected by environmental concentrations of VA, FLU and SMX. FLU and SMX only partially inhibited nitrate reduction rates at high, therapeutic concentrations by 41 and 39% respectively. The three tested antibiotics significantly enhanced the production of nitrite, an intermediate in dissimilatory nitrate reduction. Nitrite production increased 1.9 and 1.4 fold for environmental VA concentrations (107 and 187 μg L(-1) respectively), application of 58 mg L(-1) SMX resulted in a 7.5 fold increase and augmented 16 and 8.5 fold in the presence of respectively 13 μg L(-1) and 52 mg L(-1) FLU. Even though inhibition of nitrate reduction rates was observed at therapeutic antibiotic concentrations, nitrate reduction proceeded under all experimental conditions, indicating the presence of resistance toward these antibiotics among the nitrate reducing bacteria. The accumulation of nitrite suggests that the nitrite reduction step was more affected than the overall nitrate reduction process.

Soil layer-specific variability in net nitrification and denitrification in an acid coniferous forest.

Abstract High spatial variation in nitrification potentials has been observed in forest soils, but explanations for this variability have remained speculative. In the present study we determined whether sample treatment, sample size, denitrification or small-scale variations in abiotic properties could explain spatial variation in nitrogen transformations in the organic horizon of a pine forest soil. Net nitrate production in homogenates of the organic horizon was extremely variable. Sample size (60–600 cm2) had no significant effect on nitrate production. In homogenised samples no increased nitrogen production was observed compared to intact incubated cores. High small-scale variation in nitrate production was observed in the litter (L) horizon. When this stratified L layer was subdivided, high net nitrate production was observed in moss (LM) and fragmented needles, whereas no net nitrate production was found in intact needles. The addition of acetylene, inhibiting nitrous oxide reductase, led to significant nitrous oxide production in the L layer. Low nitrous oxide production was found in the LM layer and none in the fragmentation layer. These results show that denitrification can explain part of the spatial variation and plays a major role in nitrogen transformations in the L layer. The relatively higher pH and the presence of fungi are suggested as factors responsible for high denitrification rates in the L layer. As a consequence homogenisation of the organic horizon could lead to highly variable nitrate production due to denitrifying activity from the needles being introduced into other layers.

Vertical distribution of denitrification in an estuarine sediment: integrating sediment flowthrough reactor experiments and microprofiling via reactive transport modeling.

ABSTRACT Denitrifying activity in a sediment from the freshwater part of a polluted estuary in northwest Europe was quantified using two independent approaches. High-resolution N2O microprofiles were recorded in sediment cores to which acetylene was added to the overlying water and injected laterally into the sediment. The vertical distribution of the rate of denitrification supported by nitrate uptake from the overlying water was then derived from the time series N2O concentration profiles. The rates obtained for the core incubations were compared to the rates predicted by a forward reactive transport model, which included rate expression for denitrification calibrated with potential rate measurements obtained in flowthrough reactors containing undisturbed, 1-cm-thick sediment slices. The two approaches yielded comparable rate profiles, with a near-surface, 2- to 3-mm narrow zone of denitrification and maximum in situ rates on the order of 200 to 300 nmol cm−3 h−1. The maximum in situ rates were about twofold lower than the maximum potential rate for the 0- to 1-cm depth interval of the sediment, indicating that in situ denitrification was nitrate limited. The experimentally and model-derived rates of denitrification implied that there was nitrate uptake by the sediment at a rate that was on the order of 50 (± 10) nmol cm−2 h−1, which agreed well with direct nitrate flux measurements for core incubations. Reactive transport model calculations showed that benthic uptake of nitrate at the site is particularly sensitive to the nitrate concentration in the overlying water and the maximum potential rate of denitrification in the sediment.

Microbial Communities in the World's Largest Acidic Volcanic Lake, Kawah Ijen in Indonesia, and in the Banyupahit River Originating from It

A first study was made on the microbial community composition of the Indonesian crater lake Kawah Ijen (pH < 0.3) and the Banyupahit–Banyuputih river (pH 0.4–3.5) originating from it. Culture-independent, rRNA gene-based denaturing gradient gel electrophoresis was used to profile microbial communities in this natural and ancient, extremely acidic environment. Similarity in community profiles of the different sampling locations was low, indicating heterogeneity in community composition. Archaea were present at all sampling locations; archaeal diversity was low at the most acidic locations and increased at pH >2.6. Bacteria were not detected in the water column of the crater lake, but were found at all locations along the acidic river. Bacterial diversity increased with increasing pH. Eukarya were only present at pH >2.6. Retrieved rRNA gene sequences of Bacteria and Archaea were not closely related to known acidophilic species. It is concluded that tolerance to extreme acidity in this system is developed most extensively among Archaea. The acidity gradient of the Banyupahit–Banyuputih river has a clear effect on microbial community composition and biodiversity.

Potential Denitrification and Nitrous Oxide Production in the Sediments of the Seine River Drainage Network (France)

To investigate bottom sediment denitrification at the scale of the Seine drainage network, a semi-potential denitrification assay was used in which river sediments (and riparian soils) were incubated for a few hours under anaerobic conditions with non limiting nitrate concentrations. This method allowed the nitrous oxide (N(2)O) concentration in the headspace, as well as the nitrate, nitrite, and ammonium concentrations to be determined during incubation. The rates at which nitrate decreased and N(2)O increased were then used to assess the potential denitrification activity and associated N(2)O production in the Seine River Basin. We observed a longitudinal pattern characterized by a significant increase of the potential rate of denitrification from upstream sectors to large downstream rivers (orders 7-8), from approximately 3.3 to 9.1 microg NO(3)(-)-N g(-1) h(-1), respectively, while the N(2)O production rates was the highest both in headwaters and in large order rivers (0.14 and 0.09 N(2)O-N g(-1) h(-1), respectively) and significantly lower in the intermediate sectors (0.01 and 0.03 N(2)O-N g(-1) h(-1)). Consequently, the ratio N(2)O production:NO(3) reduction was found to reach 5% in headstreams, whereas it averaged 1.2% in the rest of the drainage network, an intermediate percentage being found for the riparian soils. Finally, the ignition loss of sediments, together with other redundant variables (particulate organic carbon content: g C 100 g(-1) dry weight [dw], moisture: g water 100 g(-1) dw, sediment size <50 mum: g material size <50 mum 100 g(-1) dw) were found to control these activities. However, the biodegradability of organic matter must be measured to better understand the factor controlling denitrification and its associated N(2)O production.