Microbial succession and denitrifying woodchip bioreactor performance at low water temperatures.

Mining activities are increasingly recognized for contributing to nitrogen (N) pollution and possibly also to emissions of the greenhouse gas nitrous oxide (N2O) due to undetonated, N-based explosives. A woodchip denitrifying bioreactor, installed to treat nitrate-rich leachate from waste rock dumps in northern Sweden, was monitored for two years to determine the spatial and temporal distribution of microbial communities, including the genetic potential for different N transformation processes, in pore water and woodchips and how this related to reactor N removal capacity. About 80 and 65 % of the nitrate was removed during the first and second operational year, respectively. There was a succession in the microbial community over time and in space along the reactor length in both pore water and woodchips, which was reflected in reactor performance. Nitrate ammonification likely had minimal impact on N removal efficiency due to the low production of ammonium and low abundance of the key gene nrfA in ammonifiers. Nitrite and N2O were formed in the bioreactor and released in the effluent water, although direct N2O emissions from the surface was low. That these unwanted reactive N species were produced at different times and locations in the reactor indicate that the denitrification pathway was temporally as well as spatially separated along the reactor length. We conclude that the succession of microbial communities in woodchip denitrifying bioreactors treating mining water develops slowly at low temperature, which impacts reactor performance.

Nitrogen Removal Capacity of Microbial Communities Developing in Compost- and Woodchip-Based Multipurpose Reactive Barriers for Aquifer Recharge With Wastewater.

Global water supplies are threatened by climate changes and the expansion of urban areas, which have led to an increasing interest in nature-based solutions for water reuse and reclamation. Reclaimed water is a possible resource for recharging aquifers, and the addition of an organic reactive barrier has been proposed to improve the removal of pollutants. There has been a large focus on organic pollutants, but less is known about multifunctional barriers, that is, how barriers also remove nutrients that threaten groundwater ecosystems. Herein, we investigated how compost- and woodchip-based barriers affect nitrogen (N) removal in a pilot soil aquifer treatment facility designed for removing nutrients and recalcitrant compounds by investigating the composition of microbial communities and their capacity for N transformations. Secondary-treated, ammonium-rich wastewater was infiltrated through the barriers, and the changes in the concentration of ammonium, nitrate, and dissolved organic carbon (DOC) were measured after passage through the barrier during 1 year of operation. The development and composition of the microbial community in the barriers were examined, and potential N-transforming processes in the barriers were quantified by determining the abundance of key functional genes using quantitative PCR. Only one barrier, based on compost, significantly decreased the ammonium concentration in the infiltrated water. However, the reduction of reactive N in the barriers was moderate (between 21 and 37%), and there were no differences between the barrier types. All the barriers were after 1 year dominated by members of Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria, although the community composition differed between the barriers. Bacterial classes belonging to the phylum Chloroflexi showed an increased relative abundance in the compost-based barriers. In contrast to the increased genetic potential for nitrification in the compost-based barriers, the woodchip-based barrier demonstrated higher genetic potentials for denitrification, nitrous oxide reduction, and dissimilatory reduction of nitrate to ammonium. The barriers have previously been shown to display a high capacity to degrade recalcitrant pollutants, but in this study, we show that most barriers performed poorly in terms of N removal and those based on compost also leaked DOC, highlighting the difficulties in designing barriers that satisfactorily meet several purposes.

Microbial controls on net production of nitrous oxide in a denitrifying woodchip bioreactor.

Denitrifying woodchip bioreactors are potential low-cost technologies for the removal of nitrate (NO3 - ) in water through denitrification. However, if environmental conditions do not support microbial communities performing complete denitrification, other N transformation processes will occur, resulting in the export of nitrite (NO2 - ), nitrous oxide (N2 O), or ammonium (NH4 + ). To identify the factors controlling the relative accumulation of NO2 - , N2 O, and/or NH4 + in denitrifying woodchip bioreactors, porewater samples were collected over two operational years from a denitrifying woodchip bioreactor designed for removing NO3 - from mine water. Woodchip samples were collected at the end of the operational period. Changes in the abundances of functional genes involved in denitrification, N2 O reduction, and dissimilatory NO3 - reduction to NH4 + were correlated with porewater chemistry and temperature. Temporal changes in the abundance of the denitrification gene nirS were significantly correlated with increases in porewater N2 O concentrations and indicated the preferential selection of incomplete denitrifying pathways ending with N2 O. Temperature and the total organic carbon/NO3 - ratio were strongly correlated with NH4 + concentrations and inversely correlated with the ratio between denitrification genes and the genes indicative of ammonification (Σnir/nrfA), suggesting an environmental control on NO3 - transformations. Overall, our results for a denitrifying woodchip bioreactor operated at hydraulic residence times of 1.0-2.6 d demonstrate the temporal development in the microbial community and indicate an increased potential for N2 O emissions with time from the denitrifying woodchip bioreactor.

Substrate type determines microbial activity and community composition in bioreactors for nitrate removal by denitrification at low temperature.

High levels of nitrogen originating from blasting operations, for example at mining sites or quarries, risk contaminating water bodies through leaching from waste rock dumps. Woodchip bioreactors can be a simple and cost-effective way of reducing nitrate concentrations in the leachate. In this study we investigated how bottle sedge, barley straw, and pine woodchips used as electron donors for denitrification influenced microbial community composition and nitrate removal in lab-scale bioreactors during 270 days. The reactors were operated to ensure that nitrate was never limiting and to achieve similar nitrate removal (%). Distinct bacterial communities developed due to the different substrates, as determined by sequencing of the 16S rRNA gene. Sedge and straw reactors shared more taxa with each other than with woodchips and throughout the experimental period, sedge and straw were more diverse than woodchips. Cellulose degrading bacteria like Fibrobacteres and Verrucomicrobia were detected in the substrates after 100-150 days of operation. Nitrate removal rates were highest in the sedge and straw reactors. After initial fluctuations, these reactors removed 5.1-6.3 g N m-3 water day-1, which was 3.3-4.4 times more than in the woodchip reactors. This corresponded to 48%, 42%, and 44% nitrate removal for the sedge, straw, and woodchip reactors respectively. The functional communities were characterized by quantitative PCR and denitrification was the major nitrate removing process based on genetic potential and water chemistry, although sedge and straw developed a capacity for ammonification. Gene ratios suggested that denitrification was initially incomplete and terminating with nitrous oxide. An increase in abundances of nitrous oxide reducing capacity in all substrate types towards the end increased the potential for less emissions of the greenhouse gas nitrous oxide.

External carbon addition for enhancing denitrification modifies bacterial community composition and affects CH4 and N2O production in sub-arctic mining pond sediments.

Explosives used in mining operations release reactive nitrogen (N) that discharge into surrounding waters. Existing pond systems at mine sites could be used for N removal through denitrification and we investigated capacity in tailings and clarification pond sediments at an iron-ore mine site. Despite differences in microbial community structure in the two ponds, the potential denitrification rates were similar, although carbon limited. Therefore, a microcosm experiment in which we amended sediment from the clarification pond with acetate, cellulose or green algae as possible carbon sources was conducted during 10 weeks under denitrifying conditions. Algae and acetate treatments showed efficient nitrate removal and increased potential denitrification rates, whereas cellulose was not different from the control. Denitrifiers were overall more abundant than bacteria performing dissimilatory nitrate reduction to ammonium (DNRA) or anaerobic ammonium oxidation, although DNRA bacteria increased in the algae treatment and this coincided with accumulation of ammonium. The algae addition also caused higher emissions of methane (CH4) and nitrous oxide (N2O). The bacterial community in this treatment had a large proportion of Bacteroidia, sulfate reducing taxa and bacteria known as fermenters. Functional gene abundances indicated an imbalance between organisms that produce N2O in relation to those that can reduce it, with the algae treatment showing the lowest relative capacity for N2O reduction. These findings show that pond sediments have the potential to contribute to mitigating nitrate levels in water from mining industry, but it is important to consider the type of carbon supply as it affects the community composition, which in turn can lead to unwanted processes and increased greenhouse gas emissions.

Relative importance of plant uptake and plant associated denitrification for removal of nitrogen from mine drainage in sub-arctic wetlands.

Reactive nitrogen (N) species released from undetonated ammonium-nitrate based explosives used in mining or other blasting operations are an emerging environmental problem. Wetlands are frequently used to treat N-contaminated water in temperate climate, but knowledge on plant-microbial interactions and treatment potential in sub-arctic wetlands is limited. Here, we compare the relative importance of plant uptake and denitrification among five plant species commonly occurring in sub-arctic wetlands for removal of N in nitrate-rich mine drainage in northern Sweden. Nitrogen uptake and plant associated potential denitrification activity and genetic potential for denitrification based on quantitative PCR of the denitrification genes nirS, nirK, nosZI and nosZII were determined in plants growing both in situ and cultivated in a growth chamber. The growth chamber and in situ studies generated similar results, suggesting high relevance and applicability of results from growth chamber experiments. We identified denitrification as the dominating pathway for N-removal and abundances of denitrification genes were strong indicators of plant associated denitrification activity. The magnitude and direction of the effect differed among the plant species, with the aquatic moss Drepanocladus fluitans showing exceptionally high ratios between denitrification and uptake rates, compared to the other species. However, to acquire realistic estimates of N-removal potential of specific wetlands and their associated plant species, the total plant biomass needs to be considered. The species-specific plant N-uptake and abundance of denitrification genes on the root or plant surfaces were affected by the presence of other plant species, which show that both multi- and inter-trophic interactions are occurring. Future studies on N-removal potential of wetland plant species should consider how to best exploit these interactions in sub-arctic wetlands.

Nitrogen removal and spatial distribution of denitrifier and anammox communities in a bioreactor for mine drainage treatment.

Mine drainage water may contain high levels of nitrate (NO3(-)) due to undetonated nitrogen-based explosives. The removal of NO3(-) and nitrite (NO2(-)) in cold climates through the microbial process of denitrification was evaluated using a pilot-scale fixed-bed bioreactor (27 m(3)). Surface water was diverted into the above-ground bioreactor filled with sawdust, crushed rock, and sewage sludge. At hydraulic residence times of ca.15 h and with the addition of acetate, NO3(-) and NO2(-) were removed to below detection levels at a NO3(-) removal rate of 5-10 g N m(-3) (bioreactor material) d(-1). The functional groups contributing to nitrogen removal in the bioreactor were studied by quantifying nirS and nirK present in denitrifying bacteria, nosZI and nosZII genes from the nitrous oxide - reducing community, and a taxa-specific part of the16S rRNA gene for the anammox community. The abundances of nirS and nirK were almost 2 orders of magnitude greater than the anammox specific 16S rRNA gene, indicating that denitrification was the main process involved in nitrogen removal. The spatial distribution of the quantified genes was heterogeneous in the bioreactor, with trends observed in gene abundance as a function of depth, distance from the bioreactor inlet, and along specific flowpaths. There was a significant relationship between the abundance of nirS, nirK, and nosZI genes and depth in the bioreactor, such that the abundance of organisms containing these genes may be controlled by oxygen diffusion and substrate supply in the partially or completely water-saturated material. Among the investigated microbial functional groups, nirS and anammox bacterial 16S rRNA genes exhibited a systematic trend of decreasing and increasing abundance, respectively, with distance from the inlet, which suggested that the functional groups respond differently to changing environmental conditions. The greater abundance of nirK along central flowpaths may indicate that the bioreactor design favored preferential flow along these flowpaths, away from the sides of the bioreactor. An improved bioreactor design should consider the role of preferential flowpaths and the heterogeneous distribution of the genetic potential for denitrification, nitrous oxide reduction and anammox on bioreactor function.

Survey of bromodeoxyuridine uptake among environmental bacteria and variation in uptake rates in a taxonomically diverse set of bacterial isolates.

Incorporation of 5-Bromo-2'-Deoxyuridine (BrdU) into DNA can be used to target replicating bacteria in the environment, but differential uptake capacity is a potential bias. Among 23 bacterial isolates commonly found in soils, most took up BrdU, but at up to 10-fold different cell-specific rates. Combined with results from an in silico analysis of 1000 BrdU-labeled 16S rRNA gene sequences, our results demonstrate a BrdU uptake bias with no apparent relationship between taxa affiliation and ability to incorporate BrdU.

Spatial distribution of ammonia-oxidizing bacteria and archaea across a 44-hectare farm related to ecosystem functioning.

Characterization of spatial patterns of functional microbial communities could facilitate the understanding of the relationships between the ecology of microbial communities, the biogeochemical processes they perform and the corresponding ecosystem functions. Because of the important role the ammonia-oxidizing bacteria (AOB) and archaea (AOA) have in nitrogen cycling and nitrate leaching, we explored the spatial distribution of their activity, abundance and community composition across a 44-ha large farm divided into an organic and an integrated farming system. The spatial patterns were mapped by geostatistical modeling and correlations to soil properties and ecosystem functioning in terms of nitrate leaching were determined. All measured community components for both AOB and AOA exhibited spatial patterns at the hectare scale. The patchy patterns of community structures did not reflect the farming systems, but the AOB community was weakly related to differences in soil pH and moisture, whereas the AOA community to differences in soil pH and clay content. Soil properties related differently to the size of the communities, with soil organic carbon and total nitrogen correlating positively to AOB abundance, while clay content and pH showed a negative correlation to AOA abundance. Contrasting spatial patterns were observed for the abundance distributions of the two groups indicating that the AOB and AOA may occupy different niches in agro-ecosystems. In addition, the two communities correlated differently to community and ecosystem functions. Our results suggest that the AOA, not the AOB, were contributing to nitrate leaching at the site by providing substrate for the nitrite oxidizers.

Over-expression of major colonization factors of enterotoxigenic Escherichia coli, alone or together, on non-toxigenic E. coli bacteria.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveller's diarrhea. Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. We have recently described the development of recombinant strains over-expressing CFA/I; the most prevalent CF among human clinical ETEC isolates. Here, non-toxigenic recombinant E. coli strains over-expressing Coli surface antigen 2 (CS2), CS4, CS5, and CS6, either alone, or each in combination with CFA/I were constructed by cloning the genes required for expression and assembly of each CF into expression vectors harboring a strong promoter. Immunological assays showed that recombinant strains expressing single CFs produced those in significantly larger amounts than did corresponding naturally high producing reference strains. Recombinant strains co-expressing CFA/I together with another CF also expressed significantly larger amounts of both CFs compared with the corresponding references strains. Further, when tested in mice, oral immunization with formalin-killed recombinant bacteria co-expressing one such double-expression CF pair, CFA/I+CS2, induced specific serum IgG+IgM and fecal IgA antibody responses against both CFs exceeding the responses induced by immunizations with natural reference strains expressing CFA/I and CS2, respectively. We conclude that the described type of recombinant bacteria over-expressing major CFs of ETEC, alone or in combination, may be useful as candidate strains for use in an oral whole-cell CF-ETEC vaccine.

Use of a novel nonantibiotic triple marker gene cassette to monitor high survival of Pseudomonas fluorescens SBW25 on winter wheat in the field.

Pseudomonas fluorescens SBW25 was tagged with a triple marker gene cassette containing gfp, encoding green fluorescent protein; luxAB, encoding luciferase; and telABkilA, encoding tellurite resistance, and the tagged strain was monitored in the first Swedish field release of a genetically modified microorganism (GMM). The cells were inoculated onto winter wheat seeds and the GMM cells (SBW25:tgl) were monitored in the field from September 2005 to May 2006 using plating, luminometry and microscopic analyses. Cell numbers were high on all sampling occasions and metabolically active cells were detected on all plant parts. Field results were similar to those obtained in a parallel phytotron study, although the amount of SBW25:tgl detected on shoots was significantly higher in the phytotron than in the field. After winter, cell counts were 100-fold higher on the roots and root-associated soil compared with prewinter measurements, although the cells had a lower relative metabolic activity. The wheat seeds were naturally infested with Microdochium nivale, but no treatment resulted in reduction of disease symptoms. No SWB25:tgl cells were ever found in bulk soil or uninoculated plants. The Swedish field trial results complement and contrast with prior field studies performed with the same parent organism in the United Kingdom under different soil, plant and climatic conditions.