Microbiome Structure and Function in Woodchip Bioreactors for Nitrate Removal in Agricultural Drainage Water.
ABSTRACT: Woodchip bioreactors are increasingly used to remove nitrate (NO3-) from agricultural drainage water in order to protect aquatic ecosystems from excess nitrogen. Nitrate removal in woodchip bioreactors is based on microbial processes, but the microbiomes and their role in bioreactor efficiency are generally poorly characterized. Using metagenomic analyses, we characterized the microbiomes from 3 full-scale bioreactors in Denmark, which had been operating for 4-7 years. The microbiomes were dominated by Proteobacteria and especially the genus Pseudomonas, which is consistent with heterotrophic denitrification as the main pathway of NO3- reduction. This was supported by functional gene analyses, showing the presence of the full suite of denitrification genes from NO3- reductases to nitrous oxide reductases. Genes encoding for dissimilatory NO3- reduction to ammonium were found only in minor proportions. In addition to NO3- reducers, the bioreactors harbored distinct functional groups, such as lignocellulose degrading fungi and bacteria, dissimilatory sulfate reducers and methanogens. Further, all bioreactors harbored genera of heterotrophic iron reducers and anaerobic iron oxidizers (Acidovorax) indicating a potential for iron-mediated denitrification. Ecological indices of species diversity showed high similarity between the bioreactors and between the different positions along the flow path, indicating that the woodchip resource niche was important in shaping the microbiome. This trait may be favorable for the development of common microbiological strategies to increase the NO3- removal from agricultural drainage water.
Project description:Denitrifying woodchip bioreactors (WBR), which aim to reduce nitrate (NO<sub>3</sub><sup>-</sup>) pollution from agricultural drainage water, are less efficient when cold temperatures slow down the microbial transformation processes. Conducting bioaugmentation could potentially increase the NO<sub>3</sub><sup>-</sup> removal efficiency during these specific periods. First, it is necessary to investigate denitrifying microbial populations in these facilities and understand their temperature responses. We hypothesized that seasonal changes and subsequent adaptations of microbial populations would allow for enrichment of cold-adapted denitrifying bacterial populations with potential use for bioaugmentation. Woodchip material was sampled from an operating WBR during spring, fall, and winter and used for enrichments of denitrifiers that were characterized by studies of metagenomics and temperature dependence of NO<sub>3</sub><sup>-</sup> depletion. The successful enrichment of psychrotolerant denitrifiers was supported by the differences in temperature response, with the apparent domination of the phylum <i>Proteobacteria</i> and the genus <i>Pseudomonas</i>. The enrichments were found to have different microbiomes' composition and they mainly differed with native woodchip microbiomes by a lower abundance of the genus <i>Flavobacterium</i>. Overall, the performance and composition of the enriched denitrifying population from the WBR microbiome indicated a potential for efficient NO<sub>3</sub><sup>-</sup> removal at cold temperatures that could be stimulated by the addition of selected cold-adapted denitrifying bacteria.
Project description:Woodchip bioreactor technology removes nitrate from agricultural subsurface drainage by using denitrifying microorganisms. Although woodchip bioreactors have demonstrated success in many field locations, low water temperature can significantly limit bioreactor efficiency and performance. To improve bioreactor performance, it is important to identify the microbes responsible for nitrate removal at low temperature conditions. Therefore, in this study, we identified and characterized denitrifiers active at low-temperature conditions by using culture-independent and -dependent approaches. By comparative 16S rRNA (gene) analysis and culture isolation technique, Pseudomonas spp., Polaromonas spp., and Cellulomonas spp. were identified as being important bacteria responsible for denitrification in woodchip bioreactor microcosms at relatively low temperature conditions (15°C). Genome analysis of Cellulomonas sp. strain WB94 confirmed the presence of nitrite reductase gene nirK. Transcription levels of this nirK were significantly higher in the denitrifying microcosms than in the non-denitrifying microcosms. Strain WB94 was also capable of degrading cellulose and other complex polysaccharides. Taken together, our results suggest that Cellulomonas sp. denitrifiers could degrade woodchips to provide carbon source and electron donors to themselves and other denitrifiers in woodchip bioreactors at low-temperature conditions. By inoculating these denitrifiers (i.e., bioaugmentation), it might be possible to increase the nitrate removal rate of woodchip bioreactors at low-temperature conditions.
Project description:The versatile soil bacterium Anaeromyxobacter dehalogenans lacks the hallmark denitrification genes nirS and nirK (NO2-?NO), and couples growth to NO3- reduction to NH4+ (respiratory ammonification) and to N2O reduction to N2A. dehalogenans also grows by reducing Fe(III) to Fe(II), which chemically reacts with NO2- to form N2O (i.e., chemodenitrification). Following the addition of 100 ?moles of NO3- or NO2- to Fe(III)-grown, axenic cultures of A. dehalogenans, 54 (±7) ?moles and 113 (±2) ?moles N2O-N, respectively, were produced and subsequently consumed. The conversion of NO3- to N2 in the presence of Fe(II) through linked biotic-abiotic reactions represents an unrecognized ecophysiology of A. dehalogenans The new findings demonstrate that the assessment of gene content alone is insufficient to predict microbial denitrification potential and N loss (i.e., the formation of gaseous N products). A survey of complete bacterial genomes in the NCBI Reference Sequence database coupled with available physiological information revealed that organisms lacking nirS/nirK but with Fe(III) reduction potential and genes for NO3- and N2O reduction are not rare, indicating that NO3- reduction to N2 through linked biotic-abiotic reactions is not limited to A. dehalogenans Considering the ubiquity of iron in soils and sediments and the broad distribution of dissimilatory Fe(III) and NO3- reducers, denitrification independent of NO-forming NO2- reductases (through combined biotic-abiotic reactions) may have substantial contributions to N loss and N2O flux.Importance Current attempts to gauge N loss from soils rely on the quantitative measurement of nirK and nirS genes and/or transcripts. In the presence of iron, the common soil bacterium Anaeromyxobacter dehalogenans is capable of denitrification and producing N2 without the key denitrification genes nirK/nirS Such chemodenitrifiers denitrify through combined biotic and abiotic reactions and have potentially large contributions to N loss to the atmosphere, and fill a heretofore unrecognized ecological niche in soil ecosystems. The findings emphasize that comprehensive understanding of N flux and accurate assessment of denitrification potential can only be achieved when integrated studies of interlinked biogeochemical cycles are performed.
Project description:Denitrifying woodchip bioreactors are a practical nitrogen (N) mitigation technology but evaluating the potential for bioreactor phosphorus (P) removal is highly relevant given that (1) agricultural runoff often contains N and P, (2) very low P concentrations cause eutrophication, and (3) there are few options for removing dissolved P once it is in runoff. A series of batch tests evaluated P removal by woodchips that naturally contained a range of metals known to sorb P and then three design and environmental factors (water matrix, particle size, initial dissolved reactive phosphorus (DRP) concentration). Woodchips with the highest aluminum and iron content provided the most dissolved P removal (13±2.5 mg DRP removed/kg woodchip). However, poplar woodchips, which had low metals content, provided the second highest removal (12±0.4 mg/kg) when they were tested with P-dosed river water which had a relatively complex water matrix. Chemical P sorption due to woodchip elements may be possible, but it is likely one of a variety of P removal mechanisms in real-world bioreactor settings. Scaling the results indicated bioreactors could remove 0.40 to 13 g DRP/ha. Woodchip bioreactor dissolved P removal will likely be small in magnitude, but any such contribution is an added-value benefit of this denitrifying technology.
Project description:Haloferax mediterranei (R4) belongs to the group of halophilic archaea, one of the predominant microbial populations in hypersaline environments. In these ecosystems, the low availability of oxygen pushes the microbial inhabitants toward anaerobic pathways and the presence of N-oxyanions favor denitrification. In a recent study comparing three Haloferax species carrying dissimilatory N-oxide reductases, H. mediterranei showed promise as a future model for archaeal denitrification. This work further explores the respiratory physiology of this haloarchaeon when challenged with ranges of nitrite and nitrate concentrations and at neutral or sub-neutral pH during the transition to anoxia. Moreover, to begin to understand the transcriptional regulation of N-oxide reductases, detailed gas kinetics was combined with gene expression analyses at high resolution. The results show that H. mediterranei has an expression pattern similar to that observed in the bacterial Domain, well-coordinated at low concentrations of N-oxyanions. However, it could only sustain a few generations of exponential anaerobic growth, apparently requiring micro-oxic conditions for de novo synthesis of denitrification enzymes. This is the first integrated study within this field of knowledge in haloarchaea and Archaea in general, and it sheds lights on denitrification in salty environments.
Project description:The successional ecology of nitrogen cycling in biocrusts and the linkages to ecosystem processes remains unclear. To explore this, four successional stages of natural biocrust with five batches of repeated sampling and three developmental stages of simulated biocrust were studied using relative and absolute quantified multi-omics methods. A consistent pattern across all biocrust was found where ammonium assimilation, mineralization, dissimilatory nitrite to ammonium (DNiRA), and assimilatory nitrate to ammonium were abundant, while denitrification medium, N-fixation, and ammonia oxidation were low. Mathematic analysis showed that the nitrogen cycle in natural biocrust was driven by dissolved organic N and NO<sub>3</sub> <sup>-</sup>. pH and SO<sub>4</sub> <sup>2-</sup> were the strongest variables affecting denitrification, while C:(N:P) was the strongest variable affecting N-fixation, DNiRA, nitrite oxidation, and dissimilatory nitrate to nitrite. Furthermore, N-fixation and DNiRA were closely related to elemental stoichiometry and redox balance, while assimilatory nitrite to ammonium (ANiRA) and mineralization were related to hydrological cycles. Together with the absolute quantification and network models, our results suggest that responsive ANiRA and mineralization decreased during biocrust succession; whereas central respiratory DNiRA, the final step of denitrification, and the complexity and interaction of the whole nitrogen cycle network increased. Therefore, our study stresses the changing environmental functions in the biocrust N-cycle, which are succession-dependent.
Project description:Three different woodchip forms were tested for bromide sorption including ground woodchip, unwashed woodchips, and washed woodchips. We used six varying initial bromide concentrations to conduct the bromide sorption experiments with each woodchip form. Data on the initial and equilibrium bromide concentrations, wood mass, and initial and equilibrium solution pH from each of the six experiments are presented. Seven bromide tracer tests were conducted on field-scale denitrification beds. In this paper, data from each of the tracer tests including variation of bromide concentration over time and hydraulic indices of the tracer tests are presented. Interpretation of the data can be found in the research article entitled "Efficacy of bromide tracers for evaluating the hydraulic performance of denitrification beds treating agricultural drainage water" .
Project description:Dissimilatory nitrate reduction to ammonium (DNRA), denitrification, anaerobic ammonium oxidation (anammox), and biological N<sub>2</sub> fixation (BNF) can influence the nitrogen (N) use efficiency of rice production. While the effect of N application on BNF is known, little is known about its effect on NO<sub>3</sub><sup>-</sup> partitioning between DNRA, denitrification, and anammox. Here, we investigated the effect of N application on DNRA, denitrification, anammox, and BNF and on the abundance of relevant genes in three paddy soils in Australia. Rice was grown in a glasshouse with N fertilizer (150 kg N ha<sup>-1</sup>) and without N fertilizer for 75 days, and the rhizosphere and bulk soils were collected separately for laboratory incubation and quantitative PCR analysis. Nitrogen application reduced DNRA rates by >16% in all the soils regardless of the rhizospheric zone, but it did not affect the <i>nrfA</i> gene abundance. Without N, the amount and proportion of NO<sub>3</sub><sup>-</sup> reduced by DNRA (0.42 to 0.52 μg g<sup>-1</sup> soil day<sup>-1</sup> and 45 to 55%, respectively) were similar to or higher than the amount and proportion reduced by denitrification. However, with N the amount of NO<sub>3</sub><sup>-</sup> reduced by DNRA (0.32 to 0.40 μg g<sup>-1</sup> soil day<sup>-1</sup>) was 40 to 50% lower than the amount of NO<sub>3</sub><sup>-</sup> reduced by denitrification. Denitrification loss increased by >20% with N addition and was affected by the rhizospheric zones. Nitrogen loss was minimal through anammox, while BNF added 0.02 to 0.25 μg N g<sup>-1</sup> soil day<sup>-1</sup> We found that DNRA plays a significant positive role in paddy soil N retention, as it accounts for up to 55% of the total NO<sub>3</sub><sup>-</sup> reduction, but this is reduced by N application.<b>IMPORTANCE</b> This study provides evidence that nitrogen addition reduces nitrogen retention through DNRA and increases nitrogen loss via denitrification in a paddy soil ecosystem. DNRA is one of the major NO<sub>3</sub><sup>-</sup> reduction processes, and it can outcompete denitrification in NO<sub>3</sub><sup>-</sup> consumption when rice paddies are low in nitrogen. A significant level of DNRA activity in paddy soils indicates that DNRA plays an important role in retaining nitrogen by reducing NO<sub>3</sub><sup>-</sup> availability for denitrification and leaching. Our study shows that by reducing N addition to rice paddies, there is a positive effect from reduced nitrogen loss but, more importantly, from the conversion of NO<sub>3</sub><sup>-</sup> to NH<sub>4</sub><sup>+</sup>, which is the favored form of mineral nitrogen for plant uptake.
Project description:Sulfate-rich mine water must be treated before it is released into natural water bodies. We tested ethanol as substrate in bioreactors designed for biological sulfate removal from mine water containing up to 9 g L-1 sulfate, using granular sludge from an industrial waste water treatment plant as inoculum. The pH, redox potential, and sulfate and sulfide concentrations were measured twice a week over a maximum of 171 days. The microbial communities in the bioreactors were characterized by qPCR and high throughput amplicon sequencing. The pH in the bioreactors fluctuated between 5.0 and 7.7 with the highest amount of up to 50% sulfate removed measured around pH 6. Dissimilatory sulfate reducing bacteria (SRB) constituted only between 1% and 15% of the bacterial communities. Predicted bacterial metagenomes indicated a high prevalence of assimilatory sulfate reduction proceeding to formation of l-cystein and acetate, assimilatory and dissimilatory nitrate reduction, denitrification, and oxidation of ethanol to acetaldehyde with further conversion to ethanolamine, but not to acetate. Despite efforts to maintain optimal conditions for biological sulfate reduction in the bioreactors, only a small part of the microorganisms were SRB. The microbial communities were highly diverse, containing bacteria, archaea, and fungi, all of which affected the overall microbial processes in the bioreactors. While it is important to monitor specific physicochemical parameters in bioreactors, molecular assessment of the microbial communities may serve as a tool to identify biological factors affecting bioreactor functions and to optimize physicochemical attributes for ideal bioreactor performance.
Project description:For a recirculating aquaculture system (RAS), a passive water treatment system was designed for efficient discharge nutrient removal and water reuse in RAS production. Denitrification in a woodchip bioreactor filled with birch wood (Betula pendula) followed by sand filtration was introduced into a side-loop of an experimental RAS rearing rainbow trout (Oncorhynchus mykiss). Denitrification efficiency remained high (96%) throughout the experiment and reached a nitrogen removal rate of 15 g NO<sub>3</sub>-N m<sup>-3</sup> per day. Sand filtration was used to remove dissolved and particulate matter and improve water quality before being returned to water circulation. To ensure the absence of harmful substances in the system, heavy metals were quantified. Additionally, off-flavor-inducing compounds were quantified in the circulating water and in fish flesh. Significantly higher concentrations of geosmin (GSM) (p<0.05) were observed in the controls compared to side-looped systems, but a similar effect was not observed in the case of 2-methylisoborneol (MIB). Among heavy metals, concentrations of Co (30 μg L<sup>-1</sup>), Ni (40 μg L<sup>-1</sup>), and Pb (140 μg L<sup>-1</sup>) decreased to below 10 μg L<sup>-1</sup> in the side-loop water after the start-up of the system. Only low concentrations of Cu (5-30 μg L<sup>-1</sup>) were found in the rearing tank water, in both the side-loop and controls. The results indicated that this type of process design is suitable for safely producing fish of high quality.