Gross nitrification in soils - Contribution of nitrification to N-gas emission from soils

This work contributes to developing a better understanding of nitrification in soils as an important source of N gas emissions from soils. Therefore, the nitrification process as well as N gas produced by nitrification are considered. The work described the common methods and new developed approach for determining the gross nitrification rate. Both measuring and quantifying nitrification in soils have been shown to achieve the objective. One focus is to differentiate the sources of N gases and to quantify the contribution of nitrification to N gas emission from soils. The separation of N gas production into source-related pathways that simultaneously operate in soils requires comprehensive experiments with complex analyses. Therefore a new analytical approach and calculates the fractions of ammonia oxidation, Norg oxidation and denitrification for total soil NO and N2O released from a soil probes at different oxygen states (2.5, 1.2 and 0 % O2) is presented and tested for a five loamy Spanish forest soils. Whereas the relation between ammonia oxidation and denitrification as sources of soil N2O gas release appear to be consistent, which is commonly accepted, the contribution of Norg oxidation was unexpectedly high (up to 76%). Also two model approaches to model the N-gas production in soils are parametrised on experimental data from laboratory studies. The findings are discussed in view of choosing the best approach to predict N2O production during nitrification. and an approach to combine response functions in modelling is presented and tested on field data. The advantage against the conventional combining approaches (multiplicative or min/max approaches) is discussed. N2O production data related to nitrification and nitrification rates were collected and multiple linear regression analysis between the soil properties and N2O product ratios were applied to this dataset to identify functional relationships. Future works to support the development of sufficient model approaches are needed, and in particular, the nitrite and oxygen concentrations in soils are the most important factors for N2O production.Diese Arbeit möchte zu einem besseren Verständnis über den Prozess der Nitrifikation als eine wichtige Quelle der N-Gasemission aus Böden beitragen. Daher werden einleitend die verschiedenen Prozesspfade der Nitrifikation und der Spurengasbildung beschrieben und bildlich dargestellt. Verfahren zur Messung der Nitrifikation und Versuche zu Bestimmung der Umsatzraten werden in der Arbeit vorgestellt. Dabei liegt der Fokus auf der Separation der verschiedenen Quellen von NO und N2O und beschreibt die dafür notwendigen komplexen Versuche inclusive mathematischer Verfahren zu deren Analyse. Mit Hilfe dieser Tools werden die Anteile der Ammoniakoxidation (erster Schritt der autotrophen Nitrifikation), der direkten Oxidation von organischem Stickstoff und der Denitrifikation bei unterschiedlichen Sauerstoffpartialdrücken (2.5, 1.2 und 0 % O2) bestimmt und in einem weiteren Schritt die Methoden auf fünf spanische Waldstandorte angewendet. Interessanterweise sind die Anteile der direkten Oxidation von organischem Stickstoff sehr hoch und auch relativ konstant bei verschiedenen Sauerstoffpartialdrücken. Zwei verschiedene Modellansätze zu Beschreibung der N-Spurengasproduktion in Böden werden vorgestellt und an Labordaten parametrisiert. Die beiden Ansätze und ihre Implikationen für die Bildungswege der N-Spurengasproduktion werden ausgiebig diskutiert. Zusätzlich wird für die Anwendung in Ökosystemmodellen ein auf dem harmonischen Mittel beruhenden Ansatz vorgeschlagen, um verschiedene Responsefunktionen (z.B. die für die Temperatur- und die für die Bodenfeuchteabhängigkeit) miteinander zu verbinden. Im letzten Abschnitt der Arbeit werden die Daten der zuvor beschriebenen Experimente sowie in der Literatur verfügbare Daten zur Bruttonitrifikation und der nitrifikatorischen N2O-Produktion systematisch zusammengetragen, daraus das N2O-Produktion/Nitrifikation-Verhältnis (N2O product ratio) berechnet und dieses mittels multipler lineare Regression gegenüber den Bodeneigenschaften analysiert. Es deutet sich an, dass besonders der aktuelle Sauerstoffpartialdruck und die Nitritkonzentration starken Einfluss auf die N-Spurengasproduktion haben könnten, aber um kausale Zusammenhänge zu bestätigen, gibt es zu wenige insitu Messungen dieser beiden Faktoren in bisherigen Experimenten. Daher endet die Arbeit mit der Aufforderung zukünftig in N-Gasexperimenten immer auch Nitrit und Sauerstoff im Boden zu messen

Microbial nitrogen cycling in permafrost soils: implications for atmospheric chemistry

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Greenhouse Gas Emissions from Advanced Nitrogen-Removal Onsite Wastewater Treatment Systems

Advanced onsite wastewater treatment systems (OWTS) designed to remove nitrogen from residential wastewater play an important role in protecting environmental and public health. Nevertheless, the microbial processes involved in treatment produce greenhouse gases (GHGs) that contribute to global climate change, including CO2, CH4, N2O. We measured GHG emissions from 27 advanced N-removal OWTS in the towns of Jamestown and Charlestown, Rhode Island, USA, and assessed differences in flux based on OWTS technology, home occupancy (year-round vs. seasonal), and zone within the system (oxic vs. anoxic/hypoxic). We also investigated the relationship between flux and wastewater properties. Flux values for CO2, CH4, and N2O ranged from −0.44 to 61.8, −0.0029 to 25.3, and −0.02 to 0.23 μmol GHG m−2 s−1, respectively. CO2 and N2O flux varied among technologies, whereas occupancy pattern did not significantly impact any GHG fluxes. CO2 and CH4 – but not N2O – flux was significantly higher in the anoxic/hypoxic zone than in the oxic zone. Greenhouse gas fluxes in the oxic zone were not related to any wastewater properties. CO2 and CH4 flux from the anoxic/hypoxic zone peaked at ~22-23 °C, and was negatively correlated with dissolved oxygen levels, the latter suggesting that CO2 and CH4 flux result primarily from anaerobic respiration. Ammonium concentration and CH4 flux were positively correlated, likely due to inhibition of CH4 oxidation by NH4+. N2O flux in the anoxic/hypoxic zone was not correlated to any wastewater property. We estimate that advanced N-removal OWTS contribute 262 g CO2 equivalents capita−1 day−1, slightly lower than emissions from conventional OWTS. Our results suggest that technology influences CO2 and N2O flux and zone influences CO2 and CH4 flux, while occupancy pattern does not appear to impact GHG flux. Manipulating wastewater properties, such as temperature and dissolved oxygen, may help mitigate GHG emissions from these systems

Trace metal requirements for microbial enzymes involved in the production and consumption of methane and nitrous oxide

Fluxes of greenhouse gases to the atmosphere are heavily influenced by microbiological activity. Microbial enzymes involved in the production and consumption of greenhouse gases often contain metal cofactors. While extensive research has examined the influence of Fe bioavailability on microbial CO_2 cycling, fewer studies have explored metal requirements for microbial production and consumption of the second- and third-most abundant greenhouse gases, methane (CH_4), and nitrous oxide (N_2O). Here we review the current state of biochemical, physiological, and environmental research on transition metal requirements for microbial CH_4 and N_2O cycling. Methanogenic archaea require large amounts of Fe, Ni, and Co (and some Mo/W and Zn). Low bioavailability of Fe, Ni, and Co limits methanogenesis in pure and mixed cultures and environmental studies. Anaerobic methane oxidation by anaerobic methanotrophic archaea (ANME) likely occurs via reverse methanogenesis since ANME possess most of the enzymes in the methanogenic pathway. Aerobic CH_4 oxidation uses Cu or Fe for the first step depending on Cu availability, and additional Fe, Cu, and Mo for later steps. N_2O production via classical anaerobic denitrification is primarily Fe-based, whereas aerobic pathways (nitrifier denitrification and archaeal ammonia oxidation) require Cu in addition to, or possibly in place of, Fe. Genes encoding the Cu-containing N_2O reductase, the only known enzyme capable of microbial N_2O conversion to N_2, have only been found in classical denitrifiers. Accumulation of N_2O due to low Cu has been observed in pure cultures and a lake ecosystem, but not in marine systems. Future research is needed on metalloenzymes involved in the production of N_2O by enrichment cultures of ammonia oxidizing archaea, biological mechanisms for scavenging scarce metals, and possible links between metal bioavailability and greenhouse gas fluxes in anaerobic environments where metals may be limiting due to sulfide-metal scavenging

Nitrous oxide production by ammonia oxidizers : Physiological diversity, niche differentiation and potential mitigation strategies

Funding Information: This work was financially supported by the AXA Research Fund (GWN), a Royal Society University Research Fellowship UF150571 (CGR) and all authors are members of the Nitrous Oxide Research Alliance (NORA), a Marie Skłodowska‐Curie ITN and research project under the EU's seventh framework programme (FP7).Peer reviewedPostprin

Nitrifying and Denitrifying Microbial Communities in Centralized and Decentralized Biological Nitrogen Removing Wastewater Treatment Systems

Biological nitrogen removal (BNR) in centralized and decentralized wastewater treatment systems is assumed to be driven by the same microbial processes and to have communities with a similar composition and structure. There is, however, little information to support these assumptions, which may impact the effectiveness of decentralized systems. We used high-throughput sequencing to compare the structure and composition of the nitrifying and denitrifying bacterial communities of nine onsite wastewater treatment systems (OWTS) and one wastewater treatment plant (WTP) by targeting the genes coding for ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ). The amoA diversity was similar between the WTP and OWTS, but nosZ diversity was generally higher for the WTP. Beta diversity analyses showed the WTP and OWTS promoted distinct amoA and nosZ communities, although there is a core group of N-transforming bacteria common across scales of BNR treatment. Our results suggest that advanced N-removal OWTS have microbial communities that are sufficiently distinct from those of WTP with BNR, which may warrant different management approaches

Methane-driven nitrate removal and identification of active bacterial species using RNA-SIP and Hight-throughput sequencing.

Nitrate pollution in water can potentially be mitigated by aerobic and microaerobic microbial methane oxidation coupled to nitrate removal. This research has recently attracted interest due to the key role oxygen concentration plays in the process. Although seen as potential means of removing nitrate from contaminated waters, the mechanism and microorganisms involved in the process are still poorly understood. The study seeks to investigate the nitrate removal potential of the process by answering the “who does what?” question in the process by identifying the active bacterial species by combining the RNA stable isotope probing (RNA- SIP) technique with high-throughput sequencing. In this study, methanotrophic biofilms were enriched in columns packed with 3 mm glass beads. The columns were made from acrylic tube 50 mm in diameter, and 400 mm in length and a working volume of 0.0004 m3. The CH4 and O2 gas feed into the aerobic column was 2% v/v CH4 in air while the microaerobic gas feed for the two columns was 2% CH4, 2% O2 and 96% Ar, and 2% CH4, 2% O2 and 96% N2. The gases were continuously fed into the columns and nitrate mineral salt (NMS) with a nitrate concentration of 140 mg∙L-1 NO3—N was recirculated through the columns and replaced when exhausted. The aerobic and microaerobic reactors were operated for 238 and 122 days respectively. The methane oxidation rate observed under both aerobic and microaerobic condition differed with the aerobic columns giving an average methane oxidation rate of 62.8±21.45 gCH4∙m-3hr- 1 for the aerobic column while the microaerobic columns gave an average rate of 10.67 ±3.9 (Ar) and 9.28 ±3.5 gCH4∙m-3hr-1 (N2) respectively. Nitrate removal rates observed for the columns showed that the microaerobic columns gave a maximum rate of 3.6 gN m-3h-1, while the aerobic column gave a maximum rate of 0.8 gN m-3h-1. The CH4/NO3 consumption ratio obtained from the columns showed that the average consumption ratio in the aerobic columns was 4.3 ±1.6, while the microaerobic columns gave a consumption ratio of 2.6 ±1.1 (Ar) and 2.2 ±0.8 (N2) respectively. Oxygen concentration and O2/CH4 ratio between the aerobic and microaerobic columns is thought to have played an important role in methane oxidation rates and nitrate removal rates. In addition, the production of ammonium in the aerobic column is also suggested to play a role in the low nitrate removal rate observed in the aerobic columns. RNA-SIP in combination with high-throughput sequencing identified the active bacterial species that were the key players in the methane-driven denitrification process under aerobic conditions. The bacterial genera Methylocystis and Methylosinus were identified as the main methane oxidizers under both aerobic and microaerobic conditions, producing organic intermediates such as methanol, acetate and formate that were identified. These were hypothesised to drive nitrate removal. Active denitrifiers whose 16S rRNA were enriched with 13C-labeled substrate and were considered the major players were Hyphomicrobium, Pseudoxanthomonas, Arenimonas, and Methyloversatilis

Anaerobic oxidation of methane and associated microbiome in anoxic water of Northwestern Siberian lakes

Arctic lakes emit methane (CH4) to the atmosphere. The magnitude of this flux could increase with permafrost thaw but might also be mitigated by microbial CH4 oxidation. Methane oxidation in oxic water has been extensively studied, while the contribution of anaerobic oxidation of methane (AOM) to CH4 mitigation is not fully understood. We have investigated four Northern Siberian stratified lakes in an area of discontinuous permafrost nearby Igarka, Russia. Analyses of CH4 concentrations in the water column demonstrated that 60 to 100% of upward diffusing CH4 was oxidized in the anoxic layers of the four lakes. A combination of pmoA and mcrA gene qPCR and 16S rRNA gene metabarcoding showed that the same taxa, all within Methylomonadaceae and including the predominant genus Methylobacter as well as Crenothrix, could be the major methane-oxidizing bacteria (MOB) in the anoxic water of the four lakes. Correlation between Methylomonadaceae and OTUs within Methylotenera, Geothrix and Geobacter genera indicated that AOM might occur in an interaction between MOB, denitrifiers and iron-cycling partners. We conclude that MOB within Methylomonadaceae could have a crucial impact on CH4 cycling in these Siberian Arctic lakes by mitigating the majority of produced CH4 before it leaves the anoxic zone. This finding emphasizes the importance of AOM by Methylomonadaceae and extends our knowledge about CH4 cycle in lakes, a crucial component of the global CH4 cycle

Linking Metabolic Capacity and Molecular Biology of Methylocystis sp. Strain SC2 by a Newly Developed Proteomics Workflow

Microbial methane oxidation is one of the fundamental processes in global methane cycle. Methane-oxidizing bacteria, or methanotrophs, are the major biological sink for the methane produced from anthropogenic and natural sources. Our model organism, Methylocystis sp. strain SC2, is one of the best-studied representatives of alphaproteobacterial (type IIa) methanotrophs. Proteobacterial methanotrophs possess a unique cell architecture characterized by intracytoplasmic membranes (ICMs). The cellular amount of the ICMs is increasing with methanotrophic activity. The presence of ICMs makes molecular biology approaches, but in particular global proteomics, highly challenging. In this study, we therefore aimed to develop an efficient proteomics workflow for strain SC2 and to apply this state-of-the-art tool for investigation of the strain SC2 response to environmental factors. To successfully develop the proteomics workflow, we particularly focused on an efficient solubilization and digestion of the integral membrane proteins of strain SC2 for further downstream analysis. We introduced the so-called crude-MS proteomics workflow, upon assessing and optimizing all the major steps in the proteomics workflow, including cell lysis, protein solubilization, and protein digestion. Our new SC2 proteomics workflow greatly increased not only the protein quantification accuracy (mean coefficient of variation 3.2 %) but also the proteome coverage to 62%, with up to 10-fold increase in the detection intensity of membrane-associated proteins. Previous studies have shown that the LysC/trypsin tandem digestion resulted in higher coverage of fully cleaved tryptic peptides than a trypsin-only digestion. Therefore, the development of our optimized proteomics workflow involved the application of the LysC/trypsin tandem digestion in detergent environment to increase the SC2 proteome coverage. Prior to publication of our crude-MS approach, all systematic assessments of LysC/trypsin proteolysis were conducted in chaotropic environments, like urea. As a spin-off, we therefore initiated a follow-up study to compare the efficiency of the LysC/trypsin tandem digestion in detergent environments (e.g., SDC, SLS) relative to chaotropic environments. The study revealed that the LysC/trypsin tandem digestion could be efficiently carried out not only in chaotropic environments but also in MS-compatible detergent environments. In fact, the LysC/trypsin tandem digestion in both environments resulted in a higher coverage of fully cleaved peptides than the trypsin-only digestion. After successful development of the crude-MS proteomics workflow, we used this high-throughput method to assess the molecular response of strain SC2 to the availability of hydrogen as a potentially alternative energy source. Starting point of this research was the knowledge that strain SC2 and other Methylocystis spp. possess the genetic potential to produce various hydrogenases. In fact, the addition of 2% hydrogen to the headspace atmosphere led, under limiting concentrations of methane and oxygen, to the complete hydrogen consumption by strain SC2. Concurrently, the SC2 biomass yield was significantly increased, while the methane consumption rate was significantly decreased. Global proteome analyses revealed that the addition of hydrogen induced an increase in the production of Group 1d and Group 2b [NiFe]-hydrogenases, and hydrogenase accessory proteins. Notably, the upregulation of the Group 1d, 2b [NiFe]-hydrogenases was concomitantly linked to a reconstruction of the energy metabolism in strain SC2. In another project, genome-scale metabolic modeling and growth experiments were applied to show that strain SC2 has the capacity to utilize acetate through the glyoxylate assimilation pathway. In addition, the study revealed that in type II methanotrophs, energy demand for methane oxidation is covered by complex I of the electron transport chain. In summary, our research demonstrates how to experimentally link the metabolic potential of Methylocystis sp. strain SC2 with the underlying proteome complexity. Thus, the newly developed highly reproducible SC2 proteomics workflow represents a high-throughput method that makes it possible to achieve in future research an understanding of the molecular adaptation mechanisms of strain SC2 to environmental change