17TH EUROPEAN NITROGEN-CYCLE MEETING (ENC2012)

2266--2288 SSeepptteemmbbeerr 22001122

OOsslloo,, NNoorrwwaayy

PROGRAMME AND ABSTRACT BOOK

1

17th European Nitrogen Cycle Meeting, Oslo 2012

Contents

Organisation ................................................................................................................................................ 2

Travel Information ...................................................................................................................................... 3

Programme ................................................................................................................................................. 4

Wednesday, 26 September 2012 ............................................................................................................ 4

Thursday, 27 September 2012 ................................................................................................................ 5

Friday, 28 September 2012 ..................................................................................................................... 8

Posters....................................................................................................................................................... 11

Abstracts ................................................................................................................................................... 13

Presentation abstracts .......................................................................................................................... 14

Poster abstracts .................................................................................................................................... 50

Delegate list .............................................................................................................................................. 70

2

17th European Nitrogen Cycle Meeting, Oslo 2012

Organisation

Local organising committee

Åsa Frostegård1

Natalie Lim1

Lars Bakken2

Peter Dörsch2

Norwegian University of Life Sciences 1 Department of Chemistry, Biotechnology and Food Science (IKBM)

Christian Magnus Falsens vei 1, N-1432 Ås, Norway

2 Department of Plant and Environmental Sciences (IPM)

Fougnerbakken 3, N-1430 Ås, Norway

Scientific committee

David Richardson (University of East Anglia / UK)

Rob van Spanning (VU University Amsterdam / NL)

Mike Jetten (Radboud University Nijmegen / NL)

Jeff Cole (University of Birmingham / UK)

Lars Bakken (Norwegian University of Life Sciences / NO)

Åsa Frostegård (Norwegian University of Life Sciences / NO)

Peter Dörsch (Norwegian University of Life Sciences / NO)

3

17th European Nitrogen Cycle Meeting, Oslo 2012

Travel Information

Arrival / Departure by air

Oslo has three airports (below). For information on

travel in the Oslo region:

http://www.visitoslo.com/en/transport/transport-

airport/

Oslo Airport Gardermoen (OSL)

Oslo’s main airport is located 47 km northeast of

Oslo. There are express trains with frequent

departures to the Oslo Central Station. Follow the

signs at the airport to find the express train.

Moss Airport Rygge (RYG)

Rygge airport is located 66 km south-east of Oslo.

Take “Rygge-ekspressen” to the main bus terminal

in Oslo, Oslo Bussterminal.

If you arrive on Wednesday afternoon, take either

“Rygge-ekspressen” or “Flybussekspressen”

(towards Oslo), and alight at the stop

“Korsegården” which is closest to the university.

Upon arrival, please contact one of the organisers

to be picked up there.

Sandefjord Airport Torp (TRF)

110 km south-west of Oslo, about 2 hours by

bus/train. Bus transport to the main bus terminal

in Oslo, Oslo Bussterminal, is available at the

airport.

Public transport

You can plan your travel by public transport

(including bus, train, tram, metro and ferry) on

http://ruter.no/en/. Connecting services are

shown on the website between the departure and

arrival point for any two points in Oslo and

Akershus. The website also has maps, timetables

and ticket fares.

Venue: Thon Hotel Opera

Thon Hotel Opera is situated across the street

from Oslo Central Station and 10 minutes’

walking distance from Oslo Bus Terminal (see

map below).

For more information about Thon Hotel Opera

(including driving directions), please see:

http://www.thonhotels.com/hotels/countrys/nor

way/oslo/thon-hotel-opera/

4

17th European Nitrogen Cycle Meeting, Oslo 2012

Programme

Wednesday, 26 September 2012

Location: Norwegian University of Life Sciences (UMB), Ås

1700 Shuttle bus from Thon Hotel Opera to UMB

1730 Registration, drinks and snacks

1900 Welcome address, Lars Bakken

1910

PT 01

The necessity of combining molecular biology / biochemistry with microbial ecology to

gain progress in the study of nitrogen cycling

Mary Firestone (UC Berkeley / US)

1940

PT 02

Nitrogen and atmosphere - perspectives from the European Nitrogen Assessment

(ENA)

Klaus Butterbach-Bahl (Karlsruhe Institute of Technology / DE)

2010 Dinner

2300 Departure by bus to Thon Hotel Opera, Oslo

- End of Day 1 -

5

17th European Nitrogen Cycle Meeting, Oslo 2012

Programme

Thursday, 27 September 2012

Location: Thon Hotel Opera, Oslo

0830 – 1030

Chair

Anoxic respiration, electron transport in membranes and beyond

David Richardson (University of East Anglia / UK)

0830

KN 01

Respiratory metabolism in anoxic sediments sustained by nanocables to oxic zones,

implications for N cycling

Lars Peter Nielsen (Aarhus University / DK)

0900

KN 02

Multi-heme cytochromes as electron conduits in the redox reactions of the nitrogen

and iron cycles

Tom Clarke (University of East Anglia / UK)

0930

CP 01

The last step of the denitrification pathway: Nitrous oxide reductase

Sofia Pauleta (REQUIMTE/CQFB, Universidade Nova de Lisboa / PT), et al.

0945

CP 02

Link between the C/N ratio and iron regulation in the alkaliphilic cyanotroph bacterium

Pseudomonas pseudoalcaligenes CECT 5344

Gracia Becerra (University of Extremadura / ES), et al.

1000

CP 03

Genomic analysis illuminates the evolution and metabolism of the major marine nitrite

oxidizer Nitrospina gracilis

Sebastian Lücker (University of Vienna / AT), et al.

1015

CP 04

Novel players in the cycling of methylated amines in the Movile Cave

Daniela Wischer (University of East Anglia / UK), et al.

1030 – 1115 Posters and light refreshments

6

17th European Nitrogen Cycle Meeting, Oslo 2012

PROGRAMME – THURSDAY, 27 SEPTEMBER 2012

1115 – 1500

Chair

Regulation of denitrification, experiments and models

Lars Bakken (Norwegian University of Life Sciences / NO)

1115

KN 03

The behaviour of a denitrifying cell; from experiment to model

Rob van Spanning (VU University Amsterdam / NL)

1145

CP 05

A numerical modelling-based inspection of the kinetics of oxic and anoxic respiratory

metabolism of Paracoccus denitrificans

Junaid Hassan (Norwegian University of Life Sciences / NO), et al.

1200

CP 06

Disparate response to low oxygen tension and nitrogen oxides of Bradyrhizobium

japonicum denitrification genes. Involvement of FixK2 and NnrR.

Emilio Bueno (Estación Experimental del Zaidín [EEZ, CSIC] / ES), et al.

1215

CP 07

RegSR-dependent expression of Bradyrhizobium japonicum norCBQD genes

María Torres (Estación Experimental del Zaidín [EEZ, CSIC] / ES), et al.

1230

CP 08

Transcriptomic analysis of Paracoccus denitrificans under denitrifying conditions

Matthew Sullivan (University of East Anglia / UK), et al.

1245 – 1400 Lunch

1400

CP 09

Intermediate and end products of nitrate reduction in Bacillus vireti vary depending on

nitrate levels; a presentation of phenotypic characteristics and genome analysis

Daniel Mania (Norwegian University of Life Sciences / NO), et al.

1415

CP 10

Regulation of N2O reduction in Bradyrhizobium japonicum mutants with increased N2O

reductase activity

Cristina Sánchez (Tohoku University / JP), et al.

1430

CP 11

Production of nitrogen oxides and its regulation in Thermus thermophilus

Carlos Bricio (Universidad Autónoma de Madrid [UAM, CSIC] / ES), et al.

1445

CP 12

Contrasting denitrification regulatory phenotypes in the genus Thauera revealed by gas

kinetics, transcription analysis and whole genome sequencing

Binbin Liu (Norwegian University of Life Sciences / NO), et al.

1500 – 1545 Posters and light refreshments

7

17th European Nitrogen Cycle Meeting, Oslo 2012

PROGRAMME – THURSDAY, 27 SEPTEMBER 2012

1545 – 1730

Chair

New pathways and organisms in N red/ox transformations

Mike Jetten (Radboud University Nijmegen / NL)

1545

KN 04

Anammox biology and biochemistry

Woter Maalcke (Radboud University Nijmegen / NL)

1615

KN 05

Thaumarchaeal ammonia oxidizers, ubiquity and function

Graeme Nichols (University of Aberdeen / UK)

1645

CP 13

Marine microbial ammonium conversion under oxygen-limited conditions

Suzanne Haaijer (University Nijmegen / NL), et al.

1700

CP 14

Various scenarios for dissimilatory nitrate reduction in Bacillus inferred from genome

analysis

Kim Heylen (Ghent University / BE), Paul DeVos

1715

CP 15

Community dynamic and quantification of anammox bacteria in a landfill-leachate

treatment plant

Yu-Tzu Huang (Chung-Yuan Christian University / TW), et al.

1730 – 1830 Posters, light refreshments & snacks

1830

CP 16

Anammox based single reactor systems for nitrogen removal from opto-electronic

wastewaters

Achlesh Daverey (National Chiao Tung University / TW), et al.

1845

CP 17

A nitric oxide-producing octaheme enzyme from Kuenenia stuttgartiensis

Wouter Maalcke (University Nijmegen / NL), et al.

1900

CP 18

Comparative genomics of two independently enriched ‘Candidatus Kuenenia

stuttgartiensis’ anammox bacteria

Daan Speth (Radboud University Nijmegen / NL), et al.

1915

CP 19

Role of nitriles in cyanide assimilation

Victor Luque-Almagro (Universidad de Córdoba / ES), et al.

1930 – 2000 Free time

2000 Dinner

- End of Day 2 -

8

17th European Nitrogen Cycle Meeting, Oslo 2012

Programme

Friday, 28 September 2012

Location: Thon Hotel Opera, Oslo

0830 – 1000

Chair

NO- biology, toxicity, mechanisms of reduction, role in regulation

Jeff Cole (University of Birmingham / UK)

0830

KN 06

Anaerobic bacterial response to NO generation, damage and repair

Charlene Bradley (University of Birmingham / UK), et al.

0900

CP 20

Nitric oxide detoxification and nitrate assimilation in Bradyrhizobium japonicum

Juan Cabrera (Estación Experimental del Zaidín [EEZ, CSIC] / ES), et al.

0915

CP 21

Nitrosative stress response in Paracoccus denitrificans

Katherine Hartop (University of East Anglia / UK), et al.

0930

CP 22

The production and detoxification of a potent cytotoxin, nitric oxide, by pathogenic

enteric bacteria

Sebastian Runkel (University of East Anglia / UK), et al.

0945

CP 23

Kinetics of NO scavenging in oxic and anoxic conditions, experimental approaches to

differentiate between chemical and enzymatic degradation

Shahid Nadeem (Norwegian University of Life Sciences / NO), et al.

1000 – 1100 Posters and light refreshments

9

17th European Nitrogen Cycle Meeting, Oslo 2012

PROGRAMME – FRIDAY, 28 SEPTEMBER 2012

1100 – 1245

Chair

N2O, formation and emission

Peter Dörsch (Norwegian University of Life Sciences / NO)

1100

KN 07

Identifying and quantifying biological source and sink processes of N2O using isotope

tracing and natural abundance techniques.

Reinhard Well (Johann Heinrich von Thuenen-Institute / DE), et al.

1130

CP 24

Aeration of boreal acid sulphate soils could result in enhanced fluxes of N2O and CH4

due to stimulation of potent microbial communities present in subsoil

Miloslav Šimek (Institute of Soil Biology, Biology Centre / CZ), et al.

1145

CP 25

Effect of intensified crop production on soil nitrous oxide emission in Mid-Hills of Nepal

Nani Raut (Norwegian University of Life Sciences / NO), et al.

1200

CP 26

High N2O emissions from an N saturated subtropical forested catchment, Southwest

China

Jing Zhu (Norwegian University of Life Sciences / NO), et al.

1215

CP 27

High potential for N2O emission from intensive Chinese agriculture due to soil

acidification

Zhi Qu (Norwegian University of Life Sciences / NO), et al.

1230

CP 28

Modelling the effect of Copper availability on bacterial denitrification

Hugh Woolfenden (University of East Anglia / UK), et al.

1245 – 1400 Lunch + posters

10

17th European Nitrogen Cycle Meeting, Oslo 2012

PROGRAMME – FRIDAY, 28 SEPTEMBER 2012

1400 – 1600

Chair

N transforming communities; functions and environmental controls

Åsa Frostegård (Norwegian University of Life Sciences / NO)

1400

CP 29

Unexpected diversity of nitrite oxidoreductase genes (nxrB) in marine waters:

indications for novel NOB?

Andreas Schramm (Aarhus University / DK), et al.

1415

CP 30

Biogeochemical nitrogen cycling in Arctic snowpacks: generating hypotheses from

multi-disciplinary studies

Catherine Larose (Université de Lyon / FR), et al.

1430

CP 31

Is there a direct link between denitrifier community structure or size and

denitrification activity in arable soil?

Miriam Herold (University of Aberdeen / UK), et al.

1445

CP 32

Microbial composition of various components of closed aquaculture systems: analysis

of biofilters and indogenous microbiota of fish intestines

Maartje van Kessel (Radboud University Nijmegen / NL), et al.

1500 – 1530 Refreshments

1530

CP 33

Differences in composition of active and overall denitrifier communities in three

organic soils determine responses of denitrification to pH

Kristof Brenzinger (Max Planck Institute for Terrestrial Microbiology / DE), et al.

1545

CP 34

Unusual acid-tolerant denitrifier communities in arctic permafrost soils associated with

N2O fluxes

Katharina Palmer (University of Bayreuth / DE), Marcus Horn

1600 Summing up and closing remarks

End of conference

- End of Day 3 -

11

17th European Nitrogen Cycle Meeting, Oslo 2012

Posters

P 01 Impact of loss of norEF in Rhodobacter sphaeroides 2.4.1

Linda Bergaust (Norwegian University of Life Sciences / NO), et al.

P 02 Abundance and diversity of nitrifying prokaryotes in drinking water treatment plant

Mei-Chun Chen (National Cheng Kung University / TW), et al.

P 03 The nitrite:nitrate oxidoreductase system of the anammox bacterium Kuenenia

stuttgartiensis

Naomi de Almeida (Radboud University Nijmegen / NL), et al.

P 04 Genetic and symbiotic diversity of rhizobia isolated from Ethiopian soils

Tulu Degefu (Norwegian University of Life Sciences / NO), et al.

P 05 Diversity of nitrogen fixing microorganisms from a saline-alkaline former lake Texcoco,

México

Cristina Domínguez-Mendoza (Cinvestav / MX), et al.

P 06 Strong reduction of N2O emissions by long term liming in a drained organic soil

Peter Dörsch (Norwegian University of Life Sciences / NO), et al.

P 07 Identification of key player groups of denitrifiers in an organic agricultural soil by high-

throughput sequencing techniques

Binbin Liu (Norwegian University of Life Sciences / NO), et al.

P 08 The effect of pH and oxygen on nitrous oxide emissions from the denitrifying prokaryote

Paracoccus denitrificans 1222

George Giannopoulos (University of East Anglia / UK), et al.

P 09 Identification of controlling factors of Nitrous Oxide emission for a drained spruce forest on

peat soil, a modeling approach

Hongxing He (University of Gothenburg / SE), et al.

P 10 Study of nitrification inhibitors on ammonia oxidizing archaeal community

Yu-Zen Huang (National Cheng Kung University / TW), et al.

P 11 Regulatory effect of soil properties on N2O emission from wheat-growing season in five

soils: field and pot experiment

Ulrike Lebender (Research Center Hanninghof, YARA GmbH & Co. KG / DE), Mehmet Senbayram

12

17th European Nitrogen Cycle Meeting, Oslo 2012

POSTERS

P 12 Denitrifying bacteria produce much N2O at low pH, and we are beginning to understand

why

Åsa Frostegård (Norwegian University of Life Sciences / NO), et al.

P 13 Nitrous oxide turnover in the nitrate-ammonifying Epsilonproteobacterium Wolinella

succinogenes

Monique Luckmann (Technische Universität Darmstadt / DE), Jörg Simon

P 14 Spatial patterns of soil pH and N2O emissions in field experiments

Aina Lundon (Norwegian Institute for Agricultural and Environmental Research / NO), et al.

P 15 A nitric oxide-producing octaheme enzyme from Kuenenia stuttgartiensis

Wouter Maalcke (Radboud University Nijmegen, The Netherlands), et al.

P 16 The purple form of Marinobacter hydrocarbonoclasticus nitrous oxide reductase

Sofia Pauleta (REQUIMTE/CQFB, Universidade Nova de Lisboa, Portugal), et al.

P 17 Autotrophic carbon dioxide fixation via the Calvin-Benson-Bassham cycle by the

denitrifying anaerobic methanotroph Candidatus Methylomirabilis oxyfera

Olivia Rasigraf (Radboud University Nijmegen / NL), et al.

P 18 Isotopomer ratios of N2O produced during denitrification by fungal pure cultures and

associated oxygen exchange with water

Lena Rohe (Johann Heinrich Thuenen-Institute / DE), et al.

P 19 Regulation of denitrification: pathways to nitrous oxide and dinitrogen

Surinder Saggar (Landcare Research / NZ), et al.

P 20 Off-season N2O losses from clover-rich swards

Ievina Sturite (Norwegian Institute for Agricultural and Environmental Research / NO), et al.

13

17th European Nitrogen Cycle Meeting, Oslo 2012

Abstracts

Presentation abstracts Poster abstracts

14

17th European Nitrogen Cycle Meeting, Oslo 2012

Presentation abstracts

In order of presentation

CP 01

The last step of denitrification pathway: Nitrous oxide reductase

S.R. Pauleta1, S. Dell'Acqua1,2, J.J.G. Moura1, I. Moura1,* 1REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-

516 Caparica, Portugal 2Dipartimento di Chimica, Università di Pavia, Via Taramelli 12, 27100 Pavia, Italy

Email: isabelmoura@fct.unl.pt

The final step of bacterial denitrification, the two electron reduction of N2O to N2 is catalyzed by a

multicopper enzyme named nitrous oxide reductase (N2OR). The catalytic center of this enzyme is a

tetranuclear copper site called CuZ, which is unique in biological systems.

The structure of CuZ center opened a novel area of research in metallobiochemistry. In the last

decade, there has been progress in defining the structure of the CuZ center, characterizing the

mechanism of nitrous oxide reduction, and identifying intermediates of this reaction.

The crystal structure of N2OR from Marinobacter hydrocarbonoclasticus was solved to a resolution

of 2.4 Å. This enzyme contains one binuclear (CuA, the electron transfer center) and a tetranuclear

copper center (CuZ, the catalytic center). CuZ center is a new type of cluster, in which four copper

ions are coordinated by seven histidine residues. In addition, the determination of the structure of

the CuZ center allowed a structural interpretation of the spectroscopic data, which was supported by

theoretical calculations. The current knowledge of the structure, function, and spectroscopic

characterization of the nitrous oxide reductase will be addressed in this talk. Although many

questions have been answered about this enzyme it remains a scientific challenge, with many

hypothesis being formed.

Acknowledgements: We thank the many contributions from BIOIN and BIOPROT at REQUIMTE, and FCT-MCTES for

financial support (PTDC/QUI-BIQ/116481/2010).

References: Tavares et al. JIB 100 (2006) 2087-2100. Dell’Acqua et al. JBIC 16 (2011) 183-194. Dell’Acqua et al. JBIC 16

(2011) 1241-1254. Dell’Acqua et al. Philos. Trans. R. Soc. B 367 (2012) 1204-1212

15

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 02

Link between the C/N ratio and iron regulation in the alkaliphilic cyanotroph

bacterium Pseudomonas pseudoalcaligenes CECT 5344

Gracia Becerra, Rafael Blasco, Faustino Merchán, M. Isabel Igeño Departamento de Bioquímica y Biología Molecular y Genética. Facultad de Veterinaria. Universidad de Extremadura.

Avenida de la Universidad s/n.. 10003. Cáceres. Spain

Bacterial ferric uptake regulator (Fur) protein is the major sensor of cellular iron status. A gene

coding for a putative Fur has been identified in the genome of Pseudomonas pseudoalcaligenes CECT

5344. When iron is limiting for growth, Fur derepression increases the cellular capacity for iron

uptake and launches an iron-saving reaction mediated by the small noncoding RNA prrF, homolog to

RyhB, identified in Escherichia coli, where it has been demonstrated that negative regulation of

genes by RyhB is responsible for the positive regulation of some genes by Fur. RyhB RNA negatively

regulates some tricarboxylic acid cycle (TCA) genes, as Fe-S containing fumarase, aconitase and

succinate dehydrogenase, by pairing with their mRNA and causing rapid degradation of the mRNA

(Massé.et al, 2003 Genes Dev17 2374-2383). Computational prediction of PrrF targets in P.

pseudoalcaligenes CECT 5344 allowed to infer as potential targets gltD,B genes encoding for both

subunits of glutamate synthase iron–sulfur containing protein (GOGAT). α–ketoglutarate is a TCA

intermediate and substrate of GOGAT, thus serving as a central link between carbon and nitrogen

metabolism. In this work, we inactivated the fur gene and the resultant mutant showed a slow

growing phenotype and decreased fumarase and aconitase activities. Furthermore, prrF and fiuA

(ferrichrome receptor) expression was constitutive and independent of the iron concentration in the

culture media. Ammonia assimilatory enzymes were also analyzed in the context of Fur regulation.

Work funded by MICINN BIO2011-30026-C02-01, GR10165-CCV019, PRI07A097, FEDER 2007-2013.G. Becerra holds a

Gobierno Extremadura fellowship (Fondo Social Europeo).

16

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 03

Genomic analysis illuminates the evolution and metabolism of the major marine

nitrite oxidizer Nitrospina gracilis

Sebastian Lücker1, Boris Nowka2, Eva Spieck2, Michael Wagner1 and Holger Daims1 1Department of Microbial Ecology, University of Vienna, Austria

2Microbiology and Biotechnology, University of Hamburg, Germany

In marine systems, nitrate is the major reservoir for inorganic nitrogen. The only biological nitrate-

forming reaction is nitrite oxidation, but knowledge of the organisms catalyzing this key process of

the marine nitrogen cycle is limited. Even in oxygen minimum zones (OMZs) a large nitrification

potential is present and nitrite-oxidizing bacteria (NOB) are active and can compete for nitrite. This

recent finding was surprising since nitrite oxidation was believed to be a strictly oxygen-dependent

process. The key marine NOB is Nitrospina gracilis, an aerobic chemolithoautotrophic bacterium

isolated from ocean surface waters, but also abundant in OMZs. To date, few physiological and no

genomic data for this enigmatic organism were available and its phylogenetic affiliation was

uncertain.

To bridge this knowledge gap, we sequenced Nitrospina gracilis Nb-211. Unexpectedly, Nitrospina

lacks the classical genes for reactive oxygen defense mechanisms and utilizes the reverse carboxylate

cycle for carbon fixation. Genes for utilizing simple organic carbon sources were not identified, but

molecular hydrogen might serve as source of low potential electrons. Nitrospina further contains all

genes for gluconeogenesis, oxidative phosphorylation, and reverse electron transport. Interestingly,

comparative genomics revealed a strong evolutionary link to the genus Nitrospira and key genes

involved in nitrite oxidation are highly conserved between Nitrospina, Nitrospira, and anammox

bacteria. This indicates the occurrence of massive horizontal gene transfer at an early evolutionary

stage which spread the nitrite-oxidizing capability among these physiologically different organisms.

Further, detailed analyses using established marker genes finally shed light onto the phylogenetic

affiliation of Nitrospina within the Bacteria.

17

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 04

Novel players in the cycling of methylated amines in Movile Cave

Daniela Wischer1, Yin Chen2 and J. Colin Murrell1 1School of Environmental Sciences, University of East Anglia, Norwich, NR4 7TJ, UK

2School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK

Movile Cave in Southern Romania is an unusual underground ecosystem discovered less than thirty

years ago. Sealed off for over 5.5 million years, the cave harbours a remarkably complex population

of microorganisms, fungi and cave-adapted invertebrates, including many endemic species. In the

absence of light, and with no fixed carbon entering, all life in the cave is sustained by non-

phototrophic, microbial carbon fixation. Extensive microbial mats grow at the redox interface

between the reduced, sulfidic waters and the oxygenated atmosphere. Methylated amines are key

intermediates in the Movile Cave food web, produced during decomposition processes. In addition

to being methylotrophic substrates, methylated amines are also nitrogen source for many non-

methylotrophic bacteria. Combining DNA stable isotope probing with cultivation, we identified active

methylated amine-utilising bacteria in Movile Cave, uncovering novel methylotrophs of the genera

Catellibacterium, Mesorhizobium and Cupriavidus, as well as a diversity of non-methylotrophs

including Acinetobacter, Azospirillum and Rhodobacter as key players. Presently available functional

gene markers for methylamine-utilising bacteria target the mauA gene, encoding methylamine

dehydrogenase. These primers do not detect bacteria using methylamine as a nitrogen source only.

We designed primer sets targeting gmaS, encoding gamma-glutamylmethylamide synthetase, the

first enzyme of the recently characterised indirect methylamine oxidation pathway. The indirect

pathway was prevalent amongst both methylotrophic and non-methylotrophic isolates, as revealed

by PCR-based screening. The mauA gene was detected in a number of methylotrophic isolates,

generally in addition to gmaS. The new primers have been used successfully for identification of

methylamine-utilising bacteria from a range of environments.

18

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 05

A numerical modelling-based inspection of the kinetics of oxic and anoxic

respiratory metabolism of Paracoccus denitrificans

Junaid Hassan1*, I. David Wheat2, Lars R. Bakken1 1 Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, Ås, Norway

2 System Dynamics Group, University of Bergen, Norway

Denitrification sustains respiratory metabolism of some bacteria during anoxic spells. Since most

denitrifying organisms are non-fermentative, the denitrification enzymes must be synthesised using

the energy from oxic respiration. This implies that if the cells fail to synthesise these enzymes prior

to oxygen depletion, they may be entrapped in anoxia. This was hypothesised to be the case for the

majority of Paracoccus denitrificans cells in Bergaust et al’s (AEM 76:6387-6396) experiments. To

further investigate this, we set up a numerical model, which assumes that, once oxygen is low, the

transcription for denitrification enzymes is stochastic with a low probability for the cells to initiate it;

however, once initiated, it undergoes a fast autocatalytic process within each cell. The model

showed a good agreement between the simulated and experimental data for Pd, which lends strong

support to the Bergaust et al’s hypothesis.

The results have implications for interpretation of a number of experiments on regulatory biology of

Pd and possibly other denitrifying organisms. For instance, growth experiments with Pd have

demonstrated a long ‘lag-phase’ after rapid removal of oxygen, followed by a subsequent anoxic

growth (“diauxic lag”). Our simulations suggest that the ‘lag’ was a result of anoxic growth by a

minute fraction of the entire population.

Finally, the model-exercises generated a testable hypothesis that the initial levels of oxygen in a

medium should affect the onset as well as the rate of denitrification; if proven, this would explain

why oxygen is crucial for denitrifying organisms.

The observations indicate a ‘bet hedging strategy’ in P. denitrificans. A large part of the population

‘bets’ that the anoxic spell will be short. They save energy, but loose the bet if the anoxic spell lasts

for a long time.

19

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 06

Disparate response to low oxygen tension and nitrogen oxides of

Bradyrhizobium japonicum denitrification genes. Involvement of FixK2 and NnrR.

Emilio Bueno1, María J Torres1, Eloy F Robles1, Cristina Sánchez1, Tino Krell2, Eulogio J Bedmar1,

Socorro Mesa1, and María J Delgado1 1Departamento de Microbiología del Suelo y Sistemas Simbióticos

2Departamento de Protección Ambiental. Estación Experimental del Zaidín, CSIC, P.O. Box 419, 18080-Granada, España

Bradyrhizobium japonicum can reduce nitrate to molecular nitrogen by the denitrification process in

free-living conditions and in symbiotic association with soybean plants. In B. japonicum,

denitrification depends on napEDABC, nirK, norCBQD and nosRZDYFLX genes which encode the

enzymes nitrate, nitrite, nitric oxide and nitrous oxide reductases, respectively. It has been

previously reported that expression of nap, nirK, and nor genes under denitrifying conditions

depends on the FixLJ-FixK2-NnrR regulatory cascade. Moreover, a genome-wide transcription

profiling of a fixK2 mutant strain grown under free-living micro-oxic conditions showed that nap, nirK,

and nnrR genes, but not nor genes, are targets of the FixK2 regulatory protein. In support of these

observations, we show in this work that purified FixK2 activated transcription from nap and nirK

promoters but not from the nor promoter. In contrast, by using isothermal titration calorimetry,

NnrR bound specifically to the promoter region of the nor genes, but not to those of the nap or nirK

genes. Furthermore, -galactosidase activity of napE-lacZ, nirK-lacZ and norC-lacZ fusions, haem c-

staining analyses and nitrate and nitrite reductase activity assays of cells growing in complete

medium showed that, whereas micro-oxic conditions are sufficient to significantly induce nap and

nirK expression, nor requires low oxygen and the presence of a nitrogen oxide, presumably nitric

oxide. Taken together, these results suggest a disparate regulation of nap, nirK and nor genes of B.

japonicum with regard to their dependence on oxygen and nitrogen oxides. In this control, FixK2

activates expression of nap, nirK and nnrR genes in response to micro-oxia, and NnrR induces

expression of nor genes in response to NO.

Acknowledgements. This work was supported by Fondo Europeo de Desarrollo Regional co-financed grants from Junta

de Andalucía (Spain) [grant number CVI-3177] and from Ministerio de Economía y Competitividad (Spain) [grant

numbers AGL2010-18607 and AGL2011-23383]. Support from the Junta de Andalucıa to Group BIO-275 is also

acknowledged. Emilio Bueno was supported by a Postdoctoral contract from the Consejo Superior de Investigaciones

Cientificas, JAE Programme.

20

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 07

RegSR-dependent expression of Bradyrhizobium japonicum norCBQD genes

María J Torres, Socorro Mesa, Eulogio J Bedmar and María J Delgado

Estación Experimental del Zaidín, C.S.I.C., P. O. Box 419, 18080-Granada, Spain

Bradyrhizobium japonicum is a gram-negative soil bacterium associated symbiotically with soybean

plants which is also able to denitrify under free-living and symbiotic conditions. In this bacterium, the

napEDABC, nirK, norCBQD and nosRZDYFLX genes which encode reductases for nitrate, nitrite, nitric

oxide and nitrous oxide, respectively, are required for denitrification. B. japonicum RegSR proteins

belongs to the family of two-component regulatory systems present in a large number of

proteobacteria and they globally control gene expression, mostly in a redox-responsive manner. In

this work, we have performed transcription profiling of wild-type and regR mutant cells grown under

denitrifying conditions in minimal medium supplemented with succinate as carbon source. The

comparative analyses of WT and regR revealed that expression of nor and nos genes, among others,

is dependent on RegR. Furthermore, by using fluorescently labeled oligonucleotide extension, we

were able to identify two transcriptional start sites located at about 35 (P1) and 22 (P2) bp from the

putative translational start codon of NorC. The areas under the peaks let us to conclude that P2 is

the principal transcription start site and is modulated by RegR. Moreover, qRT-PCR experiments, β-

galactosidase activity assays of a norC-lacZ fusion and haem c staining analyses of regS and regR

mutants, revealed that the RegR control of nor genes is independent of the sensor protein RegS.

Taking together, these results clearly demonstrate the involvement of RegR in the control of nor

genes expression. Up to date, the exact mechanism how RegR exerts this control remains enigmatic.

Acknowledgements. This work was supported by Fondo Europeo de Desarrollo Regional co-financed grants from Junta

de Andalucía (Spain) [grant number CVI-3177] and from Ministerio de Economía y Competitividad (Spain) [grant numbers

AGL2010-18607 and AGL2011-23383]. Support from the Junta de Andalucıa to Group BIO-275 is also acknowledged.

Maria J. Torres was supported by a fellowship from the Consejo Superior de Investigaciones Cientificas JAE Programme.

21

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 08

Transcriptomic analysis of Paracoccus denitrificans under denitrifying conditions

Matthew J Sullivan1, Andrew J Gates1, Gary Rowley1, David J Richardson1 1 CMSB, School of Biological Sciences, University of East Anglia, Norwich Research Park, UK, NR4 7TJ

Molecule for molecule, the greenhouse gas nitrous oxide (N2O) is 300 times more effective at

insulating our climate than is CO2. N2O is very stable (~120 years) and contributes to ~ 9% of global

radiative forcing by greenhouse gases. Approximately 70% of anthropogenic loading of N2O arises

from agricultural practices, primarily through the use of nitrogenous fertilizers (e.g. NH4NO3) by soil

microbes. In the absence of oxygen the model denitrifier, Paracoccus denitrificans, converts nitrate

(NO3-) to di-nitrogen (N2) via four distinct reductases, the last of which is the Cu-containing nitrous

oxide reductase (NosZ). In copper limited conditions P. denitrificans will release gaseous N2O, since

Cu is required for synthesis of functional NosZ. Using batch culture experiments and microarray

analyses, we demonstrate that the structural genes required for N2O reduction, nosZDFLYX, are

subject to a novel form of transcriptional regulation, in addition to their control by the oxygen- and

nitric oxide-sensing FnrP and NnrP. Furthermore, we reveal a sub-set of gene clusters that are up-

regulated in the presence of N2O, some of which may impact significantly on growth in anaerobic

conditions.

22

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 09

Intermediate and end products of nitrate reduction in Bacillus vireti vary depending

on nitrate levels; a presentation of phenotypic characteristics and genome analysis

Daniel Mania1, Kim Heylen2, Ines Verbaendert2, Lars Bakken3 and Åsa Frostegård1 1 Dept Chemistry, Biotechnology and Food Science. Norwegian University of Life Sciences, Norway

2 Dept Biochemistry and Microbiology, Ghent University, Belgium

3 Dept Plant and Environmental Sciences. Norwegian University of Life Sciences, Norway

Endpoint analysis showed that Bacillus vireti LMG 21834Tproduces N2 when grown anaerobically in

presence of nitrate. A more detailed analysis of the gas kinetics using a robotized incubation system

demonstrated that the proportion of nitrate reduced to N2 increased with increasing initial nitrate

levels. At 5 mM initial nitrate, <10% of the nitrate was reduced to N2 while the main part was

reduced to ammonium. The highest proportion of nitrate reduced to N2 was 50%, found at 20 mM

initial nitrate concentration. The cultures reduced all available nitrate to nitrite (up to 50 mM NO2-)

before initiating a reduction of NO2-. In all cases, growth slowed down once all nitrate was respired to

nitrite. B. vireti has an apparently stringent control of NO production with a short peak of NO never

exceeding 800 nM in the liquid. Production of N2O was less controlled and the proportion of N2O

increased with NO3- concentration in the medium. N2O was the predominant endpoint product when

initial NO3- concentrations exceeded 30 mM. Whole genome sequencing confirmed the presence of

the general transcriptional regulator fnr, the narGHJI operon for nitrate reduction and narK encoding

nitrate uptake/nitrite excretion, and narX and narL encoding nitrate sensing regulators. None of the

typical denitrification genes for reduction of nitrate (nir) and NO (nor) were found. The genes

encoding the nitrite reductase subunits nrfA and nrfH, characteristic of DNRA organisms, may release

NO under stress conditions like high nitrite concentrations. An hmp gene was identified, and we

expect that it is involved in the reduction of NO to N2O. This is supposedly a non-energy yielding

process, which would explain the slowdown of growth after reduction of nitrate to nitrite. B. vireti

carries nosZ, encoding N2O reductase, and several genes involved in the maturation of this enzyme,

thereby allowing a kind of full denitrification from nitrate to N2. We are at present investigating to

what extent the N2O reduction may sustain growth by this organism.

23

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 10

Regulation of N2O reduction in Bradyrhizobium japonicum mutants with increased

N2O reductase activity

Cristina Sánchez, Manabu Itakura, Yusuke Uetake, Shima Eda, Hisayuki Mitsui and Kiwamu

Minamisawa

Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan

Nitrous oxide (N2O) is a key atmospheric green house effect gas that not only affects global warming but

also leads to the destruction of the ozone layer. Emissions from agricultural fields are a major source of

N2O through the microbial transformation of nitrogen in soil. Previous studies indicated that soybean

nodules emit N2O under field conditions in the late growth period.

In the strain USDA110 of the soybean symbiont Bradyrhizobium japonicum, the genes encoding N2O

respiration functions are clustered in a single locus: nosRZDFYLX. Previously, a B. japonicum mutant

(5M09) with increased N2O reductase (N2OR) activity was isolated by introducing a plasmid containing a

mutated B. japonicum dnaQ gene (impairing proofreading activity) and then performing enrichment

culture under selection pressure for N2O respiration.

Free-living and symbiotic cells of strain 5M09 showed higher N2O reductase activity (5- and 2-times,

respectively) than USDA110 strain under N2O-respiring conditions. Expression of nosZ (which encodes

N2OR) was analyzed in strain 5M09 under free-living aerobic and anaerobic conditions (anaerobic,

nitrate-denitrifying or N2O-respiring conditions). The results showed that constitutive expression of nosZ

gene (aerobic conditions) was increased in strain 5M09. Similarly, under anaerobic and N2O-respiring

conditions, expression of nosZ was higher in strain 5M09. These results suggest an alteration in the

regulatory mechanisms of nosZ expression in strain 5M09, perhaps by derepression. In cells grown

anaerobically in the presence of nitrate, no difference in nosZ expression was observed between strains

USDA110 and 5M09, suggesting a similar response to nitrogen-oxides.

The mechanisms underlying the induction of N2OR expression and activity in strain 5M09 are currently

being investigated.

24

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 11

Production of nitrogen oxides and its regulation in Thermus thermophilus

Carlos Bricio, Laura Álvarez, Alba Blesa, Carolina E. César, Aurelio Hidalgo, José Berenguer Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-CSIC. 28049-Madrid, Spain

Several strains of Thermus thermophilus are able to reduce nitrogen oxides under anaerobic

conditions to gain energy in a process that frequently ends up with the production of gaseous forms

of nitrogen that escapes to the atmosphere. Reduction of nitrate is encoded by a conjugative

element (NCE) (Cava et al., 2007), which contains the genes for a tetrameric nitrate reductase, one

or two nitrate/nitrite transporters, a NADH dehydrogenase and a sensory system that signals the cell

to switch between aerobic and anaerobic metabolism. Reduction of nitrite and nitric oxide is

managed by a cd1 type nitrite reductase (Nir) and a nitric oxide reductase (Nor) encoded

downstream of the NCE by a nor-nir cluster, which also includes a group of putative regulators of the

denitrification pathway (Alvarez et al., 2011; Bricio et al., 2011). When present, both the NCE and the

nor-nir clusters are unevenly associated to a highly variable region of a megaplasmid pTT27 like of

different T. thermophilus from which it can be mobilized to other strains of the species. Up to now,

no homologues to conventional nitrous oxide reductase (NosZ) has been found in the genome of

denitrificant strains of this species, suggesting that nitrous oxide is the likely final product of the

process.

For a better understanding of the whole denitrification pathway in the species T. thermophilus, 7

partial or complete denitrificant strains were sequenced. Production and consumption of nitric oxide

in living cells or with different cell extracts, and insertional mutagenesis have been developed to

analyze and confirm or sequence-based hypothesis.

25

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 12

Contrasting denitrification regulatory phenotypes in the genus Thauera revealed by

gas kinetics, transcription analysis and whole genome sequencing

Binbin Liua, Yuejian Maoa,d, Linda Bergausta,b, James P Shapleighc, Lars Reier Bakkenb and

Åsa Frostegårda a Department of Chemistry, Biotechnology and Food Sciences, Norwegian University of Life Sciences, Ås, Norway

b Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, Ås, Norway

c Department of Microbiology, Cornell University, Ithaca, USA

d Key Laboratory of Microbial Metabolism, Ministry of Education, College of Life Science and Biotechnology, Shanghai

Jiaotong University, Shanghai, China

Denitrifiers differ in how they handle transition from oxic to anoxic respiration, with consequences

for NO and N2O emissions. Systematic studies of different organisms are scarce. To enable stringent

comparisons we defined parameters to describe the Denitrification Regulatory Phenotypes (DRP)

based on accumulation of NO2-, NO and N2O, oxic/anoxic growth and transcription of functional

genes. We here present a comparison of eight Thauera strains. The strains divided into two distinct

DRP types. Four strains were characterized by a rapid, complete onset (RCO) of all denitrification

genes and no detectable nitrite accumulation. The others showed progressive onset (PO) of the

different denitrification genes. The PO-group reduced practically all available nitrate (2 mM) to

nitrite prior to transcription of nirS (coding for nitrite reductase), suggesting that nitrate has a role in

the regulating transcription of nirS. Organisms with this phenotype are likely sources of N2O

emissions in natural environments, under conditions allowing aerobic denitrification. All eight strains

controlled NO at nanomolar concentrations, possibly reflecting the importance of strict control for

survival. Transient N2O accumulation differed by two orders of magnitude, indicating that control of

N2O is less essential. No correlation was seen between phylogeny (based on 16S rRNA and functional

genes) and DRP. Whole genomes were sequenced for three RCO strains and three PO strains. Both

paired end and mate pair libraries were constructed, which produced around 120 million reads. The

genome sizes of the 6 strains are in the range of 4.1~4.9 million base pairs. About 3700~4500 CDSs

(coding sequences) were identified for each strain based on the RAST (Rapid Annotation using

Subsystem Technology) pipeline. Some distinct differences were found between the RCO and PO

groups with respect to regulatory genes involved in denitrification, as well as genes involved in

metabolism of aromatic compounds.

26

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 13

Marine microbial ammonium conversion under oxygen-limited conditions

Suzanne C.M. Haaijer1, Jia Yan1, Jaap S. Sinninghe Damsté2, Mike S.M. Jetten1, Stefan Schouten2,

Huub J.M. Op den Camp1

1Microbiology IWWR Radboud University Nijmegen, NL

2Biogeochemistry, NIOZ Texel NL

This project focuses on marine microbial ammonium oxidation under oxygen-limited conditions.

Under these conditions ammonium may be partially converted by aerobic ammonium oxidizers,

yielding nitrite which may serve as a substrate for anaerobic ammonium oxidizers. Although

nitrification was previously assumed to be mediated by bacteria, archaeal ammonium oxidizers

(AOA) have been postulated as key players.

Interactions and coexistence of aerobic and anaerobic ammonium oxidizers were investigated using

bioreactor experiments under oxygen limitation which resulted in 2 stable cocultures. The first

culture [1] consisted of respectively aerobic ammonium-oxidizing bacteria (AOB, Nitrosomonas sp.),

nitrite-oxidizing bacteria (NOB, Nitrospira sp) and Scalindua [2] anammox bacteria. The second

consisted of AOB, AOA (N. maritimus strain SCM1 [3]) and anammox bacteria. These experiments

indicated that AOA are able to coexist with anammox bacteria as well as that competition between

AOA and AOB is likely substrate-level dependent. In addition, enrichments of marine nitrifiers from

the North Sea were established. Highly enriched (>80%) cultures of marine ammonium-oxidizing

bacteria (AOB; a new Nitrosomonas species) and nitrite-oxidizing bacteria (NOB, a new species of

Nitrospira sp) were obtained. In addition, a North Sea AOA enrichment was obtained containing N.

maritimus-like AOA. This batch flask enrichment served as the inoculum for a bioreactor culture

operated under low oxygen and ammonium conditions. This bioreactor is expected to yield sufficient

North Sea AOA biomass to allow more in-depth physiological characterization (e.g. pH, T optima,

affinity constants for oxygen and ammonium) and allow for comparison of this AOA species to N.

maritimus strain SCM1.

[1] Yan J, Op den Camp HJM, Jetten MSM, Hu YY, Haaijer SCM. (2010) Induced cooperation between marine nitrifiers and

anaerobic ammonium-oxidizing bacteria by incremental exposure to oxygen. Syst Appl Microbiol. 33: 407-415

[2] Van de Vossenberg J, Rattray JE, Geerts W, Kartal B, Van Niftrik L, Van Donselaar EG, Damste JSS, Strous M, Jetten

MSM. (2008) Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas

production. Environ. Microbiol. 10, 3120-3129.

[3] Könneke M, Bernhard AE, de La Torre JR, Walker CB, Waterbury JB, Stahl DA (2005), Isolation of an autotrophic

ammonia-oxidizing marine archaeon. Nature 437: 543-546.

27

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 14

Various scenarios for dissimilatory nitrate reduction in Bacillus inferred from

genome analysis

Kim Heylen, Paul De Vos Laboratory of Microbiology, Ghent University, Belgium

Nitrous oxide continues to accumulate in the atmosphere as a product of anthropogenic activities

and land-use changes. Denitrification is considered to be the only ecologically relevant dissimilatory

nitrate reduction pathway responsible for nitrous oxide emission (next to aerobic nitrification).

Reports on nitrous oxide production during dissimilatory nitrate reduction to ammonium (DNRA)

already date back to several decades ago, but still relatively little consideration is given to this

pathway, its key genes/enzymes and its ecological role. We are currently investigating the role of

representative from the genus Bacillus in the nitrogen cycle. And because dissimilatory nitrate

reduction phenotype of completely sequenced strains is not always clear from annotation data or

literature, six strains were selected for genome sequencing (Illumina GAIIx, assembly with CLC

Genomics Workbench 4). Output from the RAST server, KAAS and NCBI’s PGAAP were compared and

used for functional annotation and reconstruction of the dissimilatory nitrate reduction pathways.

Two genomes (B. azotoformans LMG 9581T and B. bataviensis LMG 21833T) contained the

(complete) gene inventory for denitrification, but, in addition, also had nrfA genes, indicating the

potential to perform DNRA. In another genome (B. vireti LMG 21834T), only evidence for DNRA was

found, however together with a nosZ gene. And finally, in the genomes of three distinct strains of B.

licheniformis complete narGHIJ operons and a qnorB genes were found. In conclusion, these results

suggest a potentially complex and overlapping network of dissimilatory nitrate reduction scenarios

and indicate that nitrous oxide in Bacillus can be produced via either the classical denitrification

pathway or the one-electron reduction of nitrite by NarG to nitric oxide and further detoxification

with qNORB.

28

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 15

Community dynamic and quantification of anammox bacteria in a landfill-leachate

treatment plant

Yu-Tzu Huanga*, Shiou-Shiou Chen, Jih-Gaw Linb a

Department of Bioenvironmental Engineering, Chung-Yuan Christian University, 200 Chung Pei Road, Chung-Li 32023

Taiwan, ROC b

Institute of Environmental Engineering, National Chiao Tung University, 1001 University Road, Hsinchu City 30010,

Taiwan, ROC

Anaerobic ammonium-oxidizing (anammox) bacteria participate in a microbial nitrogen cycle

pathway that connects nitrite reduction and ammonium oxidation under anoxic conditions. To

detect anammox bacteria using molecular biology technique of polymerase chain reaction (PCR),

real-time quantitative PCR (qPCR) or fluorescence in situ hybridization (FISH), biomarkers such as 16S

rRNA, hzo and nirS were studied. In order to detect the specific species of anammox bacteria and

elucidate the dynamic shift of the anammox community corresponding to the operating condition of

the reactor, specific primers were designed to target two most common anammox species,

Candidatus Kuenenia stuttgartiensis (KS) and Candidatus Brocadia anammoxidans (BA).

Granular microbial sludge samples were collected and analyzed during a four-year period from a

simultaneous nitrification, annammox, and denitrification (SNAD) treatment plant treating landfill

leachate. Anammox bacteria were kept at relatively high concentration (1.59 x 107 to 6.58 × 108

copies μg-1 DNA). By applying the newly designed KS-specific primer (KSqF3/KSqR3) and BA-specific

primer (BAqF/BAqR), the estimated amount of KS and BA is in the range of 4.12 × 105 to 5.92 × 108

and 1.13 × 105 to 4.41 × 107 copies μg-1 DNA, respectively. KS was predominate species at all well

established anammox granules and played important role in formation of granules. KS/BA ratios

were positively correlated to the ammonia nitrogen removal efficiency of the treatment plant.

29

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 16

Anammox based single reactor systems for nitrogen removal from opto-electronic

wastewaters

Achlesh Davereya, Sin-Han Sua, Yu-Tzu Huangb, Jih-Gaw Lina* a

Institute of Environmental Engineering, National Chiao Tung University, 1001 University Road, Hsinchu City 30010,

Taiwan, ROC b

Department of Bioenvironmental Engineering, Chung-Yuan Christian University, 200 Chung Pei Road, Chung-Li 32023

Taiwan, ROC

Anammox is a nitrogen removal process with many advantages over traditional nitrification and

denitrification process. Anammox bacteria use nitrite as an electron acceptor to convert ammonia

into nitrogen gas and therefore, anammox process is generally combined with partial nitrification

process. Simultaneous partial nitrification, anammox and denitrification (SNAD) and completely

autotrophic nitrogen removal over nitrite (CANON) are two single reactor anammox based

processes. In this study, SNAD and CANON processes were developed to treat two different

optoelectronic industry wastewaters in lab scale sequential batch reactors.

The characteristics of wastewater treated by SNAD process were low C/N ratio (~0.2) with

100 mg-COD/L and 567 mg NH4+-N/L. The experiment was carried out over eight months, where

nitrogen loading rate was gradually increased to 230 g-N/m3·d. The results showed that COD and

nitrogen removal rates by the SNAD system reached to 28 g COD/m3·d and 197 g NH4+-N/m3·d,

respectively in final stage of the process.

The wastewater treated by CANON process had very high ammonium concentration

(3,600 mg NH4+-N/L) with no COD. Lab scale 18 L sequential batch reactor (SBR) was used to study

the wastewater treatment over 450 d (15 months). Around 95% NH4+-N removal and 90% total

nitrogen removal were observed at 4 d HRT with nitrogen loading rate of 909 g/m3/d.

Analysis of microbial communities (nitrifiers, anammox and denitrifiers bacteria) in the reactors was

also carried out by polymerase chain reaction.

30

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 17

A nitric oxide-producing octaheme enzyme from Kuenenia stuttgartiensis

WJ Maalcke1, A Dietl2, SJ Marritt3, MSM Jetten1, JTM Keltjens1, JN Butt3, TRM Barends2, B Kartal1 1 Department of Microbiology, Radboud University Nijmegen, Heijendaalseweg 135, 6525 AJ, Nijmegen, The Netherlands

2 Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120,

Heidelberg, Germany 3 School of Chemistry, University of East Anglia, Earlham Park, Norwich, NR4 7TJ, United Kingdom

Anaerobic ammonium-oxidizing (anammox) bacteria combine ammonium and nitrite to form

dinitrogen gas. Anammox bacteria are abundantly present in the oxygen minimum zones of the

world’s oceans and have a significant role in global dinitrogen gas production. They are also

successfully applied in cost-effective and environment-friendly ways to treat industrial and municipal

wastewater. Although the physiology of anammox bacteria is quite well understood, the

biochemistry behind it is less clear. One of the persisting anammox enigmas is the presence of ten

copies of an open reading frame that resembles the octaheme enzyme hydroxylamine

oxidoreductase (HAO). HAO converts hydroxylamine to nitrite in aerobic ammonium oxidizing

bacteria. The function of one of these enzymes (kustc0694) is predicted to be hydrazine oxidation to

dinitrogen gas, whereas the functions of the other nine octaheme proteins remain to be established.

Here, a highly expressed octaheme protein was purified from native Kuenenia stuttgartiensis

biomass and characterized. MALDI-TOF mass spectrometry confirmed that the enzyme was encoded

by the open reading frame kustc1061. Enzyme kinetics, assessed by using cytochrome c-based assays

and gas chromatography coupled to mass spectrometry, showed kustc1061 to be a hydroxylamine

oxidizing enzyme that produces nitric oxide instead of nitrite. Protein film electrochemistry revealed

the midpoint potentials of the eight separate heme-groups, and elucidation of the crystal structure

enabled us to form a hypothetical mechanism of hydroxylamine oxidation to nitric oxide. As the

proposed hydrazine-oxidizing enzyme is inhibited by hydroxylamine, the physiological role of

kustc1061 is proposed to be hydroxylamine detoxification

31

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 18

Comparative genomics of two independently enriched ‘Candidatus Kuenenia

stuttgartiensis’ anammox bacteria

Daan R. Speth1, Baolan Hu2, Niek Bosch1, Jan G. Keltjens1, Henk G. Stunnenberg3, Mike S. M.

Jetten1, 4 1 Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, Nijmegen, The

Netherlands 2 Department of Environmental Engineering, Zhejiang University, Hangzhou, China.

3 Department of molecular biology, NCMLS, Radboud University Nijmegen, Nijmegen, The Netherlands

4 Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

Bacteria capable of anaerobic oxidation of ammonium (anammox) have many unique features, such

as the conversion of hydrazine, an anammoxosome compartment and ladderane lipids. Although

their core metabolic pathway is largely resolved1, many questions still remain. Data mining of the

(meta)genomes of anammox bacteria is one of the powerful methods to address these questions or

identify targets for further study. The availability of high quality reference data greatly aids such

analysis. Currently, only a single high quality reference genome is available; that of model organism

‘Kuenenia stuttgartiensis’2.

We present a comparative genomic analysis of two ‘K. stuttgartiensis’ bacteria that were

independently enriched aiming to further improve the reference genome. The two bacteria used are

‘K. stuttgartiensis’ RU1, which was originally sequenced for the reference genome, and for the

present study resequenced after seven (2002-2009) years in continuous culture. Furthermore

‘K. stuttgartiensis’ CH1, enriched from a Chinese wastewater treatment plant was used as an

independent source of genomic information.

The two different ‘Kuenenia’ bacteria showed a very high sequence identity (> 99 % at nucleotide

level) over almost the entire genome, but 31 genomic regions (average size 11 kb) were absent from

strain CH1 and 220 kb of sequence was specifically found in the CH1 assembly. Moreover,

comparative analysis of the RU1 and CH1 assemblies led to identification 49 additional genes absent

from the reference genome, including two additional ammonium transporters. Future comparison

will include the metagenomes of Scalindua profunda3 and anammox strain KSU-1.

1. Kartal, B. et al. Molecular mechanism of anaerobic ammonium oxidation. Nature 479, 127–130 (2011).

2. Strous, M. et al. Deciphering the evolution and metabolism of an anammox bacterium from a community genome.

Nature 440, 790–794 (2006).

3. van de Vossenberg, J. et al. The metagenome of the marine anammox bacterium “Candidatus Scalindua profunda”

illustrates the versatility of this globally important nitrogen cycle bacterium. Environmental Microbiology no–no

(2012).doi:10.1111/j.1462-2920.2012.02774.x

32

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 19

Role of nitriles in cyanide assimilation

Victor M. Luque-Almagro, Isabel Manso, Jessica Estepa, M. Paz Escribano, Francisco Castillo,

Conrado Moreno-Vivián, M. Dolores Roldán Departamento de Bioquímica y Biología Molecular. Campus de Rabanales. Universidad de Córdoba. Córdoba, Spain

The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 is able to grow with cyanide as

the sole nitrogen source. Under cyanotrophic conditions, an inducible cyanide-insensitive alternative

oxidase is coupled to a malate:quinone oxidoreductase activity that oxidizes L-malate to

oxaloacetate. Oxaloacetate reacts chemically with cyanide to produce a cyanohydrin (2-

hydroxynitrile), which may be further converted into ammonium. Two cyanide-induced proteins of

unknown function identified by proteomic analysis are encoded by the nitB and nitG genes belonging

to the nit1C gene cluster in the strain CECT5344. In addition to NitB and NitG, the predicted products

of nit1C gene cluster are a Fis-like 54-dependent transcriptional activator (NitA), a nitrilase (NitC), an

S-adenosylmethionine superfamily member (NitD), an N-acyltransferase superfamily member (NitE),

a trifunctional polypeptide of the AIRS/GARS family (NitF), and an NADH-dependent oxidoreductase

(NitH). The P. pseudoalcaligenes CECT5344 nit1C gene cluster is involved in assimilation of free and

organic cyanides (nitriles) as deduced for the inability to grow with cyanide showed by the NitA, NitB

and NitC mutant strains. The wild-type strain CECT5344 shows nitrilase activity, which enables

growth on cyanide or hydroxynitriles, but the NitB and NitC mutants have only low basal levels of

nitrilase activity that are not enough to support growth on either free cyanide or nitriles. To

summarize, P. pseudoalcaligenes CECT5344 assimilates cyanide through a novel pathway that

includes the formation of 2-hydroxynitriles that are used as N-source with the involvement of the

nitrilase-encoding nit1C gene cluster.

33

17th European Nitrogen Cycle Meeting, Oslo 2012

KN 06

Anaerobic bacterial response to NO generation, damage and repair

Charlene Bradley, Vine C, Balseny B, Wang J, Cole J School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK

Bacteria are exposed to NO generated as an immediate product of nitrite reduction by denitrifying

bacteria, from arginine by the mammalian NO synthetase, or as a by-product during nitrate

reduction to ammonia. NO binds to di-iron and iron-sulphur clusters, inactivating many enzymes

including aconitase and fumarase.

The Escherichia coli transcription factors NorR and NsrR bind NO specifically, triggering responses to

nitrosative stress. NsrR is a repressor of genes required for NO removal or damage repair, including

the flavohemoglobin, Hmp, the hybrid cluster protein, Hcp, and the di-iron repair protein, YtfE . Hmp

is an NO oxygenase. Its expression is repressed by FNR, implying that it is not required during

anaerobic growth. In contrast, Hcp expression is activated by FNR, suggesting it is important under

anaerobic conditions. Hcp contains a [4Fe-2S-2O] hybrid cluster that is so far unique in evolution.

Unique structure implies unique function and gene regulation usually reflects metabolic function: we

will demonstrate that Hcp plays a critical role in the response to nitrosative stress. Repair of

nitrosative damage inevitably focuses on how iron-sulphur cluster proteins are repaired. The repair

protein, YtfE, has been reported to repair iron-sulphur clusters damaged by nitrosative stress. We

will report experiments to establish the mechanism of repair. Data from isogenic mutants that lack

all reported NO reductases suggest that there are multiple responses to nitrosative stress in E. coli.

The responses of E. coIi to nitrosative stress during aerobic and anaerobic growth will be compared

with those of other organisms.

34

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 20

Nitric oxide detoxification and nitrate assimilation in Bradyrhizobium japonicum

Juan J Cabrera1, Cristina Sanchez1, Andrew Gates2, Eulogio J Bedmar1, David J Richardson2, Maria J

Delgado1

1 Estación Experimental del Zaidín, CSIC, P. O. Box 419, 18080-Granada, Spain

2 School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK

Bradyrhizobium japonicum is a soil bacterium that establishes nitrogen-fixing symbiotic associations

with soybean plants and is also able to denitrify under free living and symbiotic conditions. In

B. japonicum, a single-domain haemoglobin (Bjgb) has been reported to be involved in NO

detoxification under free-living conditions (Sanchez et al., 2011 Biochem Soc Transac. 39 184-188).

This Bjgb is encoded by blr2807 that is located in a gene cluster (blr2803-09) where blr2803-05

encode an ABC-type nitrate transporter, blr2806 a nitrite extrusion protein, blr2808 a flavoprotein,

and blr2809 a assimilatory nitrate reductase. Functional and regulatory analyses of these genes are

presented this work. B. japonicum blr2807, blr2808 and blr2809 mutants showed a defect in their

ability to grow anaerobically with nitrate as sole nitrogen source. Furthermore, blr2808 and blr2809

mutants were also unable to grow with nitrate as sole nitrogen source under aerobic conditions and

they recovered growth when glutamic acid was added to the medium. In addition, blr2807- and

blr2808- mutants showed greater sensitivity to NO-generating compounds than the parental strain.

Reverse transcription-PCR analysis showed that blr2806-09 genes comprise a transcriptional unit.

-galactosidase activity of a transcriptional fusion of blr2806-09 promoter region to lacZ revealed

that nitrate is required to induce expression of these genes and the presence of glutamic acid

inhibits such expression. The presence of a NO donor increases expression of the blr2806-09 operon.

Taken together, these results suggest that the B. japonicum blr2803-09 gene cluster is involved in

nitrate assimilation and NO detoxification.

Acknowledgements. This work was supported by Fondo Europeo de Desarrollo Regional co-financed grants from Junta

de Andalucía (Spain) [grant number CVI-3177] and from Ministerio de Economía y Competitividad (Spain) [grant number

AGL2010-18607]. Support from the Junta de Andalucıa to Group BIO-275 is also acknowledged. Juan Cabrera was

supported by a fellowship from the Consejo Superior de Investigaciones Cientificas JAE Programme.

35

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 21

Nitrosative stress response in Paracoccus denitrificans

Hartop, K. R., Gates, A. J., Clarke, T. A. and Richardson, D. J. Centre for Molecular and Structural Biochemistry. School of Biological Sciences, University of East Anglia, Norwich

Research Park, Norwich, NR4 7TJ, U.K.

Nitrogen based compounds highly chemically reactive in solution, termed reactive nitrogen species

(RNS), generated in concentrations greater than can be neutralised by microorganisms induce

nitrosative stress in microorganisms leading to cytotoxic effects such as inhibition of cellular growth,

disruption of biofilm attachment and cell death. Increasing nitrite concentration changes the

phenotype of aerobic growth in Paracoccus denitrificans PD1222 causing an increase in lag phase, a

decrease in exponential growth rate and reduction of final OD600nm at stationary phase, until cell

growth is not observed. A pH effect is also present; exponential growth rate µmaxapp reduction as a

function of decreased pH, therefore reducing the concentration of nitrite at which toxicity occurs.

Toxicity is therefore postulated as linked to nitrite protonation: NO2- + H+ ↔ HNO2 (pka = 3.3),

forming free nitrous acid (FNA), where higher levels of FNA are present at low solution pH as defined

by the Henderson-Hasselbalch equation. With oxygen present, the anaerobic denitrification enzyme

pathway is down-regulated, thus the fate of nitrite under aerobic growth conditions is unclear. The

decomposition and disproportionation of FNA may generate cytotoxic reactive species such as nitric

oxide (NO) detrimental to cellular function. From which cellular detoxification may form nitrous

oxide (N2O). The focus of this work is to investigate aerobic nitrosative stress response on a microbial

and transcriptional level in P. denitrificans to assess its impact on FNA, NO and N2O generation and

cellular growth in both batch and continuous chemostat cultures.

36

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 22

The production and detoxification of a potent cytotoxin, nitric oxide, by pathogenic

enteric bacteria

Sebastian Runkel, Corinne Appia-Ayme, Anke Arkenberg, Elaine Patrick, Hannah Wells,

David J. Richardson and Gary Rowley School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, Norfolk NR4 7TJ, U.K.

Escherichia coli and Salmonella are facultative anaerobes; using O2 as their preferred energy source.

However, during O2 shortage, they can switch to nitrate respiration to maintain their metabolism in a

process called denitrification. Unlike many soil bacteria, Salmonella and E. coli undergo only

truncated denitrification, where the alternative electron acceptor nitrate is converted into the

neuropharmacological agent and greenhouse gas nitrous oxide (N2O) via nitrite (NO2-) and nitric

oxide (NO). The ability to reduce NO to different compounds is crucial for their survival as NO is a

potent cytotoxin and used by macrophages to defeat invaders. Thus, E. coli and Salmonella have

evolved mechanisms to overcome the hosts’ attack and to defend themselves against their own toxic

by-products (Rowley et al., 2012 Biochemical Journal 441, 755-762). The three main NO

detoxification enzymes include: flavohaemoglobin (Hmp), flavorubredoxin (NorVW) and the

cytochrome c nitrite reductase NrfA (Mills et al., 2008 Microbiology 154, 1218-1228). Continuous

chemostat cultures and transcriptional analysis were used to investigate the importance of N2O

production and NO detoxification for the pathogenicity of enteric bacteria, using Salmonella and E.

coli as comparative pathogenic and non-pathogenic model systems (Arkenberg et al., 2011

Biochemical Society Transactions 39, 1876-1879). Results show that there are differences in N2O

production between Salmonella and selected E. coli strains, which are partially caused by differential

transcription of narG, nirB and hmp.

37

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 23

Kinetics of NO scavenging in oxic and anoxic conditions, experimental approaches to

differentiate between chemical and enzymatic degradation

Shahid Nadeem1, Andrew M. Stern4, Wenmin Long2, Peter Dörsch1, Åsa Frostegård1,

Jim Shapleigh3, Jun Zhu4 ,Binbin Liu1 and Lars R Bakken1 1

UMB Nitrogen Group, Norwegian University of Life Sciences, Norway. 2 Jiaotong Shanghai University, Shanghai, PRC,

3 Cornell University, Ithaca, NY USA,

4 Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

NO is a toxic signal molecule and an intermediate in denitrification, but NO is produced and

“scavenged” by a plethora of other enzymes and organisms. To understand its role as a toxic

compound and an inducer of denitrification in soil and N-transformations duirng host-pathogen

interactions, we need to determine the kinetics of NO scavenging by relevant organisms.

Experiments with NO are not trivial because NO is chemically unstable in the presence of oxygen due

to “autoxidation”, which is a “third order” process; V=k*[O2]*[NO]2. This happens both in the gas

phase and the liquid phase. The problem with chemical instability is escalating with high NO

concentrations, even at relatively low oxygen concentrations. We have developed an approach to

tackle these problems, by a combination of mathematical modeling and an incubation system that

monitors the concentrations of NO and O2 in a two phase system (liquid + headspace). The approach

allows a determination of the kinetic parameters for NO scavenging, be it under oxic or anoxic

conditions. The approach has been used to characterize oxic and anoxic NO scavenging in Vibrio

cholerae, Paracoccus denitrificans, Agrobacterium tumefaciens and mutants thereof, to elucidate the

role of different enzymes. The results indicate that oxic NO scavenging is very fast in some species,

and apparently absent in others, and the studies of mutants have identified some of the proteins

involved in NO scavenging.

38

17th European Nitrogen Cycle Meeting, Oslo 2012

KN 07

Identifying and quantifying biological source and sink processes of N2O using isotope

tracing and natural abundance techniques.

Reinhard Well1, Gesche Braker2, Anette Giesemann1, Dominika Lewicka-Szczebak1, Lena Rohe1,

Greta Roth1

1 Johann

Heinrich von Thuenen-Institute, Institute of Agricultural Climate Research, Braunschweig, Germany

2 Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

To understand the control of N2O fluxes to the atmosphere it is essential to elucidate the various

processes of biological N2O production and consumption. This includes production by nitrification,

nitrifier-denitrification, bacterial or fungal denitrification, co-denitrification by fungi and bacterial

reduction to N2. 15N and 18O tracing techniques can be used to quantify the contribution of the

respective pathways. Moreover, natural abundance of N2O isotopologue signatures (δ18O, average

δ15N and 15N site preference, SP = difference in δ15N between the central and peripheral N positions

of the asymmetric N2O molecule) have recently received some attention as tools to elucidate the

contribution of nitrification, bacterial and fungal denitrification to N2O fluxes as well as to estimate

N2O reduction to N2. Isotopologue signatures of individual pathways have been studied with pure

cultures of fungal and bacterial denitrifiers and of autotrophic nitrifying bacteria. Moreover, N2O

isotopologues are increasingly used as indicators of process dynamics in soil flux studies. An

overview of the techniques as well as some examples from the literature and from our own studies

will be given.

To complement previous literature data, we determined the specific isotopic signature of N2O

production from pure cultures of bacterial and fungal denitrifiers. To investigate N2O reduction in

soil, we measured isotopologue values of soil-derived N2O produced under conditions favouring

denitrification. These experiments where combined with independent estimates of denitrification

obtained from 15N tracing or incubation under N2-free atmosphere. We used an isotope fractionation

model to check if N2 production can be calculated from isotopologue data.

39

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 24

Aeration of boreal acid sulphate soils could result in enhanced fluxes of N2O and CH4

due to stimulation of potent microbial communities present in subsoil

Miloslav Šimek1,2, Alica Chroňáková1, Dana Elhottová1, Seija Virtanen3, Asko Simojoki3,

Václav Krištůfek1, Markku Yli-Halla3

1 Biology Centre AS CR, v. v. i., Institute of Soil Biology, České Budějovice, Czech Republic

2 University of South Bohemia, Faculty of Science, České Budějovice, Czech Republic

3 University of Helsinki, Department of Food and Environmental Sciences, Helsinki, Finland

Boreal acid sulphate (AS) subsoil horizons contain significant amounts of organic carbon and

nitrogen. We hypothesized that large emissions of CO2, CH4, and N2O from AS soils could occur, if the

soil conditions, e.g. aeration status, change, provided relevant microorganisms are present. We

investigated this phenomenon in Helsinki region in Finland and sampled AS and non-AS soils to

depths of 165 - 180 cm. We confirmed large carbon and nitrogen stocks (110 Mg Corg ha-1 and 15 Mg

Ntot ha-1) in the permanently flooded C horizons of the AS soil but not in the control non-AS.

Moreover, in the subsoil, high ammonium concentrations were found. We also found very high basal

and glucose-induced respiration in the same Cg2 horizon of the AS soil. Examination of microbial

communities revealed much higher abundance of methanogens in the deepest C horizons, than in

the surface Ap horizons. Nitrous oxide production determined in laboratory incubations peaked in

the BCgc and BC horizons of the AS and non-AS soils, respectively, but in the AS soil the peak value

was tenfold higher than in the non-AS soil. We conclude that surprisingly high microbial abundance

and activity, together with the abundant carbon and mineral nitrogen in the deep layers of AS soils,

may result in large gas emissions. High emissions could occur for example when the generally high

water table would be lowered due to arable farming. Additional studies of boreal AS soils are needed

to assess their potential contribution to increases in greenhouse gas emissions.

40

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 25

Effect of intensified crop production on soil nitrous oxide emission in Mid-Hills of

Nepal

Nani Raut1, Bishal K Sitaula1, Lars R Bakken2

1 Department of International Environment and Development Studies, Norwegian University of Life Sciences PO box

5003, N-1432 Aas, Norway 2

Department of Plant and Environmental Sciences, Norwegian University of Life Sciences PO box 5003, N-1432 Aas,

Norway

Dramatic changes in agricultural practices have taken place in large part of South Asia. The

traditional low input farming is replaced by intensified production systems (high fertilizer levels and

more crops per year). The high N inputs will increase the emissions of N2O, but the prospects are

potentially worse than those predicted by IPCC-methodology which assumes that N2O emission

increase linearly with N-input. The reason is that crop intensification may acidify the soils which

could increase the N2O/(N2+N2O) product ratio of denitrification. We have investigated the

phenomenon by analyzing soil samples from seven different sites, each site containing both

traditional and adjacent intensified plots. The results showed that crop intensification invariably

lowered the soil pH and increases the N2O/(N2+N2O) product ratio under standardized anoxic

incubations (Raut et al., 2012, Soil Biology & Biochemistry in press). In theory, this would imply

escalating N2O emissions beyond that predicted by IPCC. This needs to be tested by measuring N2O

emissions in field experiment, and here we present such results: Regardless of the land use

categories, intensified agricultural system had significantly higher N2O emission than traditional

agricultural system. We can estimate the emission factor for intensification: EFint = ∆N2O-N year-1/ ∆

N-input year-1, where ∆N2O-N year-1 is the estimated increase in N2O-emission by intensification, and

∆ N input year-1 is the annual input of N to Intensified plots minus that in traditional plots. For the

site with permanently drained soil, EFint was 0.13, and for the periodically flooded soil, it was 0.03. In

comparison, the emission factor for N input in the IPCC formula is 0.01-0.02. These results confirm

the predictions from standardized anoxic incubations; soil acidification by N fertilization leads to

much higher N2O emission than that predicted by the IPCC formula.

41

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 26

High N2O emissions from an N saturated subtropical forested catchment, Southwest

China

Jing Zhu, Jan Mulder, Peter Dörsch Dept. Plant and Env. Sci. Norwegian University of Life Sciences, Aas, Norway

Subtropical forests in South China, after receiving long-term elevated nitrogen (N) deposition, have

become N saturated. Previous studies found that, despite low forest growth, N leaching via stream

water was moderate, indicating important unknown N sinks. Given the monsoonal climate with hot

and wet summers and the widespread acid soil in this region, denitrification may represent a major

sink of reactive N and result in considerable emission of nitrous oxide (N2O), a potent greenhouse

gas (GHG).

Field flux measurements of N2O with static chambers carried in an N saturated subtropical forested

catchment in southwest China, Tieshanping (TSP), found high N2O fluxes in summer. Fluxes of N2O

were significantly greater on hillslopes than in a hydrologically connected groundwater discharge

zone. Thus, landscape position, which integrates different soil characteristics, affected the N2O

emission. During summer, soil moisture was the determining factor, resulting in transient N2O peaks

(up to 1730 μg N2O-N m-1 h-1) on the hillslope after rain episodes. The extrapolated annual N2O fluxes

were 0.50 and 0.41 N2O-N m-2 in 2009 and 2010, respectively. An in situ 15N-NO3- labeling experiment

confirmed that denitrification rather than nitrification was the majority pathway of N2O production.

Results from lab incubations supported these findings: soils from hillslope have high denitrification

potential with impeded N2O reduction activity as well as low nitrification potential causing NO2 -

accumulation. The latter was shown to quickly dismutase to NO under acid condition, opening for

another important pathway for N removal.

42

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 27

High potential for N2O emission from intensive Chinese agriculture due to soil

acidification

Zhi Qu1, Jingguo Wang2, Lars R Bakken1

1 Department of Plant and Environmental Sciences, Norwegian University of Life Sciences

2 College of Resources and Environmental Sciences, China Agricultural University

Chinese farmers use large amount of mineral nitrogen (N) fertilizer and manure to sustain grain

production. Excessive N inputs result in gradual soil acidification all over China, which may impair

N2O reductase and hence induce an escalating emission of N2O. We investigated this by analyzing

soils from long-term fertilization experiments in different regions of China (Gongzhuling (GZL),

Shangzhuang (SZ), Qiyang (QY), Changwu (CW) and Shouguang (SG)). The soils were incubated after

adjusting their nitrate (NO3-) concentration to 2mM NO3

- -N by flooding and drainage. The NO, N2O

and N2 gas kinetics from denitrification were monitored during anaerobic incubations, to explore the

fertilization effects on the denitrification product stoichiometry (NO/N2O/N2) during such

standardized incubations. We also investigated if the product stoichiometry could be altered by

adjusting pH prior to such anoxic incubations. The results show that:

1- High levels of mineral N acidified the soils, but not in soils with high buffer capacity;

2- High levels of mineral N led to higher N2O/(N2O+N2) product ratios, but not in soils with high

buffer capacity;

3- Adjusting the soil pH in acidified soils restored a low N2O/(N2O+N2) ratio;

4- Manure had a variable effect on soil pH;

5- Overall, we found a strong negative correlation between soil pH and the N2O/(N2O+N2) ratio

(pH range 3.7-8.0, r2=0.75).

We conclude that the excessive N fertilization input in China resulted in soil acidification, which is a

direct cause of enhanced propensity for N2O emission.

43

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 28

Modelling the effect of Copper availability on bacterial denitrification

H.C. Woolfenden1, A.J. Gates2, G. Giannopoulos2, M.J. Sullivan2, D.J. Richardson2, V. Moulton1 1

School of Computing Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, U.K. 2

School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, U.K.

When bacteria such as Paracoccus denitrificans respire anaerobically they convert nitrate (NO3- ) to

dinitrogen (N2) gas via a pathway which includes the potent greenhouse gas, nitrous oxide (N2O). The

copper-dependent enzyme Nitrous Oxide reductase (Nos) catalyses the reduction of N2O to N2.

Experiments have shown that in low-copper conditions Nos efficiency decreases, which can result in

significant emissions of N2O. A mathematical model is developed to describe the anaerobic

denitrification pathway based on Michaelis-Menten kinetics and published kinetic parameters.

Novelty is given to the model by adjusting the Nos concentration depending on the copper

concentration. The accuracy of the model can be checked by comparing the predictions with existing

experimental results from chemostats. Further, individual reductase concentrations are deduced

from simple algebraic expressions involving the kinetic parameters and the experimental data. The

relative expressions of the enzymes may then be compared to experiments. The model may also be

used as a prediction tool to estimate the output levels of the nitrogenous compounds in the

denitrification pathway, in particular nitrous oxide, for a prescribed set of initial conditions.

44

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 29

Unexpected diversity of nitrite oxidoreductase genes (nxrB) in marine waters:

indications for novel NOB?

Andreas Schramm1, Sandra Lopez1, and Sebastian Luecker2

1 Department of Bioscience, Microbiology, Aarhus University, Denmark

2 Department of Microbial Ecology, University of Vienna, Austria

Since the recent discovery of ammonia-oxidizing archaea (AOA), increasing evidence has

accumulated that AOA are the main ammonia oxidizers in the ocean, where they can be extremely

abundant and utilize minute concentrations of ammonia, which is oxidized to nitrite. However,

nitrite rarely accumulates in the ocean but is either further oxidized to nitrate or serves as important

link between aerobic and anaerobic N conversions in oxygen minimum zones (OMZ). Yet the

organisms mediating nitrite oxidation in the ocean are poorly studied, and the abundance of known

marine nitrite-oxidizing bacteria (NOB), i.e. Nitrospina, Nitrococcus, and Nitrospira, hardly correlates

with nitrite oxidation rates.

The objective of this study was to identify putative NOB in the OMZ off the coast of Chile and Peru

and to correlate their presence with chemical profiles in the water column. PCR-based approaches

were developed to target 16S rRNA genes of the known NOB genera, and the gene encoding the

beta subunit of nitrite oxidoreductase (nxrB). The only NOB-like 16S rRNA genes detected were

affiliated with Nitrospina and revealed a large diversity within this group, but also a lack of

correlation with the chemical profiles. In contrast, at least four distinct clusters of nxrB-like genes

were observed, showing conspicuous depth distributions. The possibility that these genes indicate

the presence of hitherto unknown NOB will be discussed.

45

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 30

Biogeochemical nitrogen cycling in Arctic snowpacks: generating hypotheses from

multi-disciplinary studies

Catherine Larose1, Sebastien Cécillon1, Emmanuel Prestat1, Aurélien Dommergue2,, Pascal

Simonet1 and Timothy M. Vogel1 1

Environmental Microbial Genomics, Ecole Centrale de Lyon, Université de Lyon, 36 avenue Guy de Collongue, 69134

Ecully, France 2

Université Joseph Fourier – Grenoble 1 / CNRS, LGGE, 54 rue Molière BP56, F-38402 Saint Martin d’Hères, France

Arctic snowpacks are often considered as chemical reactors for a variety of chemicals deposited

through wet and dry events, but are overlooked as potential sites for microbial metabolism of

reactive nitrogen species. The fate of deposited species is of utmost importance since melt leads to

the transfer of nutrients and contaminants to snowmelt-fed ecosystems. Here we examined the role

of microorganisms and the potential pathways involved in nitrogen cycling in the snow. Using a 16S

rRNA microarray to follow shifts in microbial community structure during a two-month springtime

field study at a high Arctic site, Svalbard, Norway (79°N) in addition to pyrosequencing, we showed

that despite the cold conditions and limited water supply, microbial communities inhabiting the

snow cover were dynamic and had the functional potential to carry out several different, but not all

pathways of the nitrogen cycle. Nitrogen cycling was shown to be a dominant process at the base of

the snowpack and seems to be among the most important functions in Arctic ecosystems.

46

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 31

Is there a direct link between denitrifier community structure or size and

denitrification activity in arable soil?

Miriam B Herold1,2, Liz M Baggs1, Tim J Daniell2

1 Institute of Biological and Environmental Sciences, University of Aberdeen, UK

2 The James Hutton Institute, UK

Denitrification is the biological process by which intermediates of the nitrogen cycle are reduced to

maintain respiration under oxygen limiting conditions. Several enzymes and their corresponding

genes have been identified in denitrifying bacteria. The reduction of nitrite to nitric oxide is catalysed

by the copper containing nitrite reductase (nirK) or the cd1 type nitrite reductase (nirS). Both nirK

and nirS were used in this study to investigate the effects of pH and cultivation management of

arable soil on the community structure and size of bacterial denitrifiers.

Soil was sampled from long-term pH plots under a cultivation gradient (SAC, Craibstone, UK). A

molecular finger printing technique (T-RFLP) was used to evaluate microbial community structure

and real-time PCR was used to estimate microbial community size. Potential denitrification rates

(PDR) were measured in order to compare denitrification activity and community structure/size.

Potential denitrification was strongly influenced by pH and cultivation with the highest PDR from

slightly acidic soil and soil under long-term ley grass. Both nirK and nirS community structures were

influenced by pH and changed significantly at acidic pH. The community structure of nirK but not the

community structure of nirS responded significantly to cultivation management (ploughing) which

suggested that bacteria with different nitrite reductases can follow different population dynamics.

Significant changes in community structures did not necessarily follow changes in gene abundance

and therefore we concluded that both community structure and size influenced denitrification

activity in arable soil.

47

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 32

Microbial composition of various components of closed aquaculture systems:

analysis of biofilters and indogenous microbiota of fish intestines

Maartje A.H.J. van Kessela,b, Arslan Arshada,b, Gert Flikb, Mike S.M. Jettena, Peter H.M. Klarenb,

Huub J.M. Op den Campa a

Department of Microbiology and b

Department of Organismal Animal Physiology, IWWR, Radboud University Nijmegen,

Heyendaalseweg 135, NL-6525 AJ Nijmegen, the Netherlands

The maintenance of an optimal water quality poses challenges for aquaculture worldwide. Especially

the accumulation of nitrogen compounds, which are excreted by fish, is a major problem in

aquaculture. Strict rules for concentrations of these compounds in the effluent water and toxicity of

these compounds force aquaculture operators to reduce nitrogen in the effluent water. This can be

accomplished in different manners: 1) Efficient removal of the nitrogenous compounds from the

water or 2) Reduction of the nitrogen input. Biofiltration is often used to remove nitrogen

compounds from the water. However, currently we know little about the contribution of various

groups of N-cycle bacteria to the removal of nitrogenous compounds in aquaculture systems. We

therefore investigated the composition of a biofilter which revealed the presence of various

members of nitrogen-cycling bacteria, including ammonium- and nitrite-oxidizing and anammox

bacteria. Reducing nitrogen accumulation by reducing the nitrogen input can be accomplished by

decreasing the protein concentration in food. Most fish are fed high protein content food to

maximize growth. Part of this protein can be exchanged for plant material, however it is important to

know if cultured fish can convert the plant components to usable metabolites. We therefore

investigated the intestinal microbiota of common carp (Cyprinus carpio) using culture-independent

methods. This revealed the presence of a large population of microorganisms able to ferment plant

components. Furthermore, members of the nitrogen cycle bacteria were found to reside in the fish’

gut pointing to new in situ possibilities to reduce nitrogen emissions in aquaculture.

48

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 33

Differences in composition of active and overall denitrifier communities in three

organic soils determine responses of denitrification to pH

Kristof Brenzinger1, Peter Dörsch2 and Gesche Braker1 1

Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, D-35043 Marburg, Germany 2

Norwegian University of Life Sciences, Aas, Norway

Denitrifier communities from three drained organic soils in Finland (FIN), Germany (GER) and

Sweden (SWE) were studied, differing in soil history and soil parameters. The soils showed marked

physiological differences in denitrification response to anoxia and low temperature. Moreover, a

direct effect of pH on denitrifier functioning had been observed when exposing bacterial consortia

extracted from these soils to two different pH levels (pH 5.4 and 7.1) (1). We hypothesized that the

denitrifier communities harbored by these soils were composed differently. Indeed, we found

differences in diversity, composition and abundance of denitrification genotypes between the soils

based on NO2--reductase (nirK/nirS) and N2O-reductase (nosZ) genes which appeared to congruent

with the communities’ ability to produce and reduce N2O (2).

In a subsequent study we explored the population dynamics and transcription of denitrification

genes in extracted communities during incubation at different pH and found both to be strongly

controlled by pH. Moreover, our results indicate that denitrifier activity may be additionally affected

by posttranslational modification of the enzymes.

We conclude that links exist between the genetic makeup of denitrifier communities and their

physiological responses to environmental triggers. Furthermore, we could show that links to

functional differences were mostly caused by differences in the composition of the active denitrifier

community in response to pH and we hypothesize that posttranslational regulation is an important

regulatory step impacting ecosystem functioning.

(1) Dörsch, P., Braker, G. and Bakken, L.R. (2012) Community specific pH response of denitrification: experiments with

cells extracted from organic soils. FEMS Microbiol. Ecol., 79, 530–541.

(2) Braker, G., Dörsch, P. and Bakken, L.R. (2012) Genetic characterization of denitrifier communities with contrasting

intrinsic functional traits. FEMS Microbiol. Ecol., 79, 542–554.

49

17th European Nitrogen Cycle Meeting, Oslo 2012

CP 34

Unusual acid-tolerant denitrifier communities in arctic permafrost soils associated

with N2O fluxes

Katharina Palmer and Marcus A. Horn

Peramfrost-affected peatlands were recentlly identified as sources of the greenhouse gas N2O.

Unvegetated tundra peat circles (PC) are nitrate-rich ‘hot spots’ of N2O emissions, while adjacent

vegetated tundra areas (UT), and moderately vegetated palsa peats (PP) emit only minor amounts of

N2O. Soil pH approximated 4 for all sites. N2O emission potentials of unsupplemented anoxic peat

soil slurries and apparent maximal nitrate-dependent maximal reaction velocities were highest for

PC, intermediate for PP, and lowest for UT. Stimulation of N2O production by nitrate-, nitrite-, and/or

acetylene suggested denitrification as major source of N2O in all soils. N2O/(N2+N2O) ratios were 30,

55, and 100% for PC, PP, and UT, respectively, when 10 µM of nitrate were supplied. Such dissimilar

physiological responses suggested dissimilar denitrifier communities in the soils. Barcoded amplicon

pyrosequencing of narG, nirK, nirS, and nosZ (encoding nitrate, nitrite, and N2O reductases,

respectively) revealed diverse denitrifiers including hitherto unknown species, and likewise

suggested that denitrifier diversity differed between the soils. Sequences affiliated with Actino-,

Alpha- and Betaproteobacteria as well as environmental sequences. Quantitative PCR of narG, nirK,

nirS, and nosZ indicated that detected nirS outnumbered nirK in all soils. Detected narG and nirK

were most abundant in PC. The collective data suggest that (i) Proteobacterial nirS-type rather than

nirK-type denitrifiers are abundant in acidic permafrost soils, and (ii) the contrasting N2O emission

patterns are associated with contrasting denitrifier communities.

50

17th European Nitrogen Cycle Meeting, Oslo 2012

Poster abstracts

Ordered alphabetically, according to first author

P 01

Impact of loss of norEF in Rhodobacter sphaeroides 2.4.1

Linda Bergaust1, Angela Hartsock2, Lars Bakken1, Åsa Frostegård1 and James P. Shapleigh2 1

UMB Nitrogen Group, Norwegian University of Life Sciences, Norway 2 Department of Microbiology, Cornell University, Ithaca, NY USA

During denitrification nitric oxide (NO) is reduced by NO reductase (Nor) to nitrous oxide (N2O).

There are two types of Nor found in denitrifying bacteria with one, qNor, receiving electrons from

quinol while the other, cNor, receives electrons from cytochrome c. Both are members of the

superfamily of heme-copper oxidases. Purified cNor is a two subunit complex. Many members of the

heme-copper superfamily contain a third subunit upon purification. A gene encoding a protein with

sequence similarity to the heme-copper subunit III is found in the denitrification clusters of many

denitrifiers. This gene has been designated norE and is nearly always associated with a second gene

designated norF. R. sphaeroides 2.4.1 contains the genes for a cNor but lacks norEF. This strain also

lacks genes for and nitrite reductase and nitrous oxide reductase but has a nitrate reductase. R.

sphaeroides 2.4.3 has both the cNor gene cluster and norEF but the latter do not form a tight genetic

cluster with the cNor operon. To learn more about the role of norEF we determined how the loss of

norEF impacted NO reduction in R. sphaeroides 2.4.1. Both 2.4.1 with nirK from 2.4.3 and 2.4.3

lacking norEF were able to reduce nitrate to N2O without accumulation of detrimentally high

concentrations of NO. However, reduction of nitrite lead to accumulation of NO consistent with an

impairment of Nor activity. Addition of norEF from 2.4.3 to the 2.4.1 strain with nirK reduced the

accumulation of NO that occurred during the reduction of nitrite. Taxis behavior of the various

strains to a nitrite source was consistent with norEF limiting nitrite toxicity, most likely because it

enables the cells to control NO accumulation. These results show that loss of norEF by 2.4.1 does

impact Nor activity. This suggests that despite the loss of norEF the Nor in 2.4.1 provides a selectable

advantage because this strain only periodically encounters exogenous NO. The rate of endogenous

NO production during denitrification prevents the loss of norEF in 2.4.3.

51

17th European Nitrogen Cycle Meeting, Oslo 2012

P 02

Abundance and diversity of nitrifying prokaryotes in drinking water treatment plant

Mei-Chun Chen1, Toshikazu Fukushima2, Sin-Ming Liu3, Pei-Chu Chen3, Liang-Ming Whang1 1

Department of Environmental Engineering, National Cheng Kung University, Tainan, Taiwan 2 Division of Environmental Engineering, Hokkaido University, Japan

3 Taiwan Water Corporation, Taichung, Taiwan

In order to better understand nitrification in drinking water treatment processes, this study was

motivated to investigate abundance and diversity of nitrifying prokaryotes in filtration processes in

three drinking water treatment plants (plant A, B-1 and B-2) using real-time PCR and terminal

restriction fragment length polymorphism. In general, real-time PCR results showed that AOA and

Nitrospira were dominant ammonia and nitrite oxidizers in the filtration processes, except for the

plant B-1 which practiced pre-chlorination.

The ratio of AOA to AOB in filter media in plants A and B-2 were 5-10 times higher in winter than

those in summer samples. It was presumed that temperature may be affect ammonia oxidizers

community in drinking water treatment process. Besides, the ratio of raw water and filtration was

difference. This implied that community of nitrifying in raw water did not affect community structure

in treatment process. Besides, Nitrobactor below the detect limit on raw water and media. Terminal

restriction fragment (T-RF) 220 bp was dominated ammonia-oxidizing archaea in our study, while

previous study reported that T-RF 169 was dominant in drinking water treatment plant in Japan, the

phenomenon would be interesting.

According to those finding, the contribution of nitrification suggest that ammonia-oxidizing archaea

and Nitrospira may account for most of the ammonia and nitrite oxidation. Furthermore, pre-

chlorination should be avoided to achieve good nitrification. The T-RF 220 bp necessary to do

sequencing in the future.

52

17th European Nitrogen Cycle Meeting, Oslo 2012

P 03

The nitrite:nitrate oxidoreductase system of the anammox bacterium

Kuenenia stuttgartiensis

Naomi M. de Almeida1, Hans J. C. T. Wessels2, Wouter J. Maalcke1, Jan T. Keltjens1,

Mike S. M. Jetten1, Boran Kartal1 1

IWWR, Microbiology, Radboud University Nijmegen, The Netherlands 2

Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre,

Nijmegen, The Netherlands

The genome of the anaerobic ammonium-oxidizing (anammox) bacterium ‘Candidatus Kuenenia

stuttgartiensis’ encodes for a nitrite:nitrate oxidoreductase system that oxidizes nitrite to nitrate,

yielding two high potential electrons. Although the gene cluster kustd1699 – kustd1713 has initially

been annotated as a nitrate reductase, a closer look at the amino acid sequence suggests a more

elaborate role.

According to previous results, the gene products form two main subcomplexes. The soluble

subcomplex catalyzing the nitrite oxidation reaction comprises of three proteins, namely NarG, NarH

and a heme b containing protein. These together contain a molybdenum cofactor, 5 Fe-S clusters

and two heme b’s, all tuned on amino acid level to have the high redox potential needed for the

oxidation reaction of nitrite. In addition, the gene cluster contains a set of genes coding for a broad

repertoire of electron carriers. This includes six putative heme-containing proteins, two putative

blue-copper proteins and a membrane-bound polypeptide showing homology to the quinol-binding

subunit of a cytochrome bd oxidase.

These allow the electrons obtained from nitrite oxidation to be wired either towards the quinone

pool via the membrane bound subunit, or towards different reducing reactions in the cell via the

putative blue copper and heme containing proteins.

Current work includes the purification and characterization of the catalytic subcomplex and the

functional expression of the two blue-copper containing proteins. Further, we investigated the

possible interactions of the catalytic subcomplex with other protein complexes with protein

correlation profiling of purified membranes using LC-MS/MS data from consecutive blue native gel

slices.

53

17th European Nitrogen Cycle Meeting, Oslo 2012

P 04

Genetic and symbiotic diversity of rhizobia isolated from Ethiopian soils

Tulu Degefu1, Endalkachew Wolde-meskel1,2, Binbin Liu1, Anne Willems3, Ilse Cleenwick3 and

Åsa Frostegård1 1Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, NO-

1432 Ås, Norway 2School of Plant and Horticultural Sciences, Hawassa University, P.O. Box 5, Hawassa, Ethiopia

3Laboratorium voor Microbiologie (WE10), Universiteit Gent, KL Ledeganckstraat 35, B-9000

Gent, Belgium

Leguminous trees play an important role in agroforestry in Ethiopia partly due to their ability to

establish a symbiosis with rhizobia for biological nitrogen-fixation (BNF). BNF plays a substantial role

in linking the biological nitrogen cycle between the atmosphere and the earth and hence is a key

factor in agricultural sector and in the rehabilitation of degraded soil by improving nutrient cycling

and other soil physical conditions. By virtue of these, isolation and characterization of the legume

associated rhizobia is crucial to develop inocula and thereby utilize their use for sustainable

agriculture. As the first step to such an approach, 240 rhizobial strains were isolated from root

nodules of a large number of legumes species growing in diverse agro-ecological zones in southern

Ethiopia and characterized based on multilocus sequence analyses of several housekeeping and

symbiosis-related genes. Cross-inoculation experiments were also conducted on a range of legume

species to determine the host ranges. The core gene sequence-based phylogenetic analyses showed

that all test strains represent the branches designating the Rhizobium (5 genospecies),

Mesorhizobium (3 genospecies), Ensifer (7 genospecies) and Bradyrhizobium (5 genospecies), with

the majority of the test strains occupying distinct positions by excluding any defined species in each

of the genus. Further characterizations of a selected strains belonging to the Mesorhizobium genus

from the same collection using DNA-DNA hybridization, cellular fatty acid analyses and additional

housekeeping genes revealed the presence of novel lineages, hence we described three new species

within the genus. Our results while showing discordant phylogeny between housekeeping and

symbiotic gene in some genera like Ensifer and Rhizobium also revealed similar pattern of grouping

in some other genus like Mesorhizobium. The phylogenetic relationship based on the symbiosis-

related genes were not similar to those shown by core genes for the strains represented in Ensifer

and Rhizobium genus suggesting differences in the evolutionary history between the chromosomal

and symbiotic genes. The cross-inoculation experiment, conducted to evaluate the symbiotic

performance of the test strains on a range of legumes species, indicated the potential to improve N

fixation through host selection. Our study confirms a high diversity of rhizobia in Ethiopia and, while

contributing to the general knowledge of the biodiversity within the rhizobial genera, also highlights

the need to focus on previously less explored bio-geographical regions to unravel as yet unidentified

rhizobial resources.

54

17th European Nitrogen Cycle Meeting, Oslo 2012

P 05

Diversity of nitrogen fixing microorganisms from a saline-alkaline former lake

Texcoco, México

Cristina Domínguez-Mendoza, Carolina Castro-Silva, Rocío Alcántara-Hernández, Rodolfo Marsch,

Luc Dendooven* Laboratory of Soil Ecology, Cinvestav, México

*Author for correspondence: Luc Dendooven, Tel. +52 55 5747 33 19, Fax. +52 55 5747 33 13, E-mail:

dendooven@me.com

Soil of the former lake Texcoco is an ‘extreme’ alkaline saline environment with pH>10 and

electrolytic conductivity >10 dS m-1. Although this environment is adverse for N2 fixing

microorganisms, earlier studies showed their presence in this soil. In this study nitrogenase activity

was measured and related with a nifH libraries with the objective to compare the diversity and

phylogenetic relationships of nitrogen fixing microorganisms.

55

17th European Nitrogen Cycle Meeting, Oslo 2012

P 06

Strong reduction of N2O emissions by long term liming in a drained organic soil

Peter Dörsch1, Linn Hovlandsdal1,2, Samson Øpstad2 and Lars Bakken1 1 Norwegian University of Life Sciences (Postboks 5003, 1432 Ås, peter.doersch@umb.no)

2 Bioforsk West Fureneset (6967 Hellevik)

Drained peatlands have been reported to be among the strongest sources for soil-borne N2O.

Cultivated organic soils derived from peats are often limed or mixed with mineral soil to raise the

naturally low pH. We previously found that soil acidity strongly inhibits N2O reductase and

hypothesised that long-term liming would reduce N2O emissions by changing the product

stoichiometry of denitrification in favour of N2. Here we report N2O emission fluxes measured in a

liming experiment in Norway (Fjaler, Outer Sunnfjord) where different rates of mineral addition

(shell sand or moraine soil) had been applied more than 30 years ago resulting in distinct soil

pH(H2O) values (4.5, 5.3, 6.5, 7.2 and 7.4). N2O emission fluxes were measured by closed chamber

technique directly after fertilisation (90 kg N ha-1 as NPK) following the first harvest in 2010 and

throughout autumn when soil mineral N concentrations declined. Precipitation after fertilisation

elicited N2O emission fluxes of up to 7000 µg N m-2 h-1, which are among the highest fluxes reported

from land ecosystems ever. The control soil (no liming, pH 4.5) lost 6 kg N2O-N within the first 19

days after fertilisation equalling 6.4% of the applied nitrogen. N2O-N loss was significantly lower in

limed treatments (1.9 kg at pH 7.4), resulting in a significant negative correlation between soil pH

and N2O emission (r2 = 0.6, p<0.05). No such correlation was found after the fertiliser peak in mineral

N had levelled off and N2O emissions decreased to background fluxes. Our data suggest that long-

term liming of acid soils can reduce annual N2O emissions in crop production, supposedly by

increasing the N2/N2O ratio during periods of high denitrification activity.

56

17th European Nitrogen Cycle Meeting, Oslo 2012

P 07

Identification of key player groups of denitrifiers in an organic agricultural soil by

high-throughput sequencing techniques

Binbin Liu1, Xiaojun Zhang3, Lars R. Bakken2 and Åsa Frostegård1 1

Department of Chemistry, Biotechnology and Food Sciences, Norwegian University of Life Sciences, Ås, Norway 2

Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, Ås, Norway 3

Key Laboratory of Microbial Metabolism, Ministry of Education, College of Life Science and Biotechnology, Shanghai

Jiaotong University,Shanghai, China

Our earlier studies of denitrification gene expression in soil show that transcription occurs only

during a few hours after oxygen depletion. DGGE analysis indicated sequential triggering of gene

expression by different populations. We used a robotized incubation system, and guided by the gas

kinetics we sampled soil for mRNA isolation at time intervals (1, 3 and 6 h after O2 depletion).

Functional genes (narG, napA, nirS, nirK, qnorB, nosZ) were amplified from both DNA and cDNA using

ID tagged primers. A total of 210,000 sequences were analyzed, mostly representing Proteobacteria.

The cDNA libraries confirmed that some groups initiated transcription earlier than others. The same

groups were detected both in cDNA and DNA libraries, but in different proportions; those

dominating the DNA library were often less abundant in the cDNA and vice versa. For example,

Dechloromonas sequences constituted 4 and 6% of the total sequences in nirS and napA DNA and

56% and 22% in the corresponding cDNA; Cupriavidus constituted 5% in nirS DNA and 22% in the

cDNA libraries; and Pseudomonas increased from 15% in nosZ DNA to >50% in cDNA while, in

contrast, Herbaspirillum decreased from 58% in DNA to 16% in cDNA. The findings reveal key-player

denitrifiers and are currently used for targeted isolation of these groups.

57

17th European Nitrogen Cycle Meeting, Oslo 2012

P 08

The effect of pH and Oxygen on nitrous oxide emissions from the denitrifying

prokaryote Paracoccus denitrificans 1222.

G. Giannopoulos1, N. Watmough1, P. Poole2 and D. Richardson1 1

School of Biological Sciences, University of East Anglia, Norwich, UK 2 John Innes Center, Norwich, UK

Denitrification forms a part of the bio-geo-chemical cycle in which nitrate is reduced to nitrite, nitric

oxide, nitrous oxide and finally to di-nitrogen. Prokaryotic organisms possess specific

metalloenzymes that reduce nitrogen species; a periplasmic and an inner membrane bound nitrate

reductase, a cytochrome or copper containing nitrite reductase, a cytochrome bc complex nitric

oxide reductase and a copper containing nitrous oxide reductase.

Paracoccus denitrificans PD1222 is a model heterotrophic denitrifying bacterium, capable of nitrate

reduction to di-nitrogen in anaerobic conditions, utilising nitrate as alternative electron acceptor and

succinate as an electron donor. However, in aerobic conditions with nitrate available, P.denitrificans

utilizes oxygen as the final electron acceptor with succinate as an electron donor.

Comparisons of aerobic and anaerobic denitrification as well as the effects of pH in anaerobic

denitrification were undertaken in continuous culture bioreactors, with focus on metabolic profiling,

enzymatic assay and transcriptional regulation. This was carried out using nitrate, nitrite, nitrous

oxide monitoring, immunoblotting, RNA extraction and microarray analysis. The postulated

mechanisms of the denitrification pathway will be discussed.

58

17th European Nitrogen Cycle Meeting, Oslo 2012

P 09

Identification of controlling factors of Nitrous Oxide emission for a drained spruce

forest on peat soil, a modeling approach

He, H.1; Kasimir Klemedtsson, Å.1; Jansson, P.-E.2; Nylinder, J.1; Meyer, A.3; Klemedtsson, L.3 1 Department of Earth Sciences, University of Gothenburg, Gothenburg

2 Land and Water Resources Engineering, Royal Institute of Technology (KTH), Stockholm 3 Department of Plant and

Environmental Sciences, University of Gothenburg, Gothenburg

High nitrous oxide (N2O) emission potential has recently been identified in hemiboreal forest on

drained Histosols (Maljanen et al. 2003; Von Arnold et al. 2005; Ernfors et al. 2007). However, the

environmental factors regulating the emissions in the ecosystems were unclear. To investigate the

importance of different factors on the N2O emission, a modeling approach was accomplished, using

the CoupModel together with Monti-Carlo based multi-criteria calibration method. The model was

made to represent a forest on drained peat soil in south-west Sweden where data of fluxes

combined with basic background information on soil and plant conditions were used. The model

outcome was consistent with measurements of abiotic (soil temperature, net radiation, groundwater

level and soil moisture) and biotic responses (net ecosystem exchange and soil respiration) from the

site. Also, both dynamics and magnitude of N2O emissions were well simulated compared to

measurements. A result for the robustness of the system was the reasonable nitrogen budget

simulated. The informal likelihoods for an ensemble of accepted simulations of N2O emission

dynamics (coefficient of determination, r2) and magnitudes (mean error, ME) were correlated to

calibrated parameters related to nitrification process and drainage depth. By applying Hornberger-

Spear-Young generalized sensitivity analysis, 12 out of 50 parameters were identified as sensitive

parameters. Parameters (Zp, gmfracN2O, nhrateNH4 and cnm) showed both correlation and

sensitivity to N2O emission within the behavior models selected. Nitrification process was identified

as the most important process regulating the N2O emissions for present forest soil. In addition,

drainage of fertile afforested peat soil had a potential increase of N2O emissions, which suggested

one practical mitigation option for N2O emission reduction is to lower drainage pipes or abandon

drainage.

Key words: N2O emission modeling; Peat soil, forest; CoupModel; GLUE

#Contact author: Hongxing He (hongxing.he@gvc.gu.se)

59

17th European Nitrogen Cycle Meeting, Oslo 2012

P 10

Study of nitrification inhibitors on ammonia oxidizing archaeal community

Yu-Zen Huang1, Toshikazu Fukushima2, Liang-Ming Whang1

1 Department of Environmental Engineering, National Cheng Kung University, Tainan, Taiwan

2 Division of Environmental Engineering, Hokkaido University, Japan

Abstract category: Archaea: important players in diverse microbial ecosystems

Ammonia oxidation is one of the critical pathways in the global nitrogen cycle. It is believed that

ammonia oxidizing bacteria (AOB) is the major group responsible for ammonia oxidation. Recently,

new ammonia-oxidizing organisms belonged to the archaeal domain have been found also involving

in ammonia oxidation. Archaea has been known to have advantage over bacteria in extreme

environments, such as harsh temperature, pH, and has resistance to many inhibitory chemicals in the

environments. Therefore, it may be considered that ammonia oxidizing archaea (AOA) may play

more important role than AOB on ammonia oxidation. Many chemicals, presenting in wastewater

treatment processes, have been reported to inhibit AOB on ammonia oxidation activity. However,

there is no such information available for AOA.

In this study, two laboratory-scale bioreactors with AOA enrichments under different salinity levels

(2.5‰ and 34‰) were used for batch experiments. Although AOA community structures were

different in these two bioreactors, AOA populations were dominant over AOB in both bioreactors.

Batch experiments were conducted to evaluate the resistance of Nitrosomonas europaea pure

culture and AOA enriched cultures to allythiourea and dimethylsulfide. In addition, ampicillin was

added to inhibit AOB activity in batch experiments using AOA enriched cultures. The results

indicated that dimethylsulfide inhibited ammonia oxidation activity of Nitrosomonas europaea, as

well as AOA enrichments in both bioreactors. Addition of 10 mg/L of allythiourea totally inhibited

AOB activity, but resulted in only 50％ and 84％ inhibition on ammonia oxidation of AOA

enrichments in low and high salinity bioreactors, respectively. The results indicated that AOA

enrichments had a higher resistance to allythiourea than Nitrosomonas europaea, and suggested

that AOA may play an important role in bioreactors treating wastewaters containing chemicals that

are known as inhibitors of AOB.

60

17th European Nitrogen Cycle Meeting, Oslo 2012

P 11

Regulatory effect of soil properties on N2O emission from wheat-growing season in

five soils: field and pot experiment

Lebender, U.1, Senbayram M. 1,2 1Research Center Hanninghof, Yara International , Dülmen, Germany

2IAPN – Institute of Applied Plant Nutrition, Göttingen, Germany

Soils are a major source of N2O emission and the two biological processes (nitrification and

denitrification) are responsible for its production. Chemical and physical properties of soils such as

texture and total carbon (C) are important variables which control the formation of N2O from these

processes. However, the impact of these variables on regulatory mechanisms of N2O formation is still

poorly understood. The first objective of this study is to compare five German soil types for their N2O

emission during the wheat-growing season under field conditions. In addition to the field

experiment, the potential denitrification and respiration rate of the soils have been tested in

incubation experiments under standardized anoxic conditions.

Present field and incubation experiments showed that a higher level of available carbon content of

soils (rather than total soil organic matter content) induced higher N2O emission and denitrification

rate. There was a weak correlation between potential denitrification rate and cumulative N2O

emitted under field conditions over a whole season. However, maximum daily N2O fluxes correlated

significantly with the denitrification potential of soils which may indicate that N2O emissions during

high peak events were derived mainly from denitrification.

61

17th European Nitrogen Cycle Meeting, Oslo 2012

P 12

Denitrifying bacteria produce much N2O at low pH, and we are beginning to understand why

Åsa Frostegård1, Binbin Liu1and Lars Bakken2

1 Department of Chemistry, Biotechnology and Food Sciences, Norwegian University of Life Sciences, Ås, Norway

2 Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, Ås, Norway

Denitrification in soil is a major source of atmospheric N2O, a potent greenhouse gas and ozone-

depleting compound. Denitrifying prokaryotes use NOx as terminal electron acceptors in response to

oxygen depletion. The process emits a mixture of NO, N2O and N2, depending on the relative activity

of the enzymes catalyzing the stepwise reduction of NO3- to N2O and finally to N2. Soil pH emerges as

a master variable determining the microbial community composition as well as its denitrification

product ratio (N2O/N2). Several studies demonstrate that this ratio is higher in acid than in alkaline

soil. It is therefore likely that emissions of N2O from agro-ecosystems will increase in large parts of

the world where soil pH is decreasing due to intensified management and increased use of chemical

fertilizers. Considering its immense implications, surprisingly few attempts have been made to

unravel the mechanisms involved in the pH-control of the product stoichiometry of denitrification.

We investigated the kinetics of gas transformations (O2, NO, N2O and N2) and transcription of

functional genes in intact soil samples from long-term liming experiments. Expression of nirS

(encoding nitrite reductase) and nosZ (encoding N2OR) was generally lower at pH6 compared to pH8,

but the nosZ /nirS transcript ratios were similar or even higher at pH6. These results were largely

corroborated in refined experiments using Nycodenz-extracted bacteria from the same soils, which

allowed us to better control the pH levels experienced by the cells. The findings indicated that the

higher N2O/N2 product ratios at pH6 were due to a post-transcriptional effect. Experiments with P.

denitrificans demonstrated N2O/N2 ratios ≤ 10-3 at pH7 while N2O reduction was severely inhibited

by suboptimal pH (~100% N2O production at pH=6). This inhibition occurred during protein

synthesis/assembly rather than at the transcription level since the relative transcription rate of nosZ

versus nirS and norB was unaffected by pH, and since low pH had a moderate effect on the N2OR

activity in cells with a denitrification proteome assembled at pH 7. Ongoing investigations of the

N2OR protein structure will show if this hypothesis is correct. The results are important for the

understanding of main factors affecting N2O emissions from terrestrial ecosystems.

62

17th European Nitrogen Cycle Meeting, Oslo 2012

P 13

Nitrous oxide turnover in the nitrate-ammonifying Epsilonproteobacterium

Wolinella succinogenes

Monique Luckmann1 and Jörg Simon1 1Microbial Energy Conversion and Biotechnology, Department of Biology, Technische Universität Darmstadt, Germany

Global warming is moving more and more to the public consciousness. Besides the commonly

mentioned carbon dioxide and methane, nitrous oxide (N2O) is one of the most important

greenhouse gases and accounts for about 10% of the anthropogenic greenhouse effect.

Several microbial energy converting processes appear to be the most important sources of N2O

emission and the responsible organisms comprise denitrifiers and ammonifiers of nitrate as well as

nitrifying bacteria and archaea. Interestingly, some respiratory nitrate-ammonifying

Epsilonproteobacteria are able to reduce nitrous oxide to dinitrogen via an unconventional

cytochrome c nitrous oxide reductase (cNosZ). The energy metabolism of one of these bacteria,

Wolinella succinogenes, has been characterized thoroughly in the past. The cells are able to use

formate or hydrogen gas as electron donors together with typical terminal electron acceptors like,

for example, fumarate, polysulfide, nitrate, nitrite or nitrous oxide.

Despite the presence of cNosZ there is neither a typical nitric oxide-producing nitrite reductase (NirS,

NirK) nor a nitric oxide reductase of the Nor-type present in W. succinogenes. It is speculated that

N2O might be produced during nitrite and/or NO detoxification by the flavodiiron protein Fdp. Using

different ratios of the electron acceptor nitrate and the donor formate the production and

consumption of nitrous oxide during nitrate respiration of W. succinogenes and various mutant

strains was characterized. Furthermore, a model of the electron transport chain of W. succinogenes

nitrous oxide respiration will be presented.

63

17th European Nitrogen Cycle Meeting, Oslo 2012

P 14

Spatial patterns of soil pH and N2O emissions in field experiments

Aina R. Lundon1, Lars Bakken2, Audun Korsæth1& Peter Dörsch2 1

Bioforsk – Norwegian Institute for Agricultural and Environmental Research. 2 Department of Plant and Environmental Sciences, Norwegian University of Life Sciences.

Emissions of N2O form agricultural soils represent a significant part of the anthropogenic emission of

nitrous oxide (N2O). pH might be an important factor regulating the magnitude of emission form

arable land. Three plots with wheat stubble with similar texture but different levels of pH in a field

trial were selected for measurements of N2O emissions in the off season between September 2010

and May 2011. The amount of N2O-N in µg m-2 h-1 were calculated from analysed gas samples

obtained from static flux chambers form September 2010 till May 2011. Four static flux chambers

were placed within each plot and the levels of pH in the total 12 chamber locations varied between

5.38 and 5.95 (pH measured in CaCl2 solution). The results showed a decrease of emissions in

samples from the plot with average pH 5.80 compared to the plot with average pH 5.48. The plot

with average pH 5.54 did not differ from the other two plots. The emissions varied throughout the

period of sampling with generally low emissions, but with a distinct peak during snowmelt in April.

The results suggest that even marginal increases in soil pH may indeed reduce the emission of N2O.

64

17th European Nitrogen Cycle Meeting, Oslo 2012

P 15

A nitric oxide-producing octaheme enzyme from Kuenenia stuttgartiensis

WJ Maalcke1, A Dietl2, SJ Marritt3, MSM Jetten1, JTM Keltjens1, JN Butt3, TRM Barends2, B Kartal1 1 Department of Microbiology, Radboud University Nijmegen, Heijendaalseweg 135, 6525 AJ, Nijmegen, The Netherlands

2 Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120,

Heidelberg, Germany 3 School of Chemistry, University of East Anglia, Earlham Park, Norwich, NR4 7TJ, United Kingdom

Anaerobic ammonium-oxidizing (anammox) bacteria combine ammonium and nitrite to form

dinitrogen gas. Anammox bacteria are abundantly present in the oxygen minimum zones of the

world’s oceans and have a significant role in global dinitrogen gas production. They are also

successfully applied in cost-effective and environment-friendly ways to treat industrial and municipal

wastewater. Although the physiology of anammox bacteria is quite well understood, the

biochemistry behind it is less clear. One of the persisting anammox enigmas is the presence of ten

copies of an open reading frame that resembles the octaheme enzyme hydroxylamine

oxidoreductase (HAO). HAO converts hydroxylamine to nitrite in aerobic ammonium oxidizing

bacteria. The function of one of these enzymes (kustc0694) is predicted to be hydrazine oxidation to

dinitrogen gas, whereas the functions of the other nine octaheme proteins remain to be established.

Here, a highly expressed octaheme protein was purified from native Kuenenia stuttgartiensis

biomass and characterized. MALDI-TOF mass spectrometry confirmed that the enzyme was encoded

by the open reading frame kustc1061. Enzyme kinetics, assessed by using cytochrome c-based assays

and gas chromatography coupled to mass spectrometry, showed kustc1061 to be a hydroxylamine

oxidizing enzyme that produces nitric oxide instead of nitrite. Protein film electrochemistry revealed

the midpoint potentials of the eight separate heme-groups, and elucidation of the crystal structure

enabled us to form a hypothetical mechanism of hydroxylamine oxidation to nitric oxide. As the

proposed hydrazine-oxidizing enzyme is inhibited by hydroxylamine, the physiological role of

kustc1061 is proposed to be hydroxylamine detoxification

65

17th European Nitrogen Cycle Meeting, Oslo 2012

P 16 The purple form of Marinobacter hydrocarbonoclasticus nitrous oxide reductase

Sofia R. Pauleta1,*, Simone Dell'Acqua1,2, Ana Rute Nunes1, Susana Ramos1, Oliver Einsle3, José J.G.

Moura1, Isabel Moura1 1REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-

516 Caparica, Portugal. 2Dipartimento di Chimica, Università di Pavia, Via Taramelli 12, 27100 Pavia, Italy.

3Institute of Organic Chemistry and Biochemistry and BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-

Universität Freiburg, 79104 Freiburg, Germany.

Email: srp@fct.unl.pt

Nitrous oxide reductase catalyses the final step of the denitrification pathway, the reduction of

nitrous oxide to nitrogen. The catalytic centre, CuZ, is a unique tetranuclear copper centre bridged

by inorganic sulphur in a tetrahedron arrangement that can have different oxidation states.

Previously, Marinobacter hydrocarbonoclasticus nitrous oxide reductase was isolated with CuZ

centre as CuZ*, in the [1Cu2+:3Cu+] redox state, which is redox inert and requires a prolonged

incubation under reductive conditions to be activated. Here, we report the isolation of nitrous oxide

reductase from Marinobacter hydrocarbonoclasticus in the “purple” form, in which CuZ centre is in

the oxidized [2Cu2+:2Cu+] redox state and is redox active. This form of the enzyme was isolated in the

presence of oxygen from a microaerobic growth in the presence of nitrate and also from a strictly

anaerobic growth. The purple form of the enzyme was biochemically characterized and was shown

to be a redox active species, though it is still catalytically non-competent, as its specific activity is

lower than the one of the activated fully reduced enzyme and comparable to the one of the enzyme

with CuZ centre in either the [1Cu2+:3Cu+] redox state or in the redox inactive CuZ* state.

Acknowledgements: We thank FCT-MCTES for the financial support (PTDC/QUI-BIQ/116481/2010), and CRUP-DAAD

Luso-German Integrated Actions.

References: Dell’Acqua et al. JBIC 16 (2011) 183-194. Dell’Acqua et al. JBIC 16 (2011) 1241-1254. Dell’Acqua et al. Philos.

Trans. R. Soc. B 367 (2012) 1204-1212.

66

17th European Nitrogen Cycle Meeting, Oslo 2012

P 17

Autotrophic carbon dioxide fixation via the Calvin-Benson-Bassham cycle by the

denitrifying anaerobic methanotroph Candidatus Methylomirabilis oxyfera

O Rasigraf, DM Kool, MSM Jetten, KF Ettwig

Anaerobic oxidation of methane coupled to nitrite reduction has recently been shown to be

performed by a member of the NC10 candidate division, Methylomirabilis oxyfera. Though living

under anoxic conditions, M. oxyfera oxidizes methane via the 'conventional' aerobic pathway

starting with particulate methane monooxygenase. This is accomplished by the intracellular

dismutation of nitric oxide to molecular oxygen and nitrogen.

In contrast to proteobacterial methanotrophs, however, M. oxyfera does not encode complete

pathways to assimilate carbon from methane (serine and ribulose monophosphate pathways) in its

genome. In line with this, no significant incorporation of carbon from 13C-labelled methane into

biomass could be demonstrated. Like acidiphilic methanotrophs of the phylum Verrucomicrobia,

M. oxyfera possesses the whole gene set encoding the Calvin-Benson-Bassham (CBB) cycle, which is

also being expressed.

Here we provide evidence for autotrophic carbon dioxide fixation by M. oxyfera via the CBB cycle.

Cell-free extracts from the enrichment culture consisting of ~80% of M. oxyfera bacteria exhibited a

specific ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity of ~0.2 nmol CO2 mg-1

protein min-1. This activity accounted for up to 10% of the total methane oxidation activity. In

addition, stable isotope studies with whole cells in batch incubations supplied with 13C-labelled

methane and carbon dioxide were performed. The results showed a significant enrichment in 13C of

putatively specific M. oxyfera-like lipids after the incubation with labeled carbon dioxide and

unlabeled methane but not vice versa. Altogether, these results indicate that CBB cycle is active and

plays a major role in carbon assimilation by M. oxyfera bacteria, and show that autotrophy might be

more widespread among methanotrophs than previously believed.

67

17th European Nitrogen Cycle Meeting, Oslo 2012

P 18

Isotopomer ratios of N2O produced during denitrification by fungal pure cultures and

associated oxygen exchange with water

Rohe, L.1*, Braker, G2, Well, R.1, Giesemann, A.1, Anderson, T.-H.1, Wrage, N.3

1 Johann

Heinrich von Thuenen-Institute, Institute of Agricultural Climate Research, Braunschweig, Germany

2 Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

3 Faculty of Life Sciences, Agricultural Sciences, Rhine-Waal University of Applied Sciences, Kleve, Germany

* Corresponding author: lena.rohe@vti.bund.de; +49 (0)531 5962655

N2O from soil denitrification originates from bacteria and - to an unknown extent - also from fungi. In

pure culture studies, bacterial and fungal denitrification showed differences in isotopomer ratios of

N2O. Isotopomer ratios of N2O can be expressed as 15N site preference (SP), i.e. the difference

between δ15N of the central and terminal N-position of the asymmetric N2O molecule and might be

suitable to distinguish between bacterial and fungal N2O under denitrifying soil conditions.

Oxygen exchange takes place between H2O and intermediates during the denitrification process.

Several studies showed that O-exchange of bacterial denitrifiers varies, but information about fungal

O-exchanges is lacking.

The objectives of the study presented are i) to verify SP of fungal N2O reported for two strains from

pure culture studies and ii) to analyze the oxygen exchange between intermediates and H2O during

denitrification in fungal pure cultures. Six different fungal pure cultures known to be capable of

denitrification were incubated under anaerobic conditions, either with nitrite or nitrate. Gas samples

were analysed for concentration and isotopic signatures (SP, average δ15N, δ18O) of N2O. To

investigate the oxygen exchange, both treatments were also established in a tracer experiment

where 18O-labelled water was used in the medium.

The fungal strains evaluated so far indicate similar SP as reported previously. Concerning the amount

of N2O produced, the nitrite led to a higher yield. Results of O-exchange with 18O-labelled water

during fungal denitrification will be presented.

68

17th European Nitrogen Cycle Meeting, Oslo 2012

P 19

Regulation of denitrification: pathways to nitrous oxide and dinitrogen

S Saggar1, N Jha2, J Deslippe1, J Luo3, R Tillman2, D Giltrap1, H Jamali1 1Global Change Processes Group, Landcare Research, Private Bag 11052, Palmerston North 4442, New Zealand

2Soil & Earth Sciences Group, Institute of Natural Resources, Massey University, Private Bag 11222 Palmerston North

4442, New Zealand 3Climate, Land & Environment Section, AgResearch, Ruakura Research Centre, Private Bag 3123, Hamilton 3240, New

Zealand.

Denitrification is the primary process producing nitrous oxide (N2O) in temperate grassland soils and

it accounts globally for about 60% of total N2O emissions to the atmosphere. We lack knowledge of

reliable quantitative denitrification rates for grazed pasture soils and how these rates vary spatially

and temporally. Issues that hinder this understanding include: difficulties in measuring denitrification

rates, the large spatial and temporal heterogeneity of denitrification products, and the large

background atmospheric N2 concentration. An understanding of the biological processes, and the

soil and environmental factors that have the potential to reduce the production of N2O during

denitrification, is vital for the development of novel and effective N2O mitigation technologies. Our

objectives were to develop and test methods to identify pathways to N2O and N2 production during

denitrification such as enzyme activities, microbial biomass and microbial communities, and

determine the soil factors contributing to spatial variability in denitrification potential of dairy soils.

Experiments were conducted initially to standardise the techniques and develop the protocols for

the measurements of denitrification enzyme activity (DEA), in-situ denitrification rate (DR) in soils.

This followed collection of 10 surface (0-10 cm) and subsurface dairy pasture soils varying in texture,

mineralogy, pH, organic C, N, Olsen P and management history representing North and South Islands

of New Zealand. The DEA, DR, microbial biomass and the N2O to dinitrogen (N2) product ratio

determined using the standardised techniques showed large variations among soils. The amount of

N2O produced was correlated with soil explanatory and response variables to determine the key soil

variables contributing to denitrification potential. Characterisation of denitrifier community by

Terminal Restriction Fragment Length Polymorphism analysis (T-RFLP) of functional genes (nirS, nirK

and nosZ) and quantification of functional gene copy numbers in soils by real time quantitative

polymerase chain reaction (qPCR) has been estimated. Detailed statistical analysis is currently

underway to understand correlations between DR, DEA, soils physico-chemical properties with

denitrifier community structure and gene abundance.

69

17th European Nitrogen Cycle Meeting, Oslo 2012

P 20

Off-season N2O losses from clover-rich swards

Ievina Sturite1, Synnøve Rivedal1, Peter Dörsch2 1

Norwegian Institute for Agricultural and Environmental Research, Norway 2

Norwegian University of Life Sciences, Department of Plant and Environmental Sciences, NO-1432 Ås, Norway

In northern areas, the long winter period between cropping seasons is a challenge to keep nitrogen

(N) within the field, particularly in swards with high clover content. The hypothesis tested was that

freezing-thawing episodes cause N leakage from the standing plant biomass and clover-rich swards

therefore have higher nitrous oxide (N2O) emissions than pure grass swards during the winter.

Gaseous emissions from pure clover and grass stands and from mixed stands containing 30% clover

were measured in western and northern Norway. The sampling time lasted from October 2011 to

May 2012. In order to meet the most important periods of N2O emissions during the winter, the

sampling frequency varied at each site. Preliminary results indicate that stands with clover had

higher N2O emissions than pure grass stands before air and soil temperatures decreased below zero.

The first freezing-thawing cycle increased N2O emissions markedly for treatments containing clover.

Air temperatures around the freezing point resulting in unstable and heterogeneous snow cover led

to high but variable N2O emissions irrespective of treatment. In general, for clover-rich stands N2O

emissions were somewhat higher compared to pure grass stands over the entire seven-month

experiment. This indicates that clover-derived N from damaged plant tissue may be subject to N

pathways that lead to N2O emissions.

70

17th European Nitrogen Cycle Meeting, Oslo 2012

Delegate list

Tulu Abdi Norwegian University of Life Sciences Norway

tulu.degefu@umb.no 4746277075

Liz Baggs University of Aberdeen UK

e.baggs@abdn.ac.uk 441 224 272 691

Lars Bakken Norwegian University of Life Sciences Norway

lars.bakken@umb.no 4797021822

Gracia Becerra University of Extremadura Spain

grbecerral@unex.es 3492725700

Linda Bergaust Norwegian University of Life Sciences Norway

linda.bergaust@umb.no 4764965819

Charlene Bradley University of Birmingham UK

clb716@bham.ac.uk 7772852751

Kristof Brenziger Max Plank Institute for Terrestrial Microbiology Marburg Germany

kristof.brenzinger@mpi-marburg.mpg.de 496 421 178 732

Carlos Bricio CSIC Spain

cbricio@cbm.uam.es 341964525

Emilio Bueno CSIC Spain

emilio.bueno@eez.csic.es 958181600

Klaus Butterbach-Bahl Karlsruhe Institute of Technology Germany

klaus.butterbac-bahl@kit.edu 498 821 183 136

Tom Clarke University of East Anglia UK

Jeff Cole University of Birmingham UK

j.a.cole@bham.ac.uk 441 214 145 440

Naomi de Almeida Radboud University Nijmegen The Netherlands

almeida@science.ru.nl 31 24 3652704

Helen Decleyre Ghent University Belgium

helen.decleyre@UGent.be 3292645134

Maria J. Delgado CSIC Spain

mdelgado@eez.csic.es 958181600 (ext. 327)

Christina Domínguez CINVESTAV Mexico

cris_ale84@hotmail.com 555 539 644 424

Peter Dörsch Norwegian University of Life Sciences Norway

peter.doersch@umb.no 4 791 663 372

Stuart Ferguson University of Oxford UK

stuart.ferguson@bioch.ox.ac.uk 441 869 613 299

Mary Firestone UC Berkeley USA

mkfstone@berkeley.edu 510-6423677

Åsa Frostegård Norwegian University of Life Sciences Norway

asa.frostegard@umb.no 4764965880

Andrew Gates University of East Anglia UK

a.gates@uea.ac.uk 1603592931

71

17th European Nitrogen Cycle Meeting, Oslo 2012

George Giannopoulos University of East Anglia UK

g.giannopoulos@uea.ac.uk +44 (0) 7747336547

Suzanne Haaijer Radboud University Nijmegen The Netherlands

s.haaijer@science.ru.nl 31243652568

Jon Fredrik Hanssen Norwegian University of Life Sciences Norway

jon.hanssen@umb.no 4764965891

Katherine Hartop University of East Anglia UK

k.hartop@uea.ac.uk 7999311294

Junaid Hassan Norwegian University of Life Sciences Norway

junaid.hassan@umb.no 4746258806

Hongxing He University of Gothenburg Sweden

hongxing.he@gvc.gu.se 460 317 862 811

Miriam Herold University of Aberdeen UK

m.herold@abdn.ac.uk 441 224 272 700

Kim Heylen Ghent University Belgium

kim.heylen@ugent.be 3292645142

Marcus Horn University of Bayreuth Germany

marcus.horn@uni-bayreuth.de 49921555620

Yu-Tzu Huang Chung-Yuan Christian University Taiwan

yt_huang@cycu.edu.tw 886 3 265 4913

Yu-Zen Huang National Cheng Kung University Taiwan

louis19890628@hotmail.com 886-6-2757575-65850-37

M. Isabel Igeño University of Extremadura Spain

migeno@unex.es 3492725700 Ext:51333

Hizbullah Jamali Lancare Research New Zealand New Zealand

Jamalih@landcareresearch.co.nz 6 463 534 931

Mike Jetten Radboud University Nijmegen The Netherlands

m.jetten@science.ru.nl 31243652940

Erik Joner Bioforsk Norway

Erik.Joner@bioforsk.no 4 792 833 168

Melanie Kern Technische Universität Darmstad Germany

kern@bio.tu-darmstadt.de 49-6151165239

Catherine Larose University of Lyons France

catherine.larose@ec-lyon.fr 33669719019

Ulrike Lebender Research Center Hanninghof, YARA GmbH & Co. KG Germany

ulrike.lebender@Yara.com +492594798122

Jih-Gaw Lin National Chiao Tung University Taiwan

jglin@mail.nctu.edu.tw 886-3-5722681

Natalie Lim Norwegian University of Life Sciences Norway

natalie.lim@student.umb.no 4796839503

Binbin Liu Norwegian University of Life Sciences Norway

binbin.liu@umb.no 4764965815

Sebastian Lücker University of Vienna Austria

luecker@microbial-ecology.net +43 1 4277 57316

Monique Luckmann TU Darmstadt Germany

luckmann@bio.tu-darmstadt.de 6 151 165 239

72

17th European Nitrogen Cycle Meeting, Oslo 2012

Aina R. Lundon Bioforsk Norway

aina.lundon@bioforsk.no 4740483136

Victor M. Luque-Almagro Universidad de Córdoba Spain

b42lualv@uco.es 34957218318

Wouter Maalcke Radboud University Nijmegen The Netherlands

w.maalcke@science.ru.nl 0031-24-3652704

Daniel Mania Norwegian University of Life Sciences Norway

daniel.mania@umb.no 0047-46231353

Lars Molstad Norwegian University of Life Sciences Norway

lars.molstad@umb.no 0047-99791282

Nick Morley University of Aberdeen UK

n.morley@abdn.ac.uk +44(0)1224 272698

Isabel Moura REQUIMTE-CQFB Portugal

isabelmoura@fct.unl.pt 351212948385

Shahid Nadeem Norwegian University of Life Sciences Norway

shahna@umb.no 4746270730

Graeme Nichol University of Aberdeen UK

graeme.nicol@abdn.ac.uk 441 224 272 258

Lars Peter Nielsen Aarhus University Denmark

biolpn@biology.au.dk 4 560 202 654

Sofia Pauleta REQUIMTE-CQFB Portugal

srp@fct.unl.pt 351212948385

Zhi Qu Norwegian University of Life Sciences Norway

zhi.qu@umb.no 0047-96876850

Olivia Rasigraf Radboud University Nijmegen The Netherlands

olivia.rasigraf@gmail.com 31617073948

Nani Raut Norwegian University of Life Sciences Norway

nani.raut@umb.no 0047-96869118

David Richardson University of East Anglia UK

d.richardson@uea.ac.uk 441 603 593 250

M. Dolores Roldán Universidad de Córdoba Spain

bb2rorum@uco.es 34957218318

Sebastian Runkel University of East Anglia UK

s.runkel@uea.ac.uk 447 909 157 820

Cristina Sanchez Tohoku University Japan

cristina.sago@gmail.com 81-22-217-5687

Andreas Schramm Aarhus University Denmark

andreas.schramm@biology.au.dk 4 560 202 659

Miloslav Simek Institute of Soil Biology, Biology Centre Czech Republic

misim@upb.cas.cz 420 723 734 722

Jörg Simon Technische Universität Darmstad Germany

simon@bio.tu-darmstadt.de 491 704 354 951

Bishal Sitaula Norwegian University of Life Sciences Norway

bishal.sitaula@umb.no 0047-47888760

Daan Speth Radboud University Nijmegen The Netherlands

d.speth@science.ru.nl 31243652569

73

17th European Nitrogen Cycle Meeting, Oslo 2012

Ievina Sturite Bioforsk Norway

ievina.sturite@bioforsk.no 4 793 499 484

Matthew Sullivan University of East Anglia UK

matthew.sullivan@uea.ac.uk 447 881 490 099

Maria J. Torres CSIC Spain

mariajesus.torres@eez.csic.es 958181600 (ext. 327)

Rannei Tjåland Norwegian University of Life Sciences Norway

rannei.tjaland@umb.no 0047-64965904

Maartje van Kessel Radboud University Nijmegen The Netherlands

maartje.vankessel@science.ru.nl +31 24 3652704

Rob van Spanning VU University Amsterdam The Netherlands

rob.van.spanning@vu.nl 31650536015

Claire Vine University of Birmingham UK

c.e.vine@bham.ac.uk 441 214 145 435

Reinhard Well Institute of Agricultural Climate Research , Johann

Heinrich von Thünen-Institut

Germany

reinhard.well@vti.bund.de 495 315 962 535

Liang-Ming Whang National Cheng Kung University Taiwan

whang@mail.ncku.edu.tw 011-886-6-2757575-65837

Daniela Wischer University of East Anglia UK

D.Wischer@UEA.ac.uk 447 743 364 277

Hugh Woolfenden University of East Anglia UK

h.woolfenden@uea.ac.uk 447 796 950 963

Xiaojun Zhang Shanghai Jiao Tong University China

xjzhangsjtu@gmail.com +86 21 34204878

Jing Zhu Norwegian University of Life Sciences Norway

jing.zhu@umb.no 0047-46259348