Functional and Molecular Diversity in Nitrogen Cycle Enzymes under Contrasting Agricultural Management Systems

Jeanette Norton John Stark

Jennifer ReeveGraduate Students:

Yang OuyangAvneet Kakkar

Mussie HabteselassieGraduate Student:

Lisa Woodruff

On a global basis reactive N inputs related to human activities now exceeds natural N inputs

From Fowler, D., M. et al. 2013. The global nitrogen cycle in the twenty-first century. Philosophical Transactions of the Royal Society B-Biological Sciences 368.

Why study nitrogen mineralization and nitrification in agricultural systems?

• Typical fertilizer N use efficiencies are often less than 50% due to N loss.

• N mineralization and nitrification are key N-transformations that largely determine the availability and mobility of N in soils

• Nitrification leads to denitrifcation , N2O emissions and nitrate leaching

• Decrease the impacts of N loss and increase N fertilizer use efficiency in agricultural ecosystems, aim to match soil N supply with plant demand

Nitrification oxidizes ammonium to nitrate

NH4+ NO2

- NO3-

Ammonia monooxygenaseAMO

Ammonia oxidizers

Nitrite oxidoreductaseNXR

Nitrite oxidizers

• Both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) have the enzyme ammonia monooxygenase.

• Our understanding of the relative contribution of AOA and AOB to nitrification in different environmental conditions is limited

Utah State University SiteStrongly calcareous Millville silt loam (Typic Haploxeroll) pH 8.0, irrigated Field plots planted to silage corn, started 2011CRB 4 N source treatments x 4 repsControl (no additional N)Ammonium sulfate (AS100 & 200 kg N ha-1) Composted steer manure (200 kg N ha-1)

*

University of Georgia SiteCecil sandy loam (Fine, kaolinitic, thermic Typic Kanhapludults, pH 5.31:1 Field plots planted to corn, started 2012CRB 4 N source treatments x 4 repsControl: No Nitrogen fertilizerAmmonium sulfate (AS100), 112 kg N ha-1

Ammonium sulfate (AS200), 224 kg N ha-1

Poultry litter (PL) and cover crop N, 112 kg N ha-1

•AOA numbers higher than AOB but unchanged by treatment •AOB numbers affected by treatment and year, significant after 1 year

•AOB increase with repeated treatments of AS fertilizers

Abundance of ammonia oxidizers- Utah

Georgia- Abundance of ammonia oxidizers

•AOA numbers higher or similar to AOB, AOA unchanged by treatment •AOB numbers affected by treatment and year, significant in year 2 year

•AOB increase with repeated treatments of AS fertilizers at AS200 level

UtahAre there changes in AO community composition?

GeorgiaAre there changes in AO community composition?

Changes observed for AOB communities by treatment and year, diversity (Shannon Index) decreased for AS treatments

Nitrification potential in August-Utah

UtahHow do these changes in community relate to process rate ?Nitrification potentials in soil slurries 2014 – seasonal rates

GeorgiaHow do these changes in community relate to process rate ?Nitrification potentials in soil slurries 2014 – seasonal rates

How to evaluate the relative contribution of AOA and AOB?

(Taylor et al 2013)

AOA are resistant to octyne, while AOB are sensitive to octyne.

Octyne

Nitrification potential

Octyne-resistant nitrification potential

AOA

Octyne-sensitive nitrification potential

AOB

1-Alkyne C8

Fresh soil

NH4+

The differential inhibitor: 1-Octyne

Potential nitrification rates (~1 mM NH4

+)

compared to

Gross nitrification rates from isotope pool dilution (at ambient ammonium).

Note

scal

e 20

XAug 2014

Aug 2014

AOB and AOA activities change with increasing ammonium concentration Note scale 10X

AOB respond to additional NH4+ while AOA’s

capacity is saturated at relative low NH4+

concentration.

Nitrification kinetic parameters from Michaelis-Menten model

0 5 10 15 20 25 300.0

0.2

0.4

0.6

0.8

1.0 AOB

NH4+ (μM )

Nitr

ifica

tion

rate

(mm

ol N

kg-

1d-1

)

0 5 10 15 20 25 300.0

0.2

0.4

0.6

0.8

1.0 AOB

NH4+ (μM )N

itrifi

catio

n ra

te(m

mol

N k

g-1d

-1)

AS200 Compost

AOB: Vmax= 4.8 mmol N kg-1 d-1; km=161 μMAOA: Vmax= 0.3 mmol N kg-1 d-1; km=3.6 μM

AOB: Vmax= 0.7 mmol N kg-1 d-1; km=44 μMAOA: Vmax= 0.2 mmol N kg-1 d-1; km=2.9 μM

Vmax is the maximum rate.

Km is the half-saturation constantV=Vmax*S/(Km+S)

How AOA and AOB activities respond to temperature?

Activity and abundance of nitrite oxidizers (all Nitrospira) in 2015

Ammonium fertilizers increase nitrite oxidation, but not abundance

UtahN mineralization enzyme activities in 2014

Arginase Urease

Protease β-glucosaminidase

Take home messages• Ammonia-oxidizing bacteria were more responsive than archaea to

N source. AOB abundance and community structure were changed significantly by ammonium fertilizers.

• Ammonium availability and temperature controlled the relative contribution of AOA and AOB to nitrification.

• Controlling the activity of AOB immediately after the application of

mineral N fertilizers would be an effective strategy to reduce and delay nitrification and therefore improve N use efficiency.

• Understanding the differential response of AOB and AOA to substrate and temperature may allow the nitrifier community to be included into simulation models as dynamic components driving N flux, linking microbial communities and function

Acknowledgements

FundingAgriculture and Food Research Initiative Competitive Grants Program Grant no. 2011-67019-30178 from the USDA National Institute of Food and Agriculture. Utah Agricultural Experiment Station

Graduate Students:Yang OuyangLisa WoodruffAvneet Kakkar

Field and lab work:Danielle BarandiaranCory OrtizCraig Marlen RiceJeremiah MooreHenry Linford

Rates TreatmentsVmax (mmol N/kg/day) Km (µM)

Jun Aug Jun Aug

Octyne-sensitive

Nitrification

Control 0.41 ±0.18 a 0.32 ±0.15 a 19.4 ±23.6 a 13.58 ± 5.4 a

AS100 2.47 ±0.45 b 1.41 ±0.43 b 86.2 ± 9.0 b 58.90 ±26.1 b

AS200 4.77 ±1.33 c 2.15 ±0.39 c 160.9 ±34.7 c 89.25 ±19.6 b

Compost 0.67 ±0.09 a 0.61 ±0.16 a 44.0 ±13.8 a 31.42 ±7.80 a

Octyne-resistant

Nitrification

Control 0.20 ±0.02 0.22 ±0.01 4.1 ±3.3 3.15 ±0.66AS100 0.22 ±0.04 0.25 ±0.07 11.2 ±11.1 3.30 ±1.66AS200 0.27 ±0.12 0.33 ±0.09 3.6 ±2.46 2.88 ±1.25Compost 0.20 ±0.03 0.23 ±0.01 2.9 ±0.84 3.07 ±0.31

Nitrification kinetic parameters from the Michaelis-Menten model results

Seasonal dynamics of activity of ammonia oxidizers

Fertilizers

Fertilizers

Are there changes in nitrifying community composition?

The nitrifying community was not altered after the first year of fertilization, but was significantly shifted by four years of repeated application of ammonium fertilizers.

Treatment: p=0.001 Year: p=0.001