Emily Mastronardi, Dr. Carlos Monreal, Dr. Maria C. DeRosa

AAFC-ECORC and Carleton University

Smart release technology for improving fertilizer efficiency

• Canada has a large area of cultivated land • Leader in fertilizer production • By 2050 – Increased demand for fertilizer for

food, biomass, biofuels – Population to increase from 7 B to 9.8 B people – Close to 1 B people undernourished – Increase food production 70% for > 2 B more people

• Major economic and environmental implications – 30 to 50% efficiency – N losses to the environment

Why create nanofertilizers in Canada?

2

Fertilizer runoff can cause algal blooms

• Algal blooms deplete oxygen and secrete a neurotoxin, affecting wildlife

• Targeted release problem – Need to increase

fertilizer efficiency

http://globalnews.ca/news/1492850/it-came-from-lake-erie-why-toxic-algaes-a-nightmare-for-canada-too/

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• Synchronize the application of nutrients with crop uptake

The challenge: increase NUE

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1. Use acquired knowledge to propose and test new hypothesis

2. Create new knowledge on soil-crop ecology 3. Develop new tools and devices using

nanotechnology (i.e. DNA aptamers) 4. Integrate knowledge and nano-tools into

novel Intelligent Nanofertilizers

How do we synchronize nutrient release with uptake?

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• Last century – the 1950s and 60’s – Crop roots emit organic materials (Rovira

et al. 1966) – Different crop root species emit different

types of organic compounds (Rovira, 1956)

– Root emissions consist of sugars, amino acids, vitamins, organic acids (Rovira, 1966)

– Root emissions from cereals are similar, but different from exudates from tomato and red peppers (Vancura and Hovadik, 1965)

Interactions and communication in plant-soil systems

http://www.lesco.com/?pageid=63 6

• Early this century – Root emissions control nutrient uptake and soil

microbial growth and function (Dakora and Phillips 2002)

– Increased root emissions occur in response to decreased nitrate (NO3) availability (Darwent et al. 2003)

– The rate of nitrogen uptake in Prairie crops is associated with growth stage (Malhi et al. 2006)

Root exudation and nutrient uptake

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• Plant pores are nano-sized • Use nanotechnology to increase NUE

DeRosa, et. al. “Nanotechnology in Fertilizers” Nature Nano. 2010, 5, 91.

Utilizing nanotechnology

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• Multi-disciplinary approach mixing soil-plant ecology and nanotechnology – New knowledge of soil-plant ecology: Identification

of root exudates associated with soil N mineralization and its uptake by crops

– Development of biosensors to detect root exudates (for on-demand release)

– Development of coating polymers and 3-D nano-coating tools

– Intelligent nano-fertilizer prototypes and products

Testing the central hypothesis

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Intelligent nanofertilizer – proposed technology

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Intelligent nanofertilizer – proposed technology

Biosensor with polymer layers

Urea

- 1 nanometer = 10-9 m or billionth of a meter

(10 to 100 nm)

Root exudates

• The biosensor will be incorporated into a very thin polymer film.

• Interaction of the biosensor with the desired root exudate changes the permeability of the polymer film:

release of urea-N according to crop demand.

• Immediate goals: 1. Identify important root

exudates 2. Develop biosensor to detect

them 12

- Crops: wheat and canola (each grown twice) - Soil: Manotick, uncropped for 15 years, 0-20 com depth - Treatments: 1) Soil alone 2) Soil + crop 3) Soils + crop (no crop) (0 N) (100 kg urea-N/ha) - Weekly characterizations: soil solution composition, enzyme activities, microbial biomass, crop N uptake and yield, C and N flows in rhizosphere using 13C and 15N - Soil solution - Four techniques of Mass Spectrometry: - Py-FIMS, GC-MS, ESI-MS, LC-ESI-MS/MS

Identifying exudates important for nitrogen uptake

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• Found increased N uptake during specific growth periods

• Used Mass Spectrometry

to identify exudates in the soil during this time

kg N

/ha/

d

3 leafs unfolded

1

2

0.1

0.2

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

200 400 600Temperature in °C

Inten

sity

m / z

IF03157

71

8599126

178 222

278

294306

318

503 577651

4 leaves unfolded, September 30

Identifying exudates important for nitrogen uptake

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Canola Wheat

Identifying exudates important for nitrogen uptake

• Found exudates that track with nitrogen uptake

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Identifying exudates important for nitrogen uptake

• Identified 12 chemical signals closely associated with crop N uptake

• Some signals are specific to wheat and others to canola

• Potential for designing crop specific fertilizer

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Nano-biosensors – Aptamers

• Single-stranded oligonucleotides

• Synthesized chemically • Fold into 3D nanoscale

shapes capable of binding targets

• Can distinguish small structural differences

http://www.genelink.com/newsite/products/aptamers.asp 17

Aptamers selected through iterative process called SELEX

• Systematic Evolution of Ligands by EXponential enrichment

• In vitro technique beginning with 1012-1015 random DNA sequences

• Each round increases affinity of pool for the target

• Pool is cloned and sequenced

• Control over selection conditions

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• Alternating layers of positive/negative polyelectrolytes creates a film

• DNA aptamers can act as the negative layer, making the film responsive

Layer-by-layer deposition creates smart aptamer films

Sultan, DeRosa, Monreal Biomacromolecules 2009, 10, 1149–1154 19

• Aptamer films show higher target binding and higher permeability than control films

Mastronardi et.al. (2015) Methods

Embedded aptamers retain binding and increase permeability

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Mastronardi et.al. (2014) Sensors, 14:3156-3171

• A) Aptamer-target binding leads to change in permeability and release of payload

• B) Aptamers act as structural support for microcapsule, and target-binding leads to microcapsule rupture

Zhang et.al. (2013) ACS App. Mater. Interfaces, 3: 5500-5507 Sultan and DeRosa. (2011). Small, 7: 1219-1226.

Layer-by-layer deposition creates smart aptamer microcapsules

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• A and D: Fluorescein channel

(Aptamer)

• B and E: Rhodamine channel (Target)

• C and F: Overlaid signals - binding

Aptamer microcapsules maintain target binding

• Fluorescence co-localization study

Sultan and DeRosa. (2011). Small, 7: 1219-1226. 22

Aptamer capsules specific for a root exudate show increased dye permeability

Target molecule Diffusion coefficient (µm2/s)

Aptamer film with Exudate 0.0113± 0.0040 Aptamer film with Negative

control 0.0051± 0.0008

Zhang et.al. (2013) ACS App. Mater. Interfaces, 3: 5500-5507 Sultan and DeRosa. (2011). Small, 7: 1219-1226.

A

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• Microcapsules show target-triggered morphology changes – Morphology unaffected

without target

• Rupture controlled by target concentration and time

Aptamer capsules for exudate-triggered delivery

Zhang et.al. (2013) ACS App. Mater. Interfaces, 3: 5500-5507 24

• Developing nanofertilizers requires a multi-disciplinary approach

• Chemical signals (exudates) important for nitrogen uptake have been identified in wheat and canola

• Biosensors (Aptamers) have been developed for these exudates

• Early prototypes of smart-release nanobiosensors have been developed for use with intelligent fertilizers

Concluding remarks

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• Organizations – The Alberta Innovations Bio Solutions and AAFC for providing financial support – Grain Growers of Canada, Canola Council of Canada and Agrium Inc. – La Coop Fédérée

The DeRosa lab Carleton University

Department of Chemistry

The Monreal lab Agriculture and Agri-Food Canada

ECORC

Acknowledgements

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