Development of Intelligent Nano-Fertilizers

C.M. Monreal

AAFC-ECORC
Manitoba Special Crops Symposium
Winnipeg, Manitoba
February 8, 2012
Focus of todays presentation:

- Food, biomass and fertilizers

- The issue: NUE and leakages in Soil N cycle

- Innovation in fertilizer technology

- Intelligent Nano-Fertilizer: a conceptual model

- R&D Progress: soil-crop ecology and nanotechnology


Mother earth: the Mayan, Iroquois and Chinese legends
We know more about the movement of celestial bodies
than about the soil underfoot

Leonardo DaVinci, circa 1500
The Hour Glass Nebula
8000 light years away
Fertilizer Use and Food Production
1. Canada - leader in fertilizer production
- 12.4 Mt total production 5 Mt exported
- Exports 10 % of total world trade in N-fertilizer
2. Increased demand: fertilizer for food, biomass - 2050
- Population to increase to 9,800 Billion people
- Close to one billion people undernourished, exacerbated by price increases
- 70 % food production for > 2 Billion more people
- Cereal production to grow by 1 Bt/yr; Meat to increase > 200 Mt/yr
- 72 % of food to be consumed in developing countries
- > double the E demand for 2050
3. Major Environmental and Economic Implications
- Mitigation and adaptation to climate change
- Use and conserve natural resources (soil, water, air)
- Cost of food production

The Issue

NUE
(U
f
U
0
)
F
N
*100
NUE = Nitrogen Use Efficiency/apparent N recovery (%)
U
f
= N-uptake by N-fertilized crop (kg ha
-1
)
U
O
=N-uptake by non N-fertilized crop (kg ha
-1
)
F
N
= added N-fertilizer (kg ha
-1
)
Fertilizer-Nitrogen Use Efficiency (NUE) by crops ranges from 30 - 50 %

remainder is lost via volatilization, denitrification, leaching and stabilization
into SOM.

Time
I
n
n
o
v
a
t
i
o
n

L
e
v
e
l

0
2
4
6
8
10
12
14
16
18
20
I 1
I 2
I 3
Innovation in Fertilizers
Agronomy
Government
Regulations
Enhance N use efficiency
& Market leadership
# of Polymer CRF Patents by Year
Source: Agrium Advanced Technologies (AAT)
Published
Data
Incomplete
#

o
f

P
a
t
e
n
t
s

Total = 181
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1961 1962 1963 1964 1965 1966 1968 1969 1970 1973 1974 1975 1976 1977 1980 1981 1982 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005
1986
1961
2005
We are at the flat end of an
innovation curve that
started in 1961
- Synchronize the release of fertilizer-N with its uptake by crops

HOW?
- Using acquired knowledge to propose and test new hypothesis
- Create new knowledge of soil N and C cycling in crop rhizosphere
- Using new advances in nanotechnology (i.e., nanobiosensors)
- Integrating knowledge, tools and devices into novel nanofertilizers
The Challenge - Achieving New Level of Innovation
- Organization, Scientists and Engineers
AAFC - C. Monreal, S. Malhi, B. Ma, M. Schnitzer,
Carleton University - M. deRosa, F. Matus
University of Ottawa - P. Mayer
University of Alberta - P. Choi
Agrium Inc. - J. Jenkins, B. Gilbertson
Grain Growers of Canada
Canola Council of Canada
The Research Team - Phase I (2008-2012)
- Funding agencies
- Alberta Innovates Bio Solutions: $800 K/4 yrs
- Agriculture and Agri-food Canada: $250 K/3 yrs
- Other:
- Sussex Research - G. McRae
- Rostock University - P. Leinweber
The soil N Cycle
Soil
Organic
Matter
NO
3
-
NH
4
+

Harvest
N
2
O
N Fertilizers
Manure
Residues
CO
2

NH
3
Root Exudation as Chemical Signals
Last Century - the 1950 and 60s (Axenic
crop cultures)
Crop roots exudate or 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 than exudates from tomato and red
peppers (Vancura and Hovadik 1965)
Early this century
Root emissions - control nutrient uptake & soil microbial growth and function
(Dakora and Phillips 2002).
Increased root emissions occur in response to decreased nitrate (NO
3
)
availability as a consequence of increased root length (Darwent et al. 2003).
The rate of nitrogen uptake in Prairie crops is associated with growth stage
(i.e., mid-tiller vs. maturity or senescence; Malhi et al. 2006).
The Science - Root Exudation and Nutrient Uptake
Central Hypothesis
- Under limiting N supply, crops emit specific chemical
signals to stimulate N mineralization by soil microbes
for N uptake by plants.
Achieving N Synchronization in Soil-Crop System
- Need to understand the interactions
between plant-soil components and
processes controlling soil N cycling

- Such understanding may help manipulate
soil-plant-fertilizer systems to achieve
synchronization
Time
R
e
s
p
o
n
s
e

Root
exudation
Soil
N mineralization,
nitrification
Amt. of enzyme
or gene
Amt. of
glucose
Glucose
Necessary Confluence of
Soil Ecology with Nanotechnology
New knowledge in soil-plant ecology
Identification of root chemical signals associated
with soil N mineralization and its uptake by crops
Development of nanobiosensors to detect these
chemical signals (controlled release of drugs)
Root chemical signals, nano-biosensors and
coating polymers may be used in nanofertilizers
to synchronize crop N uptake with supply of
fertilizer-N
An Intel. Nano-fetilizer model

Syn
A Synchronization Model for Crop Nitrogen Uptake
and Nitrogen Release by Fertilizers
Organic acid
Intelligent
NanoFertilizer
Ammonia
Sugar
Intelligent NanoFertilizer - Proposed Technology
The biosensor will be
incorporated into a very thin
(< 100 nm) polymer film.

Interaction of the biosensor
with the desired root chemical
signal would lead to a change
in the permeability of the
polymer film, allowing the
release of urea-N according to
crop demand.



Biosensor with polymer layers
Urea
- 1 nanometer = 10
-9
m or billionth of a meter

(10 to 100 nm thin)
Aptamers are synthetic stretches of DNA or RNA that
can fold into nanosized shapes capable of binding to
molecular targets (i.e., root chemical signals)
Aptamers and Enzymes
H3C
CH3
Remarkable selectivity & sensitivity
Theophyline aptamer - difference of a
single methyl group leads to 10 000
times weaker binding.
- Kd values in the micromolar to sub-nanomolar range

Nano-biosensors
A Summary of Experimental Work -
- Crops: wheat and canola (each grown twice)
- Soil: Mannotik, 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
13
C and
15
N

- Soil solution - Four techniques of Mass Spectrometry:
- Py-FIMS, GC-MS, ESI-MS, LC-ESI-MS/MS
Soil Organic
Nitrogen
NH
4
+
NO
2
-
NO
3
-
CROP
Soil
organisms
Nitrification:
15
N
N
2
Immobilization:
15
N
N mineralization:
15
N
AOB
amoA
NOB
nxrA
Denitrification:
15
N
Crop N uptake and Soil N Cycling
(Using Soil DNA and Stable Isotope Tools)
Leaching:
15
N
Crop uptake:
15
N
1. Soil Enzymatic activity
These methodologies show the maximum potential activity of a
specific enzyme under optimum conditions in soil samples.

2. Detection of specific genes in soil DNA
Genetic potential of soil microbial communities to produce enzymes

3. Soil RNA for the expression of specific soil genes
It is associated with microbial activity synthesizing specific proteins.

What tools of biology can be used to examine soil N cycling processes?
Soil DNA extraction
2. Detection and quantification of specific genes in soil DNA
-Functional gene amoA codifies the enzyme AMO in ammonia oxidizing bacteria
-Functional gene nxrA codifies the enzyme NXR in nitrite oxidizing bacteria
Methodology for detection of genes
Polymerase chain reaction (PCR)
- For the detection of genes, specific primers are necessary
amoA: amoA F1, amoA R2
nxrA: F1nxrA and R2nxrA (Wertz et al, 2008).
Electrophoresis
amoA F1, amoA R2 F1nxrA, R2nxrA
500 bp
300 bp
Soil
samples
Positive control
Nitrosomonas
europaea
Soil
samples
Positive control
Nitrobacter
- It is necessary optimize the conditions like:
Primer specificity and concentration
Taq polymerase concentration
DNA template concentration
Time and temperature of the reaction
Norgene kit: total soil DNA
Use of Stable Isotopes (
13
C &
15
N)
- Identify main components and processes
- Quantify C and N fluxes in the soil-crop rhizosphere (
13
C and
15
N)
- Ascertain the origin of chemical signals in soil solution

Carbon flows: Photosynthesis and Root Exudation:
0
500
1000
1500
2000
2500
2 6

1
3
C

M
B

(

)
Labelling exposure (hours)
Shoot wheat
-50
0
50
100
150
200
0 2 6

1
3
C

M
B

(

)
Labelling exposure (hours)
Microbial biomass
a
c b
& Soil Solution (SS)
MB
MB
MB
SS
k
g

N
/
h
a
/
d

2009
3 leafs
unfolded
Canola 0 N - 2011
0.0
0.5
1.0
1.5
2.0
2.5
0 6 13 21 29 35 42 49 63 69
R
a
t
e

o
f

s
h
o
o
t

N

u
p
t
a
k
e

(
m
g
/
d
a
y
)
Canola - Rates of N Uptake
2011
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
0 6 10 21 29 35 42 49
d
e
l
t
a

1
5
N
NH4
NO3
Incorporation of
15
N-NH
4
into
15
N-NO
3
Canola 0 N - 2011
0.0
0.5
1.0
1.5
2.0
2.5
0 6 13 21 29 35 42 49 63 69
R
a
t
e

o
f

s
h
o
o
t

N

u
p
t
a
k
e

(
m
g
/
d
a
y
)
Canola - Rate of N Uptake and Soil Nitrification
Days after seeding
Canola 2011 nxrA gene copy number
(NO
2
to NO
3
)
Canola 2011
NoN=0 kg N/ha WithN= 100 kg N/ha
Tango for two: Organic compound follows crop N uptake dynamics
R
a
t
e

o
f

c
r
o
p

N

u
p
t
a
k
e

(
g

N
/
/
k
g
/
d
)

T
o
t
a
l

i
o
n

c
o
u
n
t
s

Number of weeks
t
Root signals chemical language in soil solutions
Chemical characterization - Soil Solutions
Pyrolysis Field Ionization Mass Spectrometry (Py-FIMS)
1
2
0. 1
0. 2
50 100 150 200 250 300 350 400450 500 550 600 650700 750 800 850 900
200 400 600
Tem per at ur e in C
In
te
n
s
ity
m / z
I F031
5 7
7 1
8 5
9 9
1 2 6
1 7 8 2 2 2
2 7 8
2 9 4
3 0 6
3 1 8
5 0 3
5 7 7
6 5 1
4 leaves unfolded,
September 30
Principal Component Analysis - (Py-FIMS data)
Most significant m/z in soil solutions for 4 phenological stages of wheat
- 4
- 3
- 2
- 1
0
1
2
3
4
5
6
7
- 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 0 1 2 3 4 5 6
I F0 9 3
I F0 9 3
I F0 9 3
I F0 3 1
I F0 3 1
I F0 3 1
I F1 3 7
I F1 3 7
I F1 3 7
I F1 9 6
I F1 9 6
I F1 9 6
PC1 ( 6 1 . 4 % )
P
C
2

(
2
1
.
7

%
)
7 leaves, flag leaf unfold, October 29
4 leaves, 2 tillers, Sept 30
Early milk, Nov 19
Soft dough, December 16
Nitrogen Uptake - Chemical Signals 2009
k
g

N
/
h
a
/
d

Days after seeding
n
g
/
m
l

Compound 1
n
g
/
m
l

Compound 2
Compound 3
All Compounds in Soil Solution
(confirmed by LC-ESI-MS/MS)
Soil Ecology - Chemical Signals
- So far, we have:
- Identified/verified/quantified 12 chemical signals
closely associated with crop N uptake.

- Some of the 12 chemical signals in solution are
specific to wheat and others to canola.

- Potential for designing crop specific fertilizer
Functional Nanoscale Aptamer Films
Can aptamer-target
binding influence
the properties of
these films?
Can an aptamer still
bind its target while
immobilized in a
nanoscale film?
Layer-by-Layer approach for
nanoscale polyelectrolyte films
Sultan, DeRosa, Monreal Biomacromolecules 2009, 10, 11491154
Poly(diallyldimethylammonium)
chloride
poly(sodium 4-styrene-sulfonate)
Current work involves using
these films to make aptamer
shells
Film deposition by Layer-by-Layer
approach on sacrificial templates
Sultan and DeRosa, 2010
submitted to Small
Aptamer capsules maintain target binding
Fluorescence co-localization
confirms target binding
A and D: Fluorescein
channel (Aptamer)
B and E: Rhodamine
channel (Target)
C and F: Overlaid
signals
Can aptamer binding affect
permeability of films?
Capsules show increased permeability for the
target
Microcapsule
composition
Diffusion
coefficient (m
2
/s)
PAH/PSS 0.0029 0.0019
RO without heating 0.0031 0.0004
SA

without heating 0.0055 0.0025
RO with heating 0.0024 0.0007
SA

with heating 0.019 0.011
Concluding Remarks - NanoFertilizers
Innovation in nanofertilizer requires intra- and multi-disciplinary and multi-agency
approach.
Identified chemical signals in soil solution, now constructing nanobiosensors.
Built early prototype of nano-biosensors for use with Int. Nano-Fertilizer
Integration of Soil Science with Biotechnology and Nano-biotechnology will help
Canadian agriculture and the fertilizer industry maintain its global leadership.
High risk & strategic research needs long-term public funding and modern infrastructure.
Acknowledgements
Organizations
- The Alberta Innovations Bio Solutions and AAFC for providing financial support
- Grain Growers of Canada, Canola Council of Canada and Agrium Inc. x support

Individuals
- F. Matus, B. Ma, S. Malhi, S. Wu, M. Schnitzer, Students, Volunteers - AAFC
- J. Jenkins - Agrium Inc.
- M. DeRosa & Y. Sultan - Carleton University
- G. McRae Sussex Research
- P. Mayer and S. Curtis - University of Ottawa
- P. Choi - University of Alberta
- P. Leinweber - Rostock University

QUESTIONS?