Improving Micronutrient Fluid Fertilizers using Novel Chelating Agents

Mik M L hli 1 2 d S l St 1Mike McLaughlin1,2 and Samuel Stacey1

1Earth and Environmental Sciences, The University of Adelaide, AUSTRALIA 2CSIRO L d d W t U b AUSTRALIA2CSIRO Land and Water, Urrbrae, AUSTRALIA

Areas of the world prone to micronutrient deficiency

Stubble where no zinc was applied has a dirty, grey appearance, compared with the stubbles to which zinc was addedadded

Zinc deficient crops later in ripening.Zinc deficient crops later in ripening.

Reactions of micronutrients in soils

PbpK18 0

Soils are predominantly negatively chargedbe

d

PbCu

Zn

8.08.0

9.0 negatively charged, and sorb trace elements very t l i llt s

orb

Co

Ni

9.5

9 6 strongly, especially in alkaline soils

Am

oun Ni

Mn9.6

10.6

M2+ + H2O

= MOH+ + H+

A

pH6 7 8

Reactions of micronutrients in soils

Trace elements also form very insoluble precipitates in soils, especially with phosphate, carbonate and hydroxide ions. This is a

ti l bl i h h ti f tili f l ti i lk liparticular problem in phosphatic fertiliser formulations in alkaline soils, where P concentrations in the fertilised band are high.

Compound pKsp

CaCO3 1.0 X 10-8.3

Cu(OH)2 1.6 X 10-19

CuCO 1 4 X 10-10CuCO3 1.4 X 10 10

Cu3(PO4)2 1.4 X 10-37

Mn(OH)2 2.0 X 10-13

MnCO3 2.2 X 10-11

ZnCO3 1.5 X 10-10

Zn(OH)2 1.2 X 10-17( )2

Zn3(PO4)2 9.0 X 10-23

Compatibility issues

Solubility of ZnO in polyphosphate solutions (from Mortvedt, 1991).

Polyphosphate contentFertiliser Solution pH5.0 6.0 7.0Polyphosphate content

(% of total P) ------------------ % Zn ------------------40 0.6 1.3 1.650 0.7 1.6 1.960 0.7 1.7 2.070 0.7 1.7 1.680 0.7 1.4 2.6

Chelates used to complex micronutrient ions

Organic molecules that complex micronutrient (metal) cations

Alters the charge of the fertiliser ion, which changes its fate in soil and affects absorption by plantsp y p

-

Zn2+ + EDTA4- ZnEDTA2-

soilsoil22

ZnEDTAZnEDTA22-- Electrostatic repulsion-

--

-ZnZn2+2+

Adsorption & fixation-

Problems with anionic chelates

ZnEDTAZ 2+

Charge reversal allows the cation to be mobile in the soil environment

Zn2+

the soil environment (subject to wash off/leaching)

Zn = 50nM

In nutrient solution

Plants do not absorb–vely charged metal-chelate complexes c

upta

ke

ZnT = 50nM

chelate complexes readily due to negative membrane potentials

Zinc

EDTA added to solution

Current Trace Element Fertilizers

Limitations to their effectiveness:Current trace element fertilizers are relatively ineffective on yalkaline and calcareous soils, where micronutrient deficiencies are most severe;

Synthetic chelates (e.g. EDTA) are not readily absorbed by roots due to membrane exclusion;

Negatively charged complexes may be electrostatically repelled from plant roots;

Product cost has mainly limited their use to high-value crops.

Environmental Pollution:EDTA is a persistent substance, has been found ubiquitously in the environment and has caused environmental degradation;

NTA is carcinogenic and has been banned in the USA.

Requirements for effective micronutrient fertilizers

Reduce trace micronutrient adsorption or precipitation.

Increase solution metal concentration (to aid mass flow and diffusion))

Increase labile pool of metal in soil

Membrane permeable or actively transported by root uptake mechanisms (minimises –ve charge on metal complex)

Environmentally friendlyEnvironmentally friendly

Inexpensive

Rhamnolipid: a ‘lipophilic’ chelate

Rhamnolipid is a biological chelating agent produced by bacteria;

Non-toxic, biodegradable, can be synthesised or purchased;

Product complexes trace elements (Zn, Cu, Mn etc) into a ‘lipophilic’ state;

The chelate can be readily absorbed by plant roots;

Less prone to leaching or surface runoff than EDTA and DTPALess prone to leaching or surface runoff than EDTA and DTPA.

Micelle + Trace ElementsRhamnolipid Monomer Elements

Hydrophilic HydrophobicHydrophilic HydrophobicHead tail

R1 Rhamnolipid biosurfactant

Metal complexing

Hydrophobic

complexing group

OH Hydrophobic groups

OH

R2 Rhamnolipid – second rhamnose sugar

Polyethyleneimine - PEI: a plant-available polymer?

Chelating polymer with an extremely high complexing capacity for trace elements (chelate rates could be reduced by 75%);

Used for transmembrane delivery of DNA and other pharmaceuticals

Less prone to leaching or surface runoff than EDTA;

Ethyleneamine Polyethyleneamine

Cu2+ complexing capacity of EDTA and PEIChelate

(g Cu2+/g chelate)Max. Complexing Capacity

PEIEDTA

1.490.37

Measuring lipophilicity of rhamnolipid

hydrophobic phase

Normally used to measure biomagnification potential of pesticides and other organic chemicals

[Fert] octanol

phasechemicals

Ko/w = [Fert] octanol[Fert] water

Water phase

CuSO4 CuSO4 +rhamnolipid

Lipophilic complexes with rhamnolipid

25Zn

20

ZnCuMn

15

Ko/

w

Mn

5

10K

0

5

Rhamnolipid EDTA PEI SO42-

Response of Zn deficient to rhamnolipid: Glasshouse trial on calcareous soil in Turkey (Ismail Cakmak)

Response of Zn deficient soil to PEI: Glasshouse trial in Turkey (Ismail Cakmak)

Response of Zn deficient soil to PEI: Glasshouse trial in Turkey (Ismail Cakmak)

Wheat dry matter response to rhamnolipid.Bars denote L.S.D. (p ≤ 0.05)

0.85 Durum wheat

(g/p

lant

)

0.75

0.80Bread wheat

rodu

ctio

n (

0 65

0.70

y M

atte

r pr

0.60

0.65

Dry

0.50

0.55

Rhamnolipid concentration (mg/kg)

0 2 4 6

Effect of rhamnolipid application on Zn uptake by bread wheat. Bars denote LSD (P ≤ 0.05)

/kg) 35 0.18Shoot Zn concentration

atio

n (m

g

25

30

(mg/

pot)

0.14

0.16Total Zn in shoots

conc

entra

15

20

sho

ots

(

0.10

0.12

hoot

Zn

c

5

10

15

otal

Zn

in

0 06

0.08

0.10

0 2 4 6

Dry

Sh

0

5 To

0.04

0.06

Rhamnolipid rate (mg/kg soil)Rhamnolipid concentration (mg/kg soil)

Glasshouse trial on Zn uptake by canola

The new products are more effective trace element fertilizers than EDTA on alkaline and calcareous soils.

Alkaline sodosol

Highly calcareous (39% CO )10

12

s)

Birchip soilHighly calcareous (39% CO3)

8

10

Zn/g

sho

ots Streaky Bay soil

4

6

upta

ke (µ

g

0

2Zn u

OZnSO4 EDTA Rhamnolipid PEI4

Absorption of Zn complexes by canola roots

EDTA, rhamnolipid and PEI compared as Zn chelantscompared as Zn chelants

Canola plants, kinetic uptake of chelated fertilizers Zinc speciation determined by

Anodic Stripping Voltammetry

New chelates are readily absorbed by plant roots

Both new products were readily taken up by canola roots.

Zn EDTA was excluded at the root membrane surface and not readilyZn EDTA was excluded at the root membrane surface and not readily absorbed by canola.

Uptake of Zn by canola roots in solution culture

2

2.5

/g ro

ot)

1

1.5

c Zn

( µg

Zn/

0

0.5

Sym

plas

ti

0ZnCl2 EDTA PEI Rhamno

New chelates are readily absorbed by plants

Zn PEI was readily absorbed and translocated to canola shoots.

Zn EDTA was not taken up by the plants.

0.8t)ot)

Zn EDTA was not taken up by the plants.

PEI (R2 = 0.51)

0.6

µg Z

n/g

shoo

tg

Zn/g

sho

o

EDTA (R2 = 0.77)0.2

0.4

Zn in

sho

ot ( µ

n sh

oot (

µg

00 20 40 60

ASV labile Zn (µg/L)

Z

ASV Labile Zn (µg Zn/L)

Zn i

(µg )

Chelated Zn Free Zn

Zinc distribution and speciation in canola roots

13-BM at the Advanced Photon Source, Argonne National Laboratory.

Zn mapped in root cross-sections using X-ray Microprobe fluorescence;

Zn speciated using X-ray Absorption Fine Structure Spectroscopy.

Synchrotron Zn X-ray fluorescence maps

ZnSO4 Zn signal (counts/s)

75 225 375 500 625 750

Z Rh li idZn-Rhamnolipid

Zn-EDTA

Zinc distribution and speciation in roots from different fertilizer sources

Zn signal (counts/s)

75 225 375 500 625 750Zn-PEI

65.6% Zn-phytate25.8% Zn-lysine

95 2% Zn phytate95.2% Zn-phytate3.1% Zn-glutamate

XAS speciation of Zn in ZnSO4 treated root sections

ZnSO4ZnSO4

77% Zn-phytate77% Zn phytate23% Zn-proline

Zn was predominantly stored in the root as Zn-phytate

XAS speciation of Zn in Zn-EDTA treated root sections

81.2% Zn-phytateZn-EDTA enhanced image18.8% Zn-glutamate

Zn EDTA was not detected inside the

Zn was predominantly stored in the root

Zn-EDTA was not detected inside the root symplast;

p yas Zn-phytate, even at low (environmentally relevant) Zn levels.

XAS Zn speciation in controls (no Zn)

84.4% Zn-phytate 15 6% Zn glutamate15.6% Zn-glutamate

XAS speciation of Zn in Zn-rhamnolipid treated root sections

Zn-Rhamnolipid61.3% Zn-rhamnolipidp24.4% Zn-proline14.3% Zn-phytate

80.0% Zn-rhamnolipid16.1% Zn-phytate3.9% ZnSO4 (or Zn2+)

Zn was predominantly found in the root as Zn-rhamnolipid, rather than Zn-phytate;

Intact Zn-rhamnolipid may have been absorbed by roots.

Canola field response to PEI in Southern Australia

+ PEI - PEI

Conclusions

Rhamnolipid formed lipophilic complexes with micronutrients and PEI has a high metal-complexing capacity;has a high metal complexing capacity;

Soil application of these compounds with Zn increased DM production and Zn uptake in canola and bread/durum wheat grown on calcareous Turkish and Australian soils;Turkish and Australian soils;

Rhamnolipid significantly (P≤ 0.05) increased Zn uptake by canola in ice-cold nutrient solutions despite reducing the (Zn2+) - Zn-rhamnolipid

h d i f f Z i li d i h M Zwas the predominant form of Zn in roots supplied with 5µM Zn-rhamnolipid.

PEI also dramatically increased Zn uptake by canola roots in solution culture and suggests very effect transmembrane transport

Both products show potential to improve micronutrient delivery in fluid fertilizersfertilizers

Acknowledgements