bnf in sugarcane, ghg & impact of crop management - Fapesp

BIOEN WORKSHOP 2012: Session 1:
Biomass / Impacts and Sustainability Divisions
BNF IN SUGARCANE,
GHG & IMPACT OF
CROP MANAGEMENT
Cantarella, Carmo, LaScalla, Rossetto
& Barbosa

2
List of projects
08/56.147-1 Cantarella N nutrition of sugarcane with fertilizers
or N-fixing bacteria
08/57937-6 Barbosa Study of the transference of fixed
nitrogen from the diazotrophic bacteria to
sugarcane
08/58029-6 Rossetto Concentrated vinasse applied to
sugarcane: chemical soil characteristics,
ion leaching and agronomic efficiency
08/55989-9 Carmo N2O, CO2 and CH4 emissions from agrobiofuel
production in São Paulo state,
Brazil.
08/58187-0 LaScala The impact of tillage and harvest
Bioen Workshop 2012 (Cantarella)
practices on soil CO2 emission of

N nutrition of sugarcane with
fertilizers or N-fixing bacteria
3
Evidences of BNF in sugarcane: an
inoculant containing 5 species of
bacteria was launched in 2008
Can we replace (or decrease) mineral N
fertilization?
Objectives
Determine the extent of the contribution of BNF compared
with N fertilization in a network of field experiments
Identify genetic traits of sugarcane plants related to BNF
that could be used in a breeding program
Identify new microorganisms capable of stimulating plant
growth and carry on BNF
Measure GHGs emissions as affected by N fertilization of
inoculation of N-fixing microorganisms
Bioen Workshop 2012 (Cantarella)

BNF and N fertilization: field
studies
4
Stalks, t/ha
Stalks, t/ha
125
120
115
110
105
100
90
80
70
60
50
Plant Cane - 5 sites
y = -0.0022N 2 + 0.3975N + 102.6
R² = 0.99
0 20 40 60 80 100
Applied N, kg/ha
Ratoon Cane - 2 sites
y = -0.0009N 2 + 0.2524N + 58.8
R² = 0.98
Bioen Workshop 2012 (Cantarella)
Mineral N
Inoculated
N + Inoculation
Mineral N
Inoculated
N + Inoculation
0 50 100 150
Applied N, kg/ha
Summary of Main Results:
Mean N fertilizer response: 15 t/ha stalk and 5 t/ha dry
mass
Significant response to N: 3/5 in plant cycle and ½ in
ratoon.
No significant response to inoculation of N-fixing
bacteria with or without N fertilizer in any of the
varieties or sites studied
Effect of FBN – if significant – may be already built
into the sugarcane production system (native
species)
FBN por δ 15 N: 0 to ~70% of contribution of N from
BNF
Next: 4 new fields harvested in 2012
N stock in soil (long term effect) to be evaluated
2012

New microrganisms isolated
from sugarcane fields: BNF and
PGP
5
Genetic diversity of isolates (162) by Box PCR
(From Jaú, SP, cane cultivar IAC-5000)
Isolado - espécie PMSPA
Bioen Workshop 2012 (Cantarella)
g
Redutase
Nitrato
N acum. AIA P Sol. nifH
µmol NO2 g -1 -1
mg pl
µg mg -1
protein
mg
mg -1
Delftia acidovorans 11,5a 0,05 131a 2,47 nd -
Delftia acidovorans 10,6a 0,04 143a 0,49 nd -
B Pantoea dispersa 11,0a 0,11 134a 0.04 nd -
Enterobacter cloacae 12,4a 0,03 157a 0.34 nd -
Pantoea dispersa 11,8a 0,04 142a 0,07 1,02 -
A Pantoea dispersa 13,2a 0,07 167a 0,07 1,04 -
Pantoea dispersa 11,3a 0,04 137a 3,73 2,08 -
Herbaspirillum frisingense 12,0a 0,23 152a 0,98 nd -
B Burkholderia caledonica 11,8a 0,14 137a 0,09 3,98 -
Pseudomonas sp. 11,6a 0,03 114b 5,37 nd -
A Enterobacter asburiae 13,4a 0,09 146a 1,91 nd -
B Enterobacter asburiae 11,8a 0,06 137a 1,91 nd -
Methylobacterium fujisawaense 10,4a 0,10 119b 3,43 nd -
Controle 8,0b 0,05 99b -
Main isolates will be inoculated in
micropropaged sugarcane plants
168 strains
89% with BNF capacity
59% with effect similar to auxines

Inoculation of 5-bacteria mix to
meristem micro-propagated
sugarcane plants
6
60,0
50,0
40,0
30,0
20,0
10,0
0,0
Plant height (cm) 56 days
after inoculation
sem inocluante
Bioen Workshop 2012 (Cantarella)
Response to inoculation is varietydependent.
Early stage results (PGPB) are good
but it is not clear whether or how they
could be maintained
New cane cultivars are being tested
Gene expression will be evaluated
IACSP95-5094
No inoculation (38.1 cm) Inoculated (52.0 cm)

Study of the transference of fixed nitrogen
from the diazotrophic bacteria to sugarcane
7
Several new microorganisms associated
with sugarcane have been isolated,
presenting different effects and functions
Results presented refer to most recent
research (not published yet).
Sugarcane-bacterial interactions are
promising areas, to be further studied
Bioen Workshop 2012 (Cantarella)
H. Barbosa et all.

CO-CULTURES AS A MODEL TO STUDY INTERACTIONS BETWEEN
SUGARCANE CALUSES AND DIAZOTROPHIC ENDOPHYFITIC
BACTERIA.
R. C. R. MARTINS AND H. R. BARBOSA
Callus
Co-culture
8
• Influence of callus on bacterial growth – STIMULATION x INHIBITION
Callus
log CFU.mL -1
10
9
8
7
6
5
4
3
2
1
Enterobacter sp. (ICB117)
10
9
8
Erwinia sp. (ICB409)
0
0 200 400 600 800 1000 1200
time (h)
log CFU.mL -1
log CFU.mL -1
7
6
5
4
3
2
1
0
0 200 400 600
time (h)
• Influence of bacteria on calluses– INCREASE OF PROTEIN
CONTENT-through N-fixation
Culture medium
N combined-free
Bioen Workshop 2012 (Cantarella)
protein content of calluses
600
500
400
300
200
100
Callus + Erwinia sp.
0
0 200 400 600 800 1000 1200 1400
Time (h)
Callus +
Enterobacter sp.
Callus control

Massa fresca foliar (g)
5
4
3
2
1
0
9
Comprimento foliar (cm)
Inoculation of Enterobacter sp. in sugarcane plantlets S. Ichiwaki, F. I. Ferrara I and
100
80
60
40
20
0
H. R. Barbosa
Fertilization:
Leaf lenght Root volume
a a a a
Orgânico Convencional Sem adubação
Tratamentos (60 DPI)
Orgânico Convencional Sem adubação
Tramamento (60 DPI)
Não inoculado Enterobacter
Organic x Conventional
b
a
Vol Radicular (cm³)
8
7
6
5
4
3
2
1
0
b
a
b
Orgânico Convencional Sem adubação
a
Tratamentos (60 DPI)
b
a
Clorofila
Growth parameters: organic = conventional
Protein content: Enterobacter sp. + organic = conventional
50
40
30
20
10
0
a
Chlorophyll
a a a a
Orgânico Convencional Sem adubação
Tratamentos (60 DPI)
Leaf fresh weigh Root fresh weigh Leaf dry weigh
Root dry weigh
a a a a
Control
Inoculated x Fertilized
b
Bioen Workshop 2012 (Cantarella)
c
Massa fresca radicular (g)
5
4
3
2
1
0
a
b b b
a c
Orgânico Convencional Sem adubação
Tratamento (60DPI)
Massa seca foliar (g)
1
0,8
Não 0,6 inoculado
Enterobacter
0,4
0,2
0
a a
a a
Orgânico Convencional Sem adubação
Tratamentos (60 DPI)
b
a
Massa seca radicular (g)
1
0,8
0,6
0,4
0,2
0
b
a a a a a
b
Orgânico Convencional Sem adubação
Tratamentos (60 DPI)
Growth parameters and protein content: Always the lowest
Growth parameters: Inoculated = organic and conventional
Protein content: Inoculated = Enterobacter sp. + organic
and conventional

10
Leaf size
O EO C EC S ES
Comparação entre comprimentos foliares de plântulas não inoculadas e inoculadas 60
DPI.
Clorose
O C S
Diferença na pigmentação foliar em plântulas
não inoculadas.
Foto por: Ichiwaki (2012).
Enterobacter
EC EO ES S Foto por: Ichiwaki (2012).
Diferença entre os comprimentos foliares de plântulas de canade-açúcar
inoculadas com Enterobacter sp. ICB481 e plântula
do grupo sem adubação, após 60 DPI.
EO EC ES
Ausência de amarelecimento foliar nos grupos
inoculados com Enterobacter sp. ICB481 aos 60 DPI.
Legenda: Grupos Orgânico (O), Enterobacter-orgânico (EO) / Convencional (C), Enterobacter-convencional / Sem
adubação (S) e ES= Enterobacter-sem adubação.
Foto por: Ichiwaki (2012).
Foto por: Ichiwaki (2012).

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GHGs emission: susteinability of
ethanol production
GHG emissions reduction due to
ethanol replacement of fossil fuels
51 – 65% (Boddey et al., 2008)
85% (Börjesson, 2009)
81 and 24% to 1 st and 2 nd generation (Luo et al.,
2009)
Emission associated with
fertilizer use
N 2O releasing from soils/fertilizers
could offset the benefits of avoiding
CO 2 from fossil fuels (Scharlemann &
Laurence, 2008; Crutzen et al., 2008).
High N 2O values found in other
important cane producer countries
(Denmead et al., 2009; Allen et al., 2010)
IPCC Bioen Workshop standard: 2012 (Cantarella) 1% (IPCC, 2006)

N 2O (mg N m -2 )
CH 4 (mg N m -2 )
12
GHGs emission: fertilizer X
inoculation
300
250
200
150
100
50
0
0
-10
-20
-30
-40
-50
Nitrous Oxide
N0 N0+I N100 N150
N0 N0+I N100 N150
Methane
Jaú (Burned)
Clay: 17.8%; Sand: 76.6%
Bulk density: 1.39 g cm -3
Carbon: 2114 g C m -2
Bioen Workshop 2012 (Cantarella)
N 2O (mg N m -2 )
CH 4 (mg C m -2 )
300
250
200
150
100
50
0
0
-10
-20
-30
-40
-50
Nitrous Oxide
N0 N0+I N100 N150
N0 N0+I N100 N150
Methane
Piracicaba (Unburned)
Clay: 51.9%; Sand: 31.4%
Bulk density: 1.49 g cm -3
Carbon: 4090 g C m -2
N 2O emission increased
with N fertilizer application
but was not affected by
inoculation with diazotrhopic
bacteria
Emission factor ≤ IPCC
Vargas et al, 2012)

Emission factor (fertilizer, trash,
vinasse)
13
Trash rate Vinasse
Mg ha-1 Mg ha-1 Added
N
£ Added N
emission factor
kg ha-1 kg ha (%)
-1 (%)
§ CO2
equivalent
kg CO2 eq. ha-1 kg CO2 eq. ha-1 yr-1 yr-1 0 With 142 0.59±0.29 1,289
Without 120 0.68±0.41 382
7 With 142 1.19±0.84 1,620
Without 120 0.96±0.46 540
14 With 142 1.89±1.00 3,005
Without 120 0.76±0.30 427
21 With 142 3.03±1.22 3,060
Without 120 2.03±1.15 1,141
Bioen Workshop 2012 (Cantarella)
Carmo et al. 2012 (GCB-Bioenergy)

N 2O Emission, kg N-N 2O/ha
4
3
2
1
0
N 2O = 0,0056x 2 + 0,0207x + 0,78
R² = 0,99
N 2O = 0,0496x + 0,692
R² = 0,62
0 5 10 15 20 25
Sugarcane trash, t/ha
Trash+vin
Trash
N 2O emission from N
fertilizer in sugarcane is
within or bellow the
IPPC default value but
the addition of organic
residues caused
increase N 2O emission.
Removing excess trash
from the field (for
energy production) may
avoid high N 2O emission
Increase of N emission factor for fertilizer N (ammonium nitrate surface-applied):
Trash 0,04% per t/ha of trash left on soil
Trash+vinasse 0,12% per t/ha trash left on soil

15
Nitrification inhibitor
decreases N 2O emission
Trash
(Mg ha -1 ) + N
(100 kg/ha)
Bioen Workshop 2012 (Cantarella)
Days after fertilization
N2O emission (mg N/m 2 ) Reduction in N2O
emission due to
No DCD With DCD
DCD
0 10,89 bA § 3,85 aB 65%
8 15,92 bA 5,91 aB 63%
16 32,04 aA 8,44 aB 74%
(Vargas et al, 2012)

Concentrated vinasse in sugarcane: soil
chemical attributes, ion lixiviation and
agronomic efficiency
16
Vinasse: 10 to 13 L/L ethanol
Large amounts generated: high cost of application, risk of soil and ground water
contamination with nutrients
Concentrated vinasse: high cost (and energy) to remove water. Easier and cheaper
field application
Treatments: 0, 120, 140, 360 kg/ha K 2O (as KCl, normal or concentrated vinasse)
Bioen Workshop 2012 (Cantarella)
Rossetto et al, (2012)

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Monitoring: of K, NO 3, Cl, etc...
Soil – 0-25,25-50, 50-80cm depth
Samples in each plot every 3 months
Leaching ions - soil solution extraction –
0- 80cm depth
•Diagnosis Solo –
leave
camadas
– top
0-10,10-20,
visible dewlap
até 1,20m
Macronutrients analysis
Plant – leaves, stalks (bagasse + juice), roots
Straw
Samples taken before harvest
Bioen Workshop 2012 (Cantarella)

Vinasse: main results
18
N mineralization in concentrated vinasse is slower. Half life 6
weeks (3 in conventional vinasse): longer N availability
Vinasse improved soil fertility (SOM, N, K, P, Ca). Higher rate of
CV increase K saturation up to 2.2% of CEC in topsoil
(legislation limit is 5%)
Ca, K, N and P were concentrated in the soil surface layer. Mg
was well distributed ; SO4 was more concentrated in deep
layers.
Sugarcane yield increased with vinasse application (+12
ton/ha). Enhanced nutrient extraction contributed to avoid
accumulation of nutrients in soil.
No negative effect of vinasse was observed, even at high K
rates
Bioen Workshop 2012 (Cantarella)

Tillage and harvest practices on soil CO 2
emission in sugarcane production areas
19
Bioen Workshop 2012 (Cantarella)
Burning sugarcane before
harvest is being phased
out.
The effect of such change
on soil properties &
sustainability must be
examined.
LaScalla & collaborators are studying
the effect of green cane harvesting on
soil C, along with practices such as
no-till and conventional till

20
GHG balance of cane production:
Green cane decreases CO 2 emission
Source
Synthetic Fertilizer
Vinasse
Filter Cake
Harvest Residues
Residues Burning
Liming
Diesel
Sub-Total:
C sequestration
Total:
1,173
-2500 -2000 -1500 -1000 -500 0 500 1000 1500 2000 2500 3000
Bioen Workshop 2012 (Cantarella)
Burned harvest Green harvest
CO 2 eq emission (kg ha -1 y -1 )
1,620
2,793
3,104
3,104
Conversion to green cane
could save the equivalent
of 311 kg CO 2 ha -1 yr -1 ,
or 1484 kg CO 2 ha -1 yr -1 if
soil C sequestration is
computed
2011
Figueiredo et al., 2011

21
Effect of tillage on CO 2 emission
under burned or green cane
Total Emission (kg CO 2 -C hectare -1 )
1750
1500
1250
1000
750
500
250
0
525.4
808.8
BH
953.9
1093.5
NT Cn CnLi CnLiG NT Cn CnLi CnLiG NT Cn CnLi CnLiG
Bioen Workshop 2012 (Cantarella)
698.8
GHnores
284 kg C-CO 2 ha -1 247 kg C-CO 2 ha -1
944.5
1065.0
1081.8
Treatment
= 1039 kg C-CO 2 ha -1 (3808 kg CO 2)
Em 25 days after tillage
446.4
GHres
1485.1
1266.9
1550.2
Plowing the soil
promotes large
CO 2 emissions,
especially in areas
with trash (plant
residues) of GH
BH: burned harved
GH: green cane
(with or without
residues)
NT: no till
CN: conventional
tillage Figueiredo et al., 2011

Concluding remarks
22
Inoculation of sugarcane with endophytic bacteria is
not resulting in yield gains or apparent N
substitution under field conditions
Controlled conditions: promising results, variety-dependent.
PGP properties. Practical benefits of endophytic and
rhizobacterias are still a challenge.
Sugarcane industry by-products can be sustainably
used in the field. Subject deserves more studies
GHGs emission: large volume of good quality data is
being generated.
C balance: projects under way with good prospects.
Enormous challenge.
Bioen Workshop 2012 (Cantarella)

Thank you
cantarella@iac.sp.gov.br
Thai Delegation Meeting 16-8-2012
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