microbial dynamics, fermentation, and aerobic stability of ... · ensiling whole corn > 35% dm...
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Microbial dynamics, fermentation, and
aerobic stability of conventional and BMR
corn hybrids ensiled at low moisture with or
without a combo inoculant
J.J. Romero1, Y. Joo2, J. Park2, Y. Zhao3, M.A. Balseca-Paredes4, F.
Tiezzi5, E. Gutierrez-Rodriguez6, and M.S. Castillo4
1 Animal and Veterinary Sciences, University of Maine, USA
2 Department of Animal Science, Gyeongsang National University, Korea 3 Department of Animal Nutrition, China Agricultural University, China 4 Department of Crop and Soil Sciences; 5 Department of Animal Sciences; 6 Department of Food Science, North Carolina State University, USA
J Dairy Sci., 2018
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Introduction
Ensiling whole corn > 35% DM can result in poorly compacted, ↓ quality silos due to microbial aerobic spoilage.
The microbial community of corn silages produced in suboptimal conditions have not been studied in as much detail compared with optimally stored silages (Muck, 2013).
Vorholt, 2012
Bacteria
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Evaluate the effects of inoculant and corn hybrid (high DM %) on:
Nutritional composition, fermentation, and aerobic stability
Bacterial and fungal taxonomic profiles
OTUs richness and shifts
…before and after ensiling.
Objectives
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Corn field at Piedmont Research Station
(NCDA-NCSU), Salisbury, North Carolina.
Materials and Methods
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Treatments
Factorial combination (4 x 2) of:
Four corn hybrids (HYB):
Two additive levels (ADV)
Sterile dd water (CON) or inoculant (INO) were
applied at 1 ml/kg of fresh corn.
Label Hybrid Company Type Relative
Maturity
MCN TMF2R737 Mycogen Conventional 112
MBR F2F817 Mycogen BMR 116
PCN P2089YHR Pioneer Conventional 120
PBR P1449XR Pioneer BMR 114
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Combo Inoculant
L. buchneri 40788 (log 5.6
cfu/g of fresh corn)
P. pentosaceus 12455 (log 5
cfu/g of fresh corn)
Enzymes from T. reesei.
L. buchneri
P. pentosaceus
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Corn Harvest
6 plots/hybrid were randomly located
within field.
At ~41% DM and chopped to a TLC of 1.9 cm with kernel processing.
2 replicated piles (4.3 kg each, fresh
basis) were obtained from each plot.
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Ensiling
CON or INO randomly assigned to a
pile/plot/hybrid (2×6×4= 48 piles).
Forage was packed into 7.6L mini-silos
and stored at 23°C (± 1°C) for 100 d.
Density: ~192 kg of DM/m3
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Experimental Design and Statistical Analysis
CRD with a 4 (HYB) × 2 (ADV) factorial treatment arrangement.
6 replicates per treatment.
Model contained effect of HYB, ADV, and
their interaction.
Data analyzed with Proc. GLM of SAS v9.4.
SLICE option used to analyze interactions.
Means separated by the LSD test.
Significance P ≤ 0.05.
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Results and Discussion
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Bacteria (16S V4 region)
Fungi (ITS region)
Day 0 of Ensiling
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Day 0 Results
Item MCN PCN MBR PBR SEM
DM (%) 44.0a 42.0b 38.1c 41.3b 0.665
% of DM
CP 5.96b 5.46c 6.85a 5.98b 0.182
WSC 3.32b 3.48b 5.49a 3.66b 0.356
NDF 45.5a 41.8b 44.7a 42.3b 0.94
Log cfu/fresh g
LAB 6.73c 7.06b 7.71a 7.67a 0.101
Yeasts 6.63 6.65 6.63 6.77 0.087
Molds 5.94 5.93 5.70 5.89 0.107
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Alpha diversity
Taxonomic profile
Bacteria (16S V4 region)
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Enterobacteriaceae Dominates
0
10
20
30
40
50
60
70
Rel
ati
ve
Ab
un
da
nce
, %
Mostly unidentified, Cronobacter and Erwinia
Mostly
Sphingobacterium Mostly
unidentified Mostly
Ochrobactrum Mostly
Curtobacterium
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Alpha diversity
Taxonomic profile
Fungi (ITS region)
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a
b b
b
b b
a
b
b
a a a
0
10
20
30
40
50
60
70
MCN MBR PCN PBR MCN MBR PCN PBR MCN MBR PCN PBR
Unidentified Fungi Incertae sedis
Tremellales
Debaryomycetaceae
Rel
ati
ve
Ab
un
dan
ce, %
Unidentified Fungi Dominates
Mostly
Meyerozyma
Mostly
Hannaella
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Bacteria (16S V4 region)
Fungi (ITS region)
Day 100 of Ensiling
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INO Effects
Item CON INO Change SEM
DM recovery, % 94.6 94.0 = 1.07
NH3-N, % of N 8.0 9.7 ↑ 0.39
pH 3.80B 4.02A ↑ 0.041
Lactic acid, % of DM 4.82A 2.65B ↓ 0.436
Acetic acid, % of DM 0.51B 1.69A ↑ 0.132
Yeasts 5.13A 3.78B ↓ 0.440
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Aerobic Stability: ADV × HYB
B
A,a
B
A,a
b
B
A,a
0
50
100
150
200
250
300
350
CON INO CON INO CON INO CON INO
MCN PCN MBR PBR
h
Treatments
HYB effect on WSC: MBR vs. others (2.1% vs. ~1.3%)
Acetobacter?
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Alpha diversity
Taxonomic profile
Beta diversity
Bacteria (16S V4 region)
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b, x
a
b, y
a
b, x
a
b, x
a
0
10
20
30
40
50
60
70
80
90
100
CON INO CON INO CON INO CON INO
MCN MBR PCN PBR
Rel
ati
ve
Ab
un
da
nce
, %
Lactobacillaceae Dominates
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INO ↓ Enterobacteriaceae
a
b
a
b 0
5
10
15
20
25
CON INO CON INO
Enterobacteriaceae Streptococcaceae
Rel
ati
ve A
bu
nd
an
ce,
%
Mostly unidentified,
Erwinia and Serratia
Mostly
Lactococcus
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Weighted UNIFRAC PCoA Plot
PC2 (3.04%)
PC3 (1.63%)
PC1
(92.5%)
Blue: MCN
Yellow: PCN
Red: PBR
Green: MBR
Light blue: MCN
Light yellow: PCN
Light red: PBR
Light green: MBR
Sphere:
Small (INO)
Large (CON)
Day 0
Day 100
**ANOSIM: d 0 vs. 100 (P = 0.001) and within d 100 between the CON and INO (P =
0.001)
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Alpha diversity
Taxonomic profile
Beta diversity
Fungi (ITS region)
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b
a
a
b
a
b
0
10
20
30
40
50
60
70
CON INO CON INO CON INO
Debaryomycetaceae Pichiaceae Incertae sedis
Saccharomycetales
Rel
ati
ve
Ab
un
da
nce
, %
↑ Debaryomycetaceae ↓ Pichiacea
Mostly Candida
Mostly
Meyerozyma
Mostly
Issatchenkia
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Bray-Curtis PCoA Plot
PC2 (21.8%)
PC3 (10.3%) PC1 (46.9%)
Blue: MCN
Yellow: PCN
Red: PBR
Green: MBR
Light blue: MCN
Light yellow: PCN
Light red: PBR
Light green: MBR
Sphere:
Small (INO)
Large (CON) Day 0
Day 100
**ANOSIM: d 0 vs. 100 (P = 0.001) and within d 100 between the CON and INO (P =
0.001)
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Alpha Diversity
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Item Bacteria Fungi
OTUs Evenness OTUs Evenness
Day 0
MCN 434c
~0.025 ~77.1
0.022c
PCN 444c 0.032ab
MBR 654a 0.029bc
PBR 534b 0.038a
SEM 16 0.0011 2.78 0.0033
Day 100
CON 276 ~0.016
43.6b 0.055a
INO 61 59.8a 0.033b
SEM 8.7 0.0011 2.95 0.0047
Alpha Diversity
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Conclusions
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Conclusions
Ensiled corn (100 d):
Aerobic stability was extended at least 5× for all INO-treated HYB except for MBR. Similar DM recovery vs. CON.
INO ↓ bacterial and ↑ fungal diversity across all HYB.
Bacteria: INO: Lactobacillaceae (> 98%).
CON: Lactobacillaceae (~50%), Enterobacteriaceae (~24%) Leuconostocaceae (~14%)
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Conclusions
Fungi: INO had ↑ Debaryomycetaceae, ↓ Pichiaceae and i.s.
Saccharomycetales.
Large differences exist between CON and INO bacterial and fungal community structures. INO microbial communities were closer and more
consistent.
“INO improved dry corn silage quality by a consistent shift in the microbial community
structure during ensiling across HYB, except for MBR”
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Thanks!
Questions?
Contact: [email protected] and [email protected]
• Funding: North Carolina Dairy Producer’s Association.
• Lallemand Animal Nutrition (Milwaukee, WI) provided the inoculant.