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Microbial Dynamics During Aerobic Exposure of Corn Silage Stored Under 1
Oxygen Barrier or Polyethylene Films 2
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Paola Dolci1, Ernesto Tabacco2, Luca Cocolin1, Giorgio Borreani2* 4
Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali, Agricultural 5
Microbiology and Food Technology Sector, University of Torino, Via L. da Vinci, 44 - 6
10095 Grugliasco (Torino), Italy1 7
Dipartimento di Agronomia, Selvicoltura e Gestione del Territorio, University of 8
Torino, Via L. da Vinci, 44 - 10095 Grugliasco (Torino), Italy2; 9
* Corresponding author. Mailing address: Dipartimento di Agronomia, Selvicoltura e 10
Gestione del Territorio, University of Torino, Via L. da Vinci, 44 10095 Grugliasco 11
(Torino), Italy. Phone: (39) 011 6708783. Fax: (39) 011 6708798. E-mail: 12
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Running title: Aerobic spoilage of corn silage under different films 15
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Abstract 17
The aims were to compare the effects of sealing forage maize with a new oxygen barrier 18
film or with a conventional polyethylene film. This comparison would be during both 19
ensilage and subsequent exposure of silage to air, and would include chemical, 20
microbiological and molecular (DNA and RNA) assessments. The forage was 21
inoculated with a mixture of Lactobacillus buchneri, Lb. plantarum, and Enterococcus 22
faecium and ensiled in polyethylene (PE) and oxygen barrier (OB) plastic bags. The 23
oxygen permeability of the films were: 1480 vs. 70 cm3 m-2 per 24 h at 23°C, for PE 24
and OB, respectively. The silages were sampled after 110 days of ensilage and after 2, 25
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.05050-11 AEM Accepts, published online ahead of print on 5 August 2011
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5, 7, 9, and 14 days of air exposure and analyzed for fermentation characteristics, 26
conventional microbial enumeration and bacterial and fungal community fingerprinting 27
via PCR-DGGE and RT-PCR-DGGE. The yeast counts in the PE and OB silages were 28
3.12 and 1.17 log10 CFU g-1, respectively, with corresponding aerobic stabilities of 65 29
and 152 hours. Acetobacter pasteurianus was present at both the DNA and RNA level 30
in the PE silage samples after 2 days of air exposure, whereas it was only found after 7 31
days in the OB silages. RT-PCR-DGGE revealed the activity of Aspergillus fumigatus 32
in the PE samples from the day 7 of air exposure, whereas it only appeared after 14 days 33
in the OB silages. It has been shown that the use of a oxygen barrier film can ensure a 34
longer shelf life of silage after aerobic exposure. 35
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Keywords: corn silage, aerobic deterioration, plastic film, oxygen barrier film 37
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INTRODUCTION 39
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Forage ensiling is based on the natural fermentation of plant water soluble 41
carbohydrates by lactic acid bacteria (LAB) under anaerobic conditions (24). The most 42
important single factor that can influence the preservation efficiency of forage ensiling 43
is the degree of anaerobiosis reached in the completed silo (36). Anaerobic conditions 44
are not always achieved at farm level especially in the peripheral outer layerareas of a 45
silo, because of the difficulty of sealing it efficiently (6). The aerobic deterioration of 46
silages is a significant problem for farm profitability and feed quality throughout the 47
world. All silages exposed to air deteriorate as a result of aerobic microbial activity 48
during feedout (8, 31, 35). These losses could reach 70% of the stored dry matter in the 49
peripheral areastop layer and near the sidewalls of the bunkers and are related to the 50
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depletion of the digestible carbohydrate and organic acid fractions (4). Spoilage of 51
silage due to exposure to air is undesirable, due to the lower nutritive value and to the 52
risk of negative effects on animal performance (18), which are also connected to the 53
proliferation of potentially pathogenic or otherwise undesirable microorganisms (20) 54
and mycotoxin synthesis (28). 55
Polyethylene (PE) films have been used for many years to seal bunker silos and drive-56
over piles because of their suitable mechanical characteristics and low costs. The high 57
O2 permeability of PE films seems to be one of the main reasonscan contribute to for 58
the low quality of silage in the peripheral areastop layer of horizontal silos (6). A new 59
silage sealing plastic film, which uses a new plastic formulation with an 18-fold lower 60
oxygen permeability than the PE film usually used on farms, has been recently 61
developed (7). Some different polymers from PE, such as polyamides (PA) and 62
ethylene-vinyl alcohol (EVOH), offer excellent barrier properties to oxygen, combined 63
with good mechanical characteristics (puncture resistance), and are suitable for blown 64
co-extrusion with PE to produce 45 to 200 μm thick plastic films. 65
Nowadays, Monitoring microbiota during the ensiling process has become more reliable 66
and accurate, thanks to the recently developed culture-independent methods (29, 20). In 67
recent years, DNA-based community fingerprinting techniques, such as Denaturing 68
Gradient Gel Electrophoresis (DGGE) and Terminal Restriction Fragment Length 69
Polymorphism (T-RFLP), have been applied to investigate the microbial community 70
composition of silage (9, 22). Advanced molecular biological techniques have been 71
used to help understanding the structure of complex microbial community dynamics 72
(25). However, to the best of the authors’ knowledge, no study has used a community 73
fingerprinting approach to investigate the population dynamics of corn silage during 74
aerobic deterioration. 75
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In this study, a culture-independent technique, PCR-DGGE, was used to study the 76
microbial dynamics, whereas reverse transcription (RT)-PCR-DGGE allowed to 77
investigate the metabolically active populations. These techniques were performed 78
together with conventional bacterial enumeration in order to investigate the effects of a 79
new oxygen barrier film on the bacterial community of ensiled and aerobically 80
deteriorated corn silages. 81
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MATERIALS AND METHODS 83
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Crop and ensiling 85
The trial was carried out at the experimental farm of the University of Turin in the 86
western Po plain, northern Italy (44°50’N, 7°40’E, altitude 232 m a.s.l.) in 2008 on corn 87
(Zea mays L.) harvested as a whole corn crop, at a 50% milk-line stage, and at a 333 g 88
DM kg-1 of fresh forage. The forage was chopped with a precision forage harvester to a 89
10 mm theoretical length of cut, inoculated with a mixture of Lactobacillus buchneri 90
(strain ATCC PTA2494), Lb. plantarum (strains ATCC 53187 and 55942), and 91
Enterococcus faecium (strain ATCC 55593) inoculum (11C33, Pioneer Hi-Bred 92
International, Des Moines, IA) to supply 1 x 105, 8 x 103, and 2 x 103 colony forming 93
units (CFU) per gram of fresh forage for Lb. buchneri, Lb. plantarum, and E. faecium, 94
respectively. Standard black-on-white polyethylene film, 120One hundred and twenty 95
μm thick polyethylene plastic bags (PE) and 120 μm thick SILOSTOP (Bruno Rimini 96
Ltd., London, UK), black-on-white co-extruded polyethylene-polyamide film, with an 97
enhanced oxygen barrierthick oxygen barrier plastic bags (OB) were used to produce 98
the silage bags for this experiment. Bags were heat sealed at the closed end and they 99
were equipped with a one-way valve for CO2 release. Each bag was inserted into a 100
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portion of a PVC tube (internal dimensions: 300 mm diameter and 300 mm height, 21 l 101
volume) so that just the top and the bottom of the bag had access to air. All bags were 102
then, filled with about 12 kg of fresh forage, which was compacted manually, and 103
secured with plastic ties. Four replications were prepared for each treatment. The 104
density of the silage was 576 kg FM m-3 and 192 kg DM m-3. The oxygen permeability 105
of the plastic materials, determined by thefollowing ASTM standard method D 3985-81 106
(4), differed 1480 vs. 70 cm3 m-2 per 24 h at 100 KPabar at 23°C, 0% relative humidity, 107
for PE and OB, respectively. The silos were stored at ambient temperature (18 to 22°C) 108
indoors and opened after 110 days. The final weights were recorded at silo opening, and 109
the silage was mixed thoroughly and subsequently sampled. The DM concentration (3 110
replicates) and fermentation end-products (2 replicates) were determined for each 111
sample. Microbiological counts (2 replicates) and culture-independent techniques (2 112
replicates) were also carried out. The silages were subjected to an aerobic stability test. 113
Aerobic stability was determined by monitoring the temperature increases due to the 114
microbial activity of the samples exposed to air. About three kilograms of each silo 115
were allowed to aerobically deteriorate at room temperature (22 ± 1.6°C) in 17 l 116
polystyrene boxes (290 mm diameter and 260 mm height) for 14 days. A single layer of 117
aluminum cooking foil was placed over each box to prevent drying and dust 118
contamination, but also to allow air penetration. The temperature of the room and of the 119
silage was measured each hour by a data logger. Aerobic stability was defined as the 120
number of hours the silage remained stable before rising more than 2°C above room 121
temperature (27). The silage was sampled after 0, 2, 5, 7, 9 and 14 days of aerobic 122
exposure to quantify the microbial and chemical changes of the silage during exposure 123
to air. 124
125
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Sample preparation and analyses 126
Each of the pre-ensiled samples of each herbage and the samples of silage taken from 127
each bag of silage were split into three subsamples. One subsample was oven-dried at 128
65°C to constant weight to determine the DM content, and air equilibrated, weighed, 129
and ground in a Cyclotec mill (Tecator, Herndon, VA, USA) to pass a 1 mm screen. 130
The dried samples were analyzed for total nitrogen (TN) by combustion (30), according 131
to the Dumas method, using a Nitrogen analyser Micro-N (Elementar, Hanau, 132
Germany) and for ash by complete combustion in a muffle furnace ignition toat 550°C 133
for 3 hours. A portion of the second sub-sample was extracted using a Stomacher 134
blender (Seward Ltd, UK) for 4 min in distilled water at a ratio of water to sample 135
material (fresh weight) of 9:1, whereas another portion was extracted or in H2SO4 0.05 136
mol l-1 at a ratio of acid to sample material (fresh weight) of 5:1. The nitrate (NO3) 137
contents were determined in the water extract, through semi-quantitative analysis, using 138
Merckoquant test strips (detection limit 100 mg NO3 kg-1)(7). The ammonia nitrogen 139
(NH3-N) content, determined using a specific electrode, was quantified in the water 140
extract. The lactic and monocarboxylic acids (acetic, propionic and butyric acids) were 141
determined by high performance liquid chromatography (HPLC) in the acid extract 142
(10). Ethanol, for which the HPLC was coupled to a refractive index detector was also 143
determined using a Aminex HPX-87H column (Bio-Rad Laboratories, Richmond, CA). 144
The analyses were performed isocratically under the following conditions: mobile phase 145
0.0025 mol l-1 H2SO4, flow rate 0.5 ml min-1, column temperature 37°C, injection 146
volume 100 μl. Duplicate analyses were performed for all the determined parameters. 147
The duplicates were averaged and the four means (one for each silo) were considered as 148
four observations in the statistical analysis. The water activity (aw) of the silage was 149
measured at 25°C using an AquaLab Series 3TE (Decagon Devices Inc., Pullman, WA) 150
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on a fresh sample at silo opening. The weight losses due to fermentation were calculated 151
as the difference between the weight of the plant material placed in each silo at ensiling 152
and the weight of the silage at the end of conservation. 153
A third subsample was used for the microbiological analyses. For the microbial counts, 154
30 g of sample were transferred into sterile homogenization bags, suspended 1:10 w/v 155
in peptone salt solution (PPS: 1 g of bacteriological peptone and 9 g of sodium chloride 156
per liter) and homogenized for 4 min in a laboratory Stomacher blender (Seward Ltd, 157
London, UK). Serial dilutions were prepared and the following counts were carried out: 158
i) aerobic spores after pasteurization at 80°C for 10 min followed by double layer pour 159
plating with 24.0 g l-1 Nutrient Agar (NUA, Oxoid, Milan, Italy) and incubation at 30°C 160
for 3 days; ii) mold and yeast on 40.0 g l-1 of Yeast Extract Glucose Chloramphenicol 161
Agar (YGC agar, DIFCO, West Molesey, Surrey, UK) after incubation at 25°C for 3 162
and 5 days for yeast and mold, respectively. The mean count of the duplicate sub-163
samples was recorded for the microbial counts on plates that yielded 10 to 100 CFU per 164
Petri dish. 165
166
Bacterial community fingerprinting by PCR-DGGE and RT-PCR-DGGE 167
Sampling and nucleic acid extraction 168
Two milliliters of the supernatant of the above described 1:10 diluted sample 169
suspension were collected for each sampling point and centrifuged at 13,400 x g for 5 170
min to pellet the cells. After discarding the supernatant, the pellet was submitted to 171
DNA and RNA extraction using a DNeasy Plant mini Kit and a RNeasy Plant mini Kit 172
(Qiagen, Milan, Italy), respectively, according to the manufacturer’s instructions. The 173
presence of residual DNA in the RNA samples was checked by PCR (12). 174
PCR and RT-PCR 175
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The dominant bacterial microbiota was investigated, at both DNA and RNA level, by 176
PCR-DGGE and RT-PCR-DGGE. The primers 338fGC and 518r were used to detect 177
and amplify the bacterial variable V3 region of 16S rRNA gene (1). In order to 178
investigate the dominant fungal microbiota, the D1-D2 loop of the 26S rRNA gene was 179
amplified by PCR using the primers NL1GC and LS2 (11). 180
DGGE analysis 181
The Dcode universal mutation detection system (BioRad) was used to performe DGGE 182
analysis. The amplicons obtained from the PCR and RT-PCR were applied to an 8% 183
(w/v) polyacrilamide gel (acrylamide-bis acrylamide, 37.5:1) with a 30% to 60% 184
denaturant gradient (13). Some selected DGGE bands were excised from the gels and 185
incubated overnight at 4°C in 50 µl of sterile water. The eluted DNA was reamplified 186
and analyzed in DGGE (1). The amplicons that gave a single band co-migrating with 187
the control were then amplified with a 338f primer and NL1 primer, respectively, for 188
bacterial and fungal microbiota, without a GC clamp and purified with Perfectprep Gel 189
Clean up (Eppendorf, Milan, Italy) for the sequencing. 190
Sequence analysis 191
The PCR-DGGE and RT-PCR-DGGE bands were sent for sequencing to Eurofins 192
MWG Operon (Ebersberg, Germany) and the obtained gene sequences were aligned 193
with those in the GenBank using the BLAST program (2) to establish the closest known 194
relatives of the amplicons run in DGGE. 195
196
Statistical analysis 197
All the microbial counts and hours of aerobic stability were log10 transformed to obtain 198
log-normal distributed data. The fermentative characteristics, microbial counts, pH, 199
nitrate contents, dry weight losses, and hours of aerobic stability were subjected to a 200
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one-way analysis of variance (Statistical Package for Social Science, version 16, SPSS 201
Inc., Chicago, Illinois, USA) to evaluate the statistical significance between the two 202
treatments. Between-treatment comparisons were made using the unpaired Student's t-203
test and effects were considered significant at P < 0.05.were analyzed for their statistical 204
significance via analysis of variance using the general linear model of the Statistical 205
Package for Social Science (v 16, SPSS Inc., Chicago, Illinois, USA). Significant 206
differences between means were identified from the P-values of the analysis of variance 207
and effects were considered significant at P < 0.05. When the calculated values of F 208
were significant, the Duncan range test (P < 0.05) was used to interpret any significant 209
differences among the mean values. 210
DGGE profiles were normalized and submitted to cluster analysis using the 211
BioNumerics software (Applied Maths, Kortrijk, Belgium). The Pearson product 212
moment correlation coefficient was used to calculate the similarities in DGGE patterns, 213
and dendrograms were obtained via the unweighted pair group method with arithmetic 214
averages. 215
216
RESULTS 217
218
Fermentative quality and microbial counts of the silages. The results of the chemical 219
and microbial determination of the corn forage prior to ensiling are shown in Table 1. 220
The values are typical of those of corn harvested at a 50% milk line. The fermentation 221
quality and microbial composition of the silages, after 110 days of conservation, are 222
shown in Table 2. All the silages were well fermented. The main fermentation acids 223
found were lactic and acetic acids, whereas butyric acid was below the detection limit 224
(less than 0.1 g kg-1 DM) in all the silages. The silages sealed with the PE film led to 225
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silages with higher pH (P <0.0021), and lower concentrations of lactic acid (P 226
<0.03301) in comparison to the OB silages. The nitrates in the corn crop were 227
numerically lower in the silages in comparison to the corresponding herbage. The yeasts 228
were lower below the OB film, whereas the mold count was below 2 log10 CFU g-1 229
silage in both treatments. The aw of the silages at opening had a mean value of 0.99, and 230
there was no difference between the two treatments. The weight losses were lower in 231
the OB silages than in the PE silages. The aerobic stabilities of the silages exposed to air 232
were 65 and 152 hours in the PE and in OB silages, respectively. 233
Silage quality during the air exposure test. The changes in temperature, pH, lactic 234
acid, yeast and mold count, and aerobic spores of the silages for 14 days (329 h) of 235
aerobic exposure are reported in Fig. 1. The temperature at silo opening was about 22°C 236
for both treatments. After 51 h of aerobic exposure, the temperature of the PE silages 237
started to rise, whereas the temperature in the OB silages did not increase over the first 238
6 days (145 h) of exposure to air. The PE silages showed higher temperatures than 35°C 239
after 4.7 days (113 h) and reached the highest temperature of 42.2°C after 9.5 days (229 240
h). Under the OB film, higher temperatures than 35°C were only reached after 11.4 days 241
(274 h) of air exposure. Simultaneously to the variation in silage temperatures, a pH 242
increase was observed in the two treatments. The pH was always lower in the OB than 243
in the PE silages. The lactic acid concentration started to decrease after 2 days in the PE 244
silage and after 7 days in the OB silage. The yeast count increased from the second day 245
of air exposure in both treatments and reached 6 log10 CFU g-1 silage after 5 days of air 246
exposure. The mold count remained almost constant till day 5 of air exposure and 247
started to increase at day 7, with higher values in the PE silage than in the OB silage. 248
They reached similar values after 14 days of air exposure. The aerobic spore count 249
increased with air exposure time in the PE silages, reaching a maximum value of 9.3 250
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log10 CFU g-1 after 14 days of air exposure. The OB silages remained almost constant 251
till day 9 of air exposure, and reached a value of 7.8 log10 CFU g-1 after 14 days of air 252
exposure. 253
Bacterial community fingerprinting of the silages and aerobically exposed silages. 254
The bacterial microbiota dynamics was well described through the PCR-DGGE and RT-255
PCR-DGGE profiles. The DNA and RNA gels are shown in Figs. 2 and 3 and the band 256
identification results are reported in Tables 3 and 4. The inoculated starter, Lb. buchneri 257
was present for 14 days, at the DNA level, in the OB silage samples (Fig. 2 (c) band a), 258
whereas it was found for 5 days in the PE silages (Fig. 2 (a) band a). Lb. plantarum was 259
only detected in the samples ensiled in OB between the 5th and the 14th days (Fig. 2 (c) 260
band g). Faint bands were found at RNA level for these two species. Lb. buchneri was 261
not detected beyond 7 days (Fig. 2 (d) band a) and Lb. plantarum was detected in the 262
OB samples from days 5 to 9 (Fig. 2 (d) band g). Acetobacter pasteurianus was clearly 263
present at both DNA and RNA level in the PE silage samples, except for the days 264
immediately subsequent to air exposure (Fig. 2 (a - b) band b). Ac. pasteurianus was 265
only found in OB silages after 7, 9, or 14 days from aerobic exposure (Fig. 2 (c - d) 266
band b). Faint bands referring to the Bacillus subtilis species were detected at DNA 267
level in the PE silage samples from day 5 to day 14 (Fig. 2 (a) band c), whereas B. 268
subtilis was only recovered in the DNA extracted from the OB samples and at RNA 269
level, at day 14 (Fig. 2 (b - c - d) band c). Lb. amylovorus was found at RNA level in PE 270
silage on opening (Fig. 2 (b) band d) together with a band referring to an uncultured 271
bacterium (Fig. 2 (b) band e). Finally, a more persistent band, again identified by 272
sequencing as uncultured bacterium, was observed in the same samples, from day 5 to 273
day 14. 274
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Fungal community fingerprinting of the silages and aerobically exposed silages. 275
The fungal population was detected at DNA level, in both the PE and OB silages, with a 276
band present from opening to day 7 (Fig. 3 (a - c) band i). After sequencing, the band 277
was determined to be Kazachstania exigua. Aspergillus fumigatus was found after 14 278
days of air exposure (Fig. 3 (a - c) band h). DNA bands, referring to Pichia 279
kudriavzevii, were revealed in PE silages at day 7 and day 9 (Fig. 3 (a) band l). The RT-280
PCR-DGGE revealed the activity of A. fumigatus, in particular in the PE silages, where 281
it was detected from day 7 to day 14 of air exposure (Fig. 3 (b) band h). Unlike the 282
DNA analysis, RNA identified a new band, which was sequenced as Aureobasidium 283
pullulans. This species was present for 14 d of aerobic exposure in OB silage (Fig. 3 (d) 284
band m), whereas it was only found for 5 days of air exposure in the PE silages (Fig. 3 285
(b) band m). Furthermore, a band that could not be related to any fungal species, was 286
present in OB silages from the 2nd to the 9th day (Fig. 3 (d) band n). Finally, at DNA 287
level, bands run in the middle of the lanes were observed and excised but, once 288
reamplified, unclear profiles were obtained and, thus, they were interpreted as 289
heteroduplex. No important differences in fingerprints were observed between DNA 290
and RNA, among the replicates of each treatment. 291
The cluster analysis highlighted the influence of the PE and OB films on the bacterial 292
DGGE profiles (Fig. 4). A clustering related to DNA and RNA analysis can be noticed 293
within each treatment. The DGGE profiles of the mycobiota clustered in two main 294
groups, according to the nucleic acid analyzed (Fig. 5). Clusters correlated to both the 295
sealing treatment and temporal dynamics were noted at RNA level. 296
297
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DISCUSSION 298
An anaerobic environment is the most important individual factor that can influence 299
silage conservation (36). Most of the silages at a farm level are exposed to air during 300
conservation, due to the permeability of plastic to air and difficulties in sealing the 301
peripheral outer layer of silageareas properly, or during the feed-out phase, due to an 302
inadequate amount of silage being removed and to a poor management of the exposed 303
silo surface (3). This evidence highlights risks in terms of: spoilage with losses in 304
nutritional value (33), multiplication of potentially pathogenic microorganisms, and 305
production of mycotoxins (16). Since aerobic microbial populations increase during 306
aerobic deterioration in an exponential manner, the silage from the spoiled top corner 307
peripheral areas and from the molded spots have the potential of contaminating feedout 308
silage to a great extent, even when it is included in very small amounts. To address the 309
issue of aerobic stability, inoculants containing Lb. buchneri have been used over the 310
last decade with the primary purpose of increasing the amount of acetic acid and, as a 311
consequence, of decreasing yeast counts in silages (17). Plastic oxygen barrier films are 312
also now available to cover silages and to improve the anaerobic environment during 313
conservation (6). 314
The purpose of the present study was to have a better understanding of the susceptibility 315
to aerobic deterioration of silages stored below plastic films, characterized by different 316
oxygen permeability and to monitor the changes in microbiota population over the 317
course of aerobic silage deterioration. In our study, the fermentative profile of silages, 318
stored under both OB and PE films, was typical of fermentation driven by Lb. buchneri, 319
with a relatively high content of acetic acid, lower lactic-to-acetic ratio (< 3), and the 320
presence of more than 10 g kg-1 DM of 1,2-propanediol. These values are in agreement 321
with those reported by Kleinschmit and Kung (17), who summarized the effects of Lb. 322
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buchneri on silage quality in 43 experiments. Furthermore, the low permeability to 323
oxygen of the OB film contributed to create a more anaerobic environment, and this was 324
reflected in a silage with a higher lactic acid content, and a lower pH and acetic acid 325
content, and weight losses. The better anaerobic environment under the OB film also 326
contributed to reducing yeast counts to below 2.0 log10 CFU g-1 of silage. The reduction 327
in yeasts was reflected in an increase in the aerobic stability of the OB silages, when 328
exposed to air. It is well known that lactate-assimilating yeasts (Saccharomyces, 329
Candida and Pichia spp.) are generally the main initiators of the aerobic spoilage of 330
silages (24), and, under aerobic conditions, they utilize lactic acid thus causing an 331
increase in silage temperature and pH. In our study, the dominant species of yeasts after 332
exposure to air, as observed from the DGGE profiles of fungal DNA and RNA, was 333
Kazachstania exigua, both in the PE and OB silages. The yeasts of the genus of 334
Kazachstania were previously observed in aerobically deteriorating corn silages (21). 335
Furthermore, Pichia kudriavzevii was observed in PE silages after 7 days of air 336
exposure. The yeast of the genus Pichia is usually reported to be the initial cause of 337
aerobic deterioration of different silage crops (24). P. kudriavzevii has recently been 338
found in Italian ryegrass silage treated with Lb. buchneri (19). From the DGGE profiles 339
of bacterial RNA at silage opening and during 14 days of air exposure, apart from the 340
presence of LAB, Ac. pasteurianus was also seen to be present from the second day of 341
air exposure in the PE silages, while it appeared at day 7 in the OB silages. This could 342
partially explain the more rapid degradation that occurred in the PE silage after 343
exposure to air. Spoelstra et al. (32) found that Acetobacter spp. could be involved in 344
the aerobic spoilage of corn silage, by oxidizing ethanol to acetate, or lactate and acetate 345
to carbon dioxide and water. Furthermore, the selective inhibition of yeasts, due to the 346
addition of acetic or propionic acid, could also increase the proliferation of acetic acid 347
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bacteria in silage (15). Here, the use of Lb. buchneri as a silage inoculant provoked a 348
heterolactic fermentation with an increase in the acetic acid concentration. This aspect 349
could have indirectly stimulated the activity of Ac. pasteurianus. The presence of Ac. 350
pasteurianus in silages has recently been reported by Nishino et al. (23), who identified 351
two strains of Ac. pasteurianus in whole crop corn silage that contained significant 352
amounts of acetic acid and which had been stored for 18 months. 353
When the yeasts and Acetobacter had consumed most of the lactic acid (Fig. 1) and 354
acetic acid (sdata not shown), the pH level increased and the growth of other aerobic 355
bacteria and filamentous fungi became possible, which caused further spoilage (36). 356
This secondary aerobic spoilage microbiota, which principally consist of mold and 357
bacilli, not only decreases the nutritive value of the silage, but also presents a risk to 358
animal health and the safety of milk (34). In this study, the aerobic spores increased to 359
higher levels than 8 log10 cfu g-1 from the 3rd day of air exposure and beyond in the PE 360
silage, whereas they only tended to increase in the OB silage after 9 days of air 361
exposure. Bacillus spp. counts of up to 9 log10 cfu g-1 silage have been detected in 362
deteriorating silage and from the face layer of opened bunker silages (24). The DGGE 363
profiles showed that B. subtilis was present in both the PE and OB silages. The presence 364
of A. fumigatus was observed after 7 days of aerobic exposure in the PE silages and 365
after 14 days in the OB silages, when molds exceeded 6 log10 cfu g-1 silage. A. 366
fumigatus is a well-known human and animal pathogen that causes aspergillosis and it 367
can produce gliotoxin, a toxic compound that has potent immunosuppressive, genotoxic, 368
citotoxic and apoptotic effects (14, 26). 369
Overall, the cluster analysis highlighted the influence of PE and OB films on the 370
metabolic activity of microbiota throughout aerobic exposure. At RNA level, the 371
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clusters correlated to the sealing treatment were detected for both the bacterial and 372
fungal populations. 373
In this study, it has been shown that the use of oxygen barrier plastic films for ensiling 374
can ensure a longer shelf life of silage, protecting it from spoilage. Moreover, an 375
important aspect that has been highlighted concerns the delay in growth of pathogenic 376
molds, which are able to produce potent mycotoxins that are harmful for animals and 377
humans. 378
379
380
ACKNOWLEDGEMENTS 381
382
This work was supported by the Regione Piemonte, Assessorato Qualità, Ambiente e 383
Agricoltura years 2005-2008 Project: “Influenza della zona di produzione e del tipo di 384
gestione aziendale sulla qualità del Grana Padano D.O.P. piemontese”. All the authors 385
contributed equally to the work described in this paper. 386
387
REFERENCES 388
389
1. Alessandria, V., P. Dolci , K. Rantsiou, D. Pattono, A. Dalmasso, T. Civera, 390
and L. Cocolin. 2010. Microbiota of the Planalto de Bolona: an artisanal cheese 391
produced in uncommon environmental conditions in the Cape Verde Islands. 392
World J. Microb. Biot. 26: 2211-2221. 393
2. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. 394
Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new 395
on April 22, 2020 by guest
http://aem.asm
.org/D
ownloaded from
17
generation of protein database search programs. Nucleic Acids Res. 25: 3389-396
3402. 397
3. Ashbell, G., and N. Lisker. 1988. Aerobic deterioration in maize silage stored 398
in a bunker silos under farm conditions in a subtropical climate. J. Sci. Food 399
Agric. 45:307–315. 400
4. ASTM. 1981. Standard method D 3985-81: Standard test method for oxygen gas 401
transmission rate through plastic film and sheeting using a coulometric sensor. 402
In Annual Book of Standards. West Conshohocken, Pa.: ASTM International. 403
4.5.Bolsen, K. K., J. T. Dickerson, B. E. Brent, R. N. Sonon Jr., B. S. Dalke, C. 404
Lin, and J. E. Boyer, Jr. 1993. Rate and extent of top spoilage losses in 405
horizontal silos. J. Dairy Sci. 76:2940-2962. 406
5.6.Borreani, G., E. Tabacco, and L. Cavallarin. 2007. A new oxygen barrier film 407
reduces aerobic deterioration in farm-scale corn silage. J. Dairy Sci. 90:4701–408
4706. 409
6.7.Borreani, G., and E. Tabacco. 2008. Low permeability to oxygen of a new 410
barrier film prevents outgrowth of butyric acid bacteria in farm corn silage. J. 411
Dairy Sci. 91:4272-4281. 412
7.8.Borreani, G., and E. Tabacco. 2010. The relationship of silage temperature 413
with the microbiological status of the face of corn silage bunkers. J. Dairy Sci. 414
93:2620-2629. 415
8.9.Brusetti, L., S. Borin, D. Mora, A. Rizzi, N. Raddadi, C. Sorlini, and D. 416
Daffonchio. 2006. Usefulness of length heterogeneity-PCR for monitoring lactic 417
acid bacteria succession during maize ensiling. FEMS Microbiol. Ecol. 56:154-418
164. 419
Formatted: Bullets and Numbering
on April 22, 2020 by guest
http://aem.asm
.org/D
ownloaded from
18
9.10. Canale, A., M. E. Valente, and A. Ciotti. 1984. Determination of 420
volatile carboxylic acids (C1-C5) and lactic acid in aqueous acid extracts of 421
silage by high performance liquid chromatography. J. Sci. Food Agric. 35:1178-422
1182. 423
10.11. Cocolin, L., L. F. Bisson, and D. A. Mills. 2000. Direct profiling of the 424
yeast dynamics in wine fermentation. FEMS Microbiol Lett 189, 81-87. 425
11.12. Cocolin, L., M. Manzano, C. Cantoni, and G. Comi. 2001. Denaturing 426
gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to 427
monitor dynamics changes in the bacterial population during fermentation of 428
Italian sausages. Appl. Environ. Microb. 67: 5113-5121. 429
12.13. Dolci, P., A. Barmaz, S. Zenato, R. Pramotton, V. Alessandria, L. 430
Cocolin, K. Rantsiou, and R. Ambrosoli. 2008. Maturing dynamics of surface 431
microbiota in Fontina PDO cheese studied by culture-dependent and –432
independent methods. J. Appl. Microbiol. 106: 278-287. 433
13.14. dos Santos, V. M., J. W. Dorner, and F. Carreira 2003. Isolation and 434
toxigenicity of Aspergillus fumigatus from moldy silage. Mycopathologia 435
156:133-138. 436
14.15. Driehuis, F., and P. G. van Wikselaar. 1996. Effects of addition of 437
formic, acetic or propionic acid to maize silage and low dry matter grass silage 438
on the microbial flora and aerobic stability. p. 256-257. In D.I.H. Jones, R. 439
Jones, R. Dewhurst, R. Merry, and P. M. Haigh (ed.) Proc. 11th Int. Silage 440
Conference. IGER, Aberystwyth, UK. 441
15.16. Gonzalez Pereyra, M. L., V. A. Alonso, R. Sager, M. B. Morlaco, M. 442
E. Magnoli, A. L. Astoreca, C. A. R. Rosa, S. M. Chiacchera, A. M. Dalcero, 443
on April 22, 2020 by guest
http://aem.asm
.org/D
ownloaded from
19
and L. R. Cavaglieri. 2008. Fungi and selected mycotoxins from pre- and 444
postfermented corn silage. J. Appl. Microb. 104:1034-1041. 445
16.17. Kleinschmit, D. H., and L. Kung, Jr. 2006. A meta-analysis of the 446
effects of Lactobacillus buchneri on the fermentation and aerobic stability of 447
corn and grass and small-grain silages. J. Dairy Sci. 89:4005-4013. 448
17.18. Kung, L. Jr., A. C. Sheperd, A. M. Smagala, K. M. Endres, C. A. 449
Bessett, N. K. Ranjit, and J. L. Glancey. 1998. The effect of preservatives 450
based on propionic acid on the fermentation and aerobic stability of corn silage 451
and a total mixed ration. J. Dairy Sci. 81:1322-1330. 452
18.19. Li, Y., and N. Nishino. 2011. Bacterial and fungal communities of 453
wilted Italian ryegrass silage inoculated with and without Lactobacillus 454
rhamnosus or Lactobacillus buchneri. Lett. Appl. Microbiol. In press. 455
19.20. Lindgren, S., G. Pahlow, and E. Oldenburg. 2002. Influence of 456
microbes and their metabolites on feed and food quality, p. 503-511. In J.-L. 457
Durand, J.-C. Emile, C. Huyghe, and G. Lemaire (ed.) Multi-function 458
grasslands. Proceedings of the 19th General Meeting of the European Grassland 459
Federation, La Rochelle, France. 460
20.21. Lu, H.-Z., Y. Cai, Z.-W. Wu, J.-H. Jia, and F.-Y. Bai. 2004. 461
Kazachstania aerobia sp. nov., an ascomycetous yeast species from aerobically 462
deteriorating corn silage. Int. J. Syst. Evol. Microbiol. 54:2431 - 2435. 463
21.22. McEniry, J., P. O'Kiely, N. J. W. Clipson, P. D. Forristal, and E. M. 464
Doyle. 2010. Assessing the impact of various ensilage factors on the 465
fermentation of grass silage using conventional culture and bacterial community 466
analysis techniques. J. Appl. Microbiol. 108:1584-1593. 467
on April 22, 2020 by guest
http://aem.asm
.org/D
ownloaded from
20
22.23. Nishino, N., C. Wang, Y. Li, S. Parvin, and K. Kan. 2009. Occurrence 468
and survivali in whole crop corn silage of Acetobacter pasteurianus, p. 165-166. 469
In G. A. Broderick et al. (ed.), Proceedings of 15th International Silage 470
Conference. U.S. Dairy Forage Research Center, USDA-ARS, Madison, WI. 471
23.24. Pahlow, G., R. E. Muck, F. Driehuis, S. J. W. H. Oude Elferink, and 472
S. F. Spoelstra. 2003. Microbiology of ensiling, p. 31-93. In D. R. Buxton, R. 473
E. Muck, and J. H. Harrison (ed.), Silage Science and Technology. Vol. 42. 474
ASA, CSSA, SSSA, Madison, WI. 475
24.25. Parvin, S., and N. Nishino. 2009. Bacterial community associated with 476
ensilage process of wilted guinea grass. J. Appl. Microbiol. 107:2029-2036. 477
25.26. Pena, G. A., C. M. Pereyra, M. R. Armando, S. M. Chiacchiera, C. E. 478
Magnoli, J. L. Orlando, A. M. Dalcero, C. A. R. Rosa, and L. R. Cavaglieri. 479
2010. Aspergillus fumigatus toxicity and gliotoxin levels in feedstuff for 480
domestic animals and pets in Argentina. Let. Appl. Microbiol., 50:77–81. 481
26.27. Ranjit, N. K., and L. Kung, Jr. 2000. The effect of Lactobacillus 482
buchneri, Lactobacillus plantarum, or a chemical preservative on the 483
fermentation and aerobic stability of corn silage. J. Dairy Sci. 83:526-535. 484
27.28. Richard, E., N. Heutte, V. Bouchart, and D. Garon. 2009. Evaluation 485
of fungal contamination and mycotoxin production in maize silage. Anim. Feed 486
Sci. Technol. 148:309–320. 487
28.29. Rossi, F., and F. Dellaglio. 2007. Quality of silages from Italian farms 488
as attested by number and identity of microbial indicators. J. Appl. Microbiol. 489
103:1707-1715. 490
on April 22, 2020 by guest
http://aem.asm
.org/D
ownloaded from
21
30. Schindler, F. V., and R. E. Knighton. 1999. Sample preparation for total 491
nitrogen and 15N-ratio analysis by the automated Dumas combustion method. 492
Commun. Soil Sci. Plant Anal. 30:1315–1324. 493
29.31. Schmidt, R. J., L. Kung Jr. 2010. The effects of Lactobacillus buchneri 494
with or without a homolactic bacterium on the fermentation and aerobic stability 495
of corn silages made at different locations. J. Dairy Sci. 93:1616-1624. 496
30.32. Spoelstra, S. F., M. G. Courtin, and J. A. C. van Beers. 1988. Acetic 497
acid bacteria can initiate aerobic deterioration of whole crop maize silage. J. 498
Agric. Sci. Camb. 111:127-132. 499
31.33. Tabacco, E., F. Righi, A. Quarantelli, and G. Borreani. 2011. Dry 500
matter and nutritional losses during aerobic deterioration of corn and sorghum 501
silages as influenced by different lactic acid bacteria inocula. J. Dairy Sci. 502
94:1409-1419. 503
32.34. te Giffel, M. C., A. Wagendorp, A. Herrewegh, and F. Driehuis. 504
2002. Bacterial spores in silage and raw milk. Antonie van Leeuwenhoek 81: 505
625-630. 506
33.35. Weinberg, Z. G., Y. Chen, and R. Solomon. 2009. The quality of 507
commercial wheat silages in Israel. J. Dairy Sci. 92:638–644. 508
34.36. Woolford, M. K. 1990. The detrimental effect of air on silage. J. Appl. 509
Bacteriol. 68:101-116. 510
511
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Figure legends 512
513
FIG. 1. Dynamics of silage temperature, pH, lactic acid, yeast count, mold count and 514
aerobic spore count during air exposure of silages. PE: polyethylene film; OB: oxygen 515
barrier film. 516
517
FIG. 2. DGGE profiles of bacterial DNA (a and c) and RNA (b and d) extracted from 518
silage samples exposed to the air for 0, 2, 5, 7, 9, 14 days and ensiled into polyethylene 519
plastic bag (PE)(a and b) or into oxygen barrier bag (OB)(c and d). Bands indicated by 520
letters were subjected to sequencing as described in “Materials and methods” and the 521
results are reported in Table 3. Lane M: marker. 522
523
FIG. 3. DGGE profiles of fungal DNA (a and c) and RNA (b and d) extracted from 524
silage samples exposed to the air for 0, 2, 5, 7, 9, 14 days and ensiled into polyethylene 525
plastic bag (PE)(a and b) or into oxygen barrier bag (OB)(c and d). Bands indicated by 526
letters were subjected to sequencing as described in “Materials and methods” and the 527
results are reported in Table 4. Lane M: marker. 528
529
FIG. 4. Dendrograms obtained from the cluster analysis of DGGE profiles of the 530
bacterial microflora detected on PE and OB treated silage samples, at both DNA and 531
RNA level, throughout aerobic exposure. 532
533
FIG. 5. Dendrograms obtained from the cluster analysis of DGGE profiles of the fungal 534
microflora detected on PE and OB treated silage samples, at both DNA and RNA level, 535
throughout aerobic exposure. 536
537
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TABLE 1. Chemical and microbial composition of the corn forage prior to ensiling. 538
Item*
DM (g kg-1) 333
pH 5.84
TN (g kg-1 DM) 12.6
Starch (g kg-1 DM) 262
NDF (g kg-1 DM) 443
ADF (g kg-1 DM) 248
Ash (g kg-1 DM) 39.0
Nitrate (mg kg-1 herbage) 1181
aw 0.98
Yeasts (log10 CFU g-1 herbage) 6.90
Molds (log10 CFU g-1 herbage) 6.13
Aerobic spores (log10 CFU g-1 herbage) 3.57
*ADF = acid detergent fiber; DM = dry matter; NDF = neutral detergent fiber; TN = 539
total nitrogen; WSC = water soluble carbohydrates. 540
541
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TABLE 2. Fermentation quality and microbial composition at unloading of silages 542
sealed with an oxygen barrier (OB) and standard polyethylene (PE) films after 110 days 543
of conservation. 544
Treatment* PE OB SE P value
PpH 3.78 3.73 0.011 0.002
DM (g kg-1) 297 310 8.22 0.500
Lactic acid (g kg-1 DM) 45.3 53.2 2.08 0.033
Acetic acid (g kg-1 DM) 27.6 22.7 1.23 0.019
Butyric acid (g kg-1 DM) < 0.10 < 0.10 - -
Propionic acid (g kg-1 DM) 0.45 0.76 0.164 0.402
1,2-Propanediol (g kg-1 DM) 10.5 10.4 0.706 0.981
Ethanol (g kg-1 DM) 12.3 11.2 0.651 0.443
Lactic-to-acetic acid ratio 1.64 2.34 0.165 0.004
Nitrate (mg kg-1 silage) 837 1026 156 0.603
NH3-N (g kg-1 TN) 46.8 45.8 0.112 0.746
Ash (g kg-1 DM) 40.6 40.2 0.031 0.656
aw 0.99 0.99 0.001 0.947
Yeasts (log10 CFU g-1 silage) 3.12 1.17 0.443 < 0.001
Molds (log10 CFU g-1 silage) 1.74 1.41 0.118 0.189
Aerobic spores (log10 CFU g-1 silage) 2.65 2.97 0.095 0.095
Weight loss (g kg-1 DM) 37.5 30.6 0.178 0.035
Aerobic stability (h) 65 152 19.9 0.001
* aw = water activity; C = control treatment; DM = dry matter; LAB = lactic acid 545
bacteria; PE = under polyethylene film; LP = under oxygen barrier film; NH3-N = 546
ammonia nitrogen; TN = total nitrogen. 547
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† Within row, means followed by different letter are significantly different for P<0.05. 548
549
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TABLE 3. Sequence information for fragments detected on DGGE gels obtained by 550
analyzing the bacterial population through DNA and RNA direct analysis of silage 551
samples. 552
Band Closest sequence relative Identity (%) GenBank accession no.
a Lactobacillus buchneri 99 HM162413
b Acetobacter pasteurianus 98 AP011156
c Bacillus subtilis 100 HQ009797
d Lactobacillus amylovorus 100 EF439704
e Uncultured bacterium 98 GU343612
f Uncultured bacterium 100 GQ233026
g Lactobacillus plantarum 100 HQ117897
553
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TABLE 4. Sequence information for fragments detected on DGGE gels obtained by 554
analyzing the fungal population through DNA and RNA direct analysis of silage 555
samples. 556
Band Closest sequence relative Identity (%) GenBank accession no.
h Aspergillus fumigatus 99 HM807348
i Kazachstania exigua 100 FJ468461
l Pichia kudriavzevii 99 GQ894726
m Aureobasidium pullulans 98 GQ281758
n
Synthetic construct ankyrin
repeat protein E2_17 gene,
partial cds
100 AY195852
557
558
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20
25
30
35
40
45
0 2 4 6 8 10 12 14
PE OB
Aerobic exposure (h)
Sila
ge te
mpe
ratu
re (°
C)
3.0
4.0
5.0
6.0
7.0
0 2 4 6 8 10 12 14
PEOB
Aerobic exposure (d)pH
559
0
10
20
30
40
50
60
0 2 4 6 8 10 12 14
OB PE
Aerobic exposure (d)
Lact
ic a
cid
(g k
g -1
DM
)
0
2
4
6
8
10
0 2 4 6 8 10 12 14
PEOB
Aerobic exposure (d)
Yeas
t cou
nt (l
og C
FU g
-1)
560
0
2
4
6
8
10
0 2 4 6 8 10 12 14
PEOB
Aerobic exposure (d)
Mol
d co
unt (
log
CFU
g -1
)
0
2
4
6
8
10
0 2 4 6 8 10 12 14
PEOB
Aerobic exposure (d)
Aer
obe
spor
es (l
og C
FU g
-1)
561
562
FIG. 1. 563
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0 2 5 7 9 14M
a
b
c
d
e
f
RNA
b)
0 2 5 7 9 14M
a
b
c
DNA
a)
f
g
PE films
0 2 5 7 9 14M
a
b
c
d
e
f
RNA
b)
0 2 5 7 9 14M
a
b
c
0 2 5 7 9 14M 0 2 5 7 9 14M
a
b
c
DNA
a)
f
g
PE films
564
565
0 2 5 7 9 14 M
a
b
c
g
c)
a
b
c
g
0 2 5 7 9 14 M
d)
RNADNA
OB films
0 2 5 7 9 14 M0 2 5 7 9 14 M
a
b
c
g
c)
a
b
c
g
0 2 5 7 9 14 M
a
b
c
g
0 2 5 7 9 14 M0 2 5 7 9 14 M
d)
RNADNA
OB films
566
567
FIG. 2. 568
569
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0 2 5 7 9M 14
hi
l
a)
m 0 2 5 7 9 14M
h
m
b)
RNADNA
PE films
0 2 5 7 9M 140 2 5 7 9M 14
hi
l
a)
m 0 2 5 7 9 14M 0 2 5 7 9 14M 0 2 5 7 9 14M 0 2 5 7 9 14M
h
m
b)
RNADNA
PE films
570
571
0 2 5 7 9 14 M
hi
c)
0 2 5 7 9 14 M
h
m
n
d)
RNADNA
OB films
0 2 5 7 9 14 M0 2 5 7 9 14 M
hi
c)
0 2 5 7 9 14 M0 2 5 7 9 14 M
h
m
n
d)
RNADNA
OB films
572
FIG. 3. 573
574
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Pearson correlation [0.0%-100.0%]D GGE
100
908070605040302010
DNA_OB_5d
DNA_PE_0d
DNA_OB_0dDNA_PE_2d
DNA_OB_7d
DNA_OB_9d
DNA_OB_14dDNA_OB_2d
RNA_OB_2d
RNA_OB_5d
RNA_OB_0d
RNA_OB_14d
RNA_OB_7d
RNA_OB_9dRNA_PE_0d
RNA_PE_5dRNA_PE_7d
RNA_PE_9d
RNA_PE_14d
RNA_PE_2d
DNA_PE_7d
DNA_PE_9dDNA_PE_5d
DNA_PE_14d
Pearson correlation [0.0%-100.0%]D GGE
100
908070605040302010
DNA_OB_5d
DNA_PE_0d
DNA_OB_0dDNA_PE_2d
DNA_OB_7d
DNA_OB_9d
DNA_OB_14dDNA_OB_2d
RNA_OB_2d
RNA_OB_5d
RNA_OB_0d
RNA_OB_14d
RNA_OB_7d
RNA_OB_9dRNA_PE_0d
RNA_PE_5dRNA_PE_7d
RNA_PE_9d
RNA_PE_14d
RNA_PE_2d
DNA_PE_7d
DNA_PE_9dDNA_PE_5d
DNA_PE_14d
575
576
FIG. 4. 577
578
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Pearson correlation [0.0%-100.0%]D GGE
100
9590858075706560555045403530252015105
DNA_OB_7dDNA_PE_9d
DNA_OB_5dDNA_PE_7d
DNA_OB_9d
DNA_OB_0d
DNA_PE_14d
DNA_OB_2d
DNA_PE_0dDNA_PE_2d
DNA_PE_5d
DNA_OB_14d
RNA_OB_5d
RNA_OB_7d
RNA_OB_2d
RNA_OB_0dRNA_PE_0dRNA_PE_2d
RNA_PE_5d
RNA_OB_9d
RNA_OB_14d
RNA_PE_7d
RNA_PE_9d
RNA_PE_14d
Pearson correlation [0.0%-100.0%]D GGE
100
9590858075706560555045403530252015105
DNA_OB_7dDNA_PE_9d
DNA_OB_5dDNA_PE_7d
DNA_OB_9d
DNA_OB_0d
DNA_PE_14d
DNA_OB_2d
DNA_PE_0dDNA_PE_2d
DNA_PE_5d
DNA_OB_14d
RNA_OB_5d
RNA_OB_7d
RNA_OB_2d
RNA_OB_0dRNA_PE_0dRNA_PE_2d
RNA_PE_5d
RNA_OB_9d
RNA_OB_14d
RNA_PE_7d
RNA_PE_9d
RNA_PE_14d
579
580
FIG. 5. 581
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