Impacts of some factors that effect spoilage of silage at the periphery of the exposed face of corn silage piles

Accepted Manuscript Title: Impacts of Some Factors that Effect Spoilage of Silage at the Periphery of the Exposed Face of Corn Silage Piles Authors: Y. Okatsu, N. Swanepoel, E.A. Maga, P.H. Robinson PII: DOI: Reference:

S0377-8401(18)31249-5 https://doi.org/10.1016/j.anifeedsci.2018.11.018 ANIFEE 14113

To appear in:

Animal

Received date: Revised date: Accepted date:

18 September 2018 29 October 2018 28 November 2018

Feed

Science

and

Technology

Please cite this article as: Okatsu Y, Swanepoel N, Maga EA, Robinson PH, Impacts of Some Factors that Effect Spoilage of Silage at the Periphery of the Exposed Face of Corn Silage Piles, Animal Feed Science and Technology (2018), https://doi.org/10.1016/j.anifeedsci.2018.11.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Impacts of Some Factors that Effect Spoilage of Silage at the Periphery of the Exposed Face of Corn Silage Piles

a

SC RI PT

Y. Okatsu, N. Swanepoel, E. A. Maga, P. H. Robinson*

Department of Animal Science, University of California, Davis, CA, 95616, USA

* Corresponding author. Mobile: +1 530 754-7565;

N

U

Email: [email protected]

D

M

Revised and resubmitted in October of 2018.

A

Submitted to Animal Feed Science and Technology in October of 2018.

Factors impacting spoilage in corn silage at the periphery of the exposed face in nine silage

EP



TE

Highlights

piles were investigated. A feed-out speed of 0.29 m/day was sufficient to prevent spoilage of exposed face

CC



peripheral silage.

A





Decreasing days from pile opening to feedout from 110 to 61 reduced spoilage of exposed face peripheral silage. Surface density in the range of 312 to 360 kg/m3 had little impact on spoilage of exposed face peripheral silage.

Abstract Preserving silage in large piles is popular due to its flexibility and affordability that allows storage

SC RI PT

of large amounts of silage with low facility investment. Management of silage piles during feedout impacts silage spoilage since, once the plastic covers are removed and silage near the exposed silage ‘face’ is exposed to air, undesirable microbes can proliferate to negatively impact its hygienic quality and nutritional value. Pile silage also has characteristics, such as a long feed-out phase and a large exposed face peripheral area, which makes it prone to spoilage during feed-out,

U

especially in the exposed face area. Silage samples were collected from nine corn silage piles in

N

the San Joaquin Valley of California (USA) in order to evaluate impacts of feed-out speed, days

A

after opening and surface silage density on degree of silage spoilage at the periphery of the exposed

M

face. Faster feed-out speed (i.e., 0.59 versus 0.29 m/day) suppressed the degree of spoilage, as indicated by lower temperature and pH, and higher lactic levels, as well as higher abundance of

D

lactic acid bacteria (LAB). However, because surface spoilage in both fast- and slow-speed piles

TE

was judged to be in the spoilage initiation stage, the feed-out speed of 0.29 m/day was judged

EP

sufficient to prevent substantive spoilage. Fewer days after opening (i.e., 69 versus 110 days) impacted degree of spoilage as indicated by lower mold counts and higher lactic and acetic acid

CC

levels. As spoilage in long-open piles had reached mid-stage, while spoilage in short-open piles was still in the initiation stage, days after opening of 110 was judged too long to prevent surface

A

spoilage. Higher surface density (i.e., 360 versus 312 kg/m3 wet weight) had little impact on spoilage, due to its low impact on all response parameters. Overall, faster feed-out speed and fewer days after pile opening had the most positive impacts on suppressing spoilage in corn silage at the periphery of the exposed face, while increasing surface silage density was less impactful.

Keywords: surface spoilage; temperature; pH; yeasts and molds; silage microbiome. Abbreviations: AAB, acetic acid bacteria; DM, dry matter; DNA, deoxyribonucleic acid; LAB, lactic acid bacteria; NGS, next generation sequencing; OTU, operational taxonomic unit; PCR,

SC RI PT

polymerase chain reaction; RNA, ribonucleic acid; SJV, San Joaquin Valley (California, USA); WSC, water soluble carbohydrate.

1. Introduction

Ensiling generally refers to preservation of high moisture crops based on conversion of water-

U

soluble carbohydrates (WSC) in plants, mainly sugars, into lactic acid by lactic acid bacteria

N

(LAB) under anaerobic conditions which causes a decline in pH sufficient to stop microbial growth

A

thereby stabilizing the crop mass. Of the many ensiled forages, whole crop corn is the most

M

important in many dairy areas, including the San Joaquin Valley (SJV) of California (USA). In the initial aerobic phase of ensiling, harvested forage is placed in a silo and packed by

D

appropriate equipment to exclude as much ambient air as possible. Oxygen trapped in the ensiling

TE

crop is rapidly consumed by respiration facilitated by plant enzymes which generate heat by

EP

degrading WSC into water and CO2 (Henderson, 1993). The process transitions to the fermentation phase wherein epiphytic LAB grow rapidly once the O2 trapped in the silage mass is consumed by

CC

respiration, since most LAB bacteria compete efficiently for nutrients with epiphytic obligate and facultative aerobes that were active in the initial aerobic phase. These LAB degrade fermentable

A

sugars into lactic acid, acetic acid, ethanol and CO2, in various proportions depending upon LAB species (McDonald, 1981), which causes the pH of the crop to decline into the 3.8 to 5.0 range and, together with the acids themselves, prevents growth of microbes such as Enterobacteriaceae, Clostridium and Bacillaceae which are less tolerant of an acidic environment (Muck, 2010). Establishment of a microbial community dominated by LAB during the fermentation phase is

generally accepted as key to successful preservation of plant nutrients during the storage phase when the pH is low enough to inhibit growth of bacteria, including most LAB, and the chemical composition of the silage stabilizes due to very low biological activity.

SC RI PT

When a silo is opened for feed-out, silage is exposed to ambient air, and undesirable microbes including bacteria, yeasts and molds grow by consuming residual WSC and organic acids. The importance of this process, referred to as ‘aerobic deterioration’ or ‘spoilage’, depends upon its extent. In the 1st stage of spoilage, acid-tolerant aerobic microbes such as acetic acid bacteria (AAB) and yeasts grow. Acid tolerant yeasts play an important role in early stages of spoilage as

U

they largely survive storage due to their ability to anaerobically ferment sugars into CO2 and

N

alcohols. However, under post silo opening aerobic conditions, AAB and yeasts metabolize

A

substrates by cellular respiration, which increases silage temperature (and Pahlow et al. 2003

M

defined as ‘spoilage initiation’; Table 1). Involvement of AAB and acid-tolerant yeasts in spoilage initiation was shown by studies in EU countries (Spoelstra et al., 1988), while acid-tolerant yeasts

D

are considered to be the main initiator of spoilage based upon USA studies (Muck, 2010).

TE

Regardless, as acids are degraded by microbes the higher silage pH enables proliferation of a wider

EP

range of bacteria. The Bacillaceae (obligate/facultative anaerobic bacteria often found in silage during feed-out) proliferate after spoilage initiation. However, Enterobacteriaceae, (facultative

CC

anaerobes) also increase and can outcompete LAB by consuming residual sugars and organic acids. As a result, temperature and pH increase. If sufficient acid is metabolized, strictly aerobic

A

molds proliferate by oxidizing sugars and acids thereby causing further increases in temperature and pH. Because mold count increases only become apparent after silage has deteriorated due to activity of yeasts and other bacteria, their presence is an indication that spoilage has reached the ‘advanced stage’ (Muck, 2010; Table 1).

Of the several types of silos used commercially, pile silos, which ensile forage under a plastic cover after packing with a tractor, have become popular in many areas, especially on large commercial dairy farms such as those in the SJV. Pile silos have advantages over other silo types,

SC RI PT

including lower facility costs and higher flexibility in terms of the amount of crop ensiled, as well as unloading ease. However, silage piles have a large surface area to mass ratio and potential exposure to air. In addition to being an economic loss, removal of spoiled silage is a safety issue because large silage piles can be >9 m high and climbing on them to remove surface material is hazardous. Nevertheless, in order to maintain hygienic quality of the silage that is fed to cattle it is

U

important to prevent spoilage at the exposed silo face during feed-out because substantive spoilage

N

in pile silage occurs in silage near the exposed peripheral face (Robinson et al. 2016).

A

Increasing feed-out speed of silage in piles (i.e., the ‘speed’ that an exposed silo face moves

M

back due to silage removal) has been suggested to be one of the most effective ways to minimize aerobic deterioration of silage during feed-out. In British studies, the minimum recommended

D

feed-out speed was 0.10 to 0.30 m/day, and faster during summer (Harris et al., 1966). In the

TE

Netherlands, a similar feed-out speed was recommended; 0.15 to 0.30 m/day during winter and

EP

twice as fast in summer (Vissers et al., 2007). An Italian study, which used >100 silage piles, showed that the proportion of visibly molded silage in the exposed surface area of bunker silage

CC

decreased when feed-out speed reached 0.23 to 0.29 m/day (Borreani and Tabacco, 2012). In the USA, Pitt and Muck (1993) evaluated effects of feed-out speed on aerobic deterioration and

A

reported only 30 g/kg dry matter (DM) loss at 0.15 m/day, while 90 g/kg DM loss occurred at 0.05 m/day. In another study in the USA, feed-out speed >0.30 m/day was recommended for silage in bunker silos and piles due to minimize DM loss (Muck, 2011).

The degree of aerobic deterioration is also affected by depth of penetration of ambient air into a silage mass, which is itself influenced by silage density, porosity and DM content (Wilkinson and Davies, 2013). Silages with higher density tend to have reduced depth of air penetration as air

SC RI PT

is excluded from silage particles during packing of a fresh chop mass. In order to maintain the same degree of penetration in silage with higher DM levels, more packing is needed because there is more space between particles (Honig, 1991, Muck, 2011). However, this is successful to a limited degree in high DM crops as they generally do not remain fully compressed, even after repeated packing. Indeed the DM loss of corn silage in bunker silos after 180 days of ensiling

U

declined from 200 to 100 g/kg when density increased from 500 to 1100 kg/m3 wet weight (Ruppel

N

et al. 1995). Other studies reported similar inverse relationships between bulk density and DM loss

A

(Ruppel et al., 1995, Griswold et al., 2009, Sucu et al., 2016). In the USA, >641 kg/m3 wet weight

M

density was suggested to minimize air penetration into silage and minimize aerobic deterioration (Jones et al., 2004). However there is a wide range in silage densities in different locations within

D

a silo, with silage in the surface area and edge of a bunker, having much lower density relative to

TE

deep in the silage mass (Oelberg et al., 2006). In pile silage, spoilage often occurs in surface areas

EP

near the exposed face (i.e., the silage actually fed to cattle), likely because this area tends to have low density and is exposed to air under the plastic covers throughout feed-out.

CC

In addition to feed-out speed and surface density, the number of days after silo opening could affect the degree of surface silage spoilage. For example, Holmes (1998) reported higher DM losses

A

in bunker and pile silage relative to bagged silage with a small exposed face. Although silage piles are covered by plastic covers and weights during storage and feed-out, it is functionally not possible to completely prevent air penetration between the cover and silage. That Robinson and Swanepoel (2016), observed increase in mold and yeast count in silage 7 m back of the exposed

face suggests spoilage in the exposed face peripheral area progresses into the pile as the number of days after silo opening increases. Because silage fermentation is largely based on microbial activities, qualification of the

SC RI PT

microbial population during ensiling is important if the objective is to direct fermentation to create a desirable silage fermentation profile. Until ~2001, culture-dependent media methods were standard in silage microbiology studies, even though microorganisms that were compatible with such laboratory techniques only accounted for a small proportion of the total silage microbial population (Stewart, 2012). Since ~2010, next-generation sequencing (NGS) techniques have increased relative abundance of Lactobacilli, and reduced Proteobacteira and

U

shown

N

Bacteroidetes bacteria in lab-scale grass silage inoculated with Lactobacillus buchneri CD034

A

(Eikmeyer et al., 2013). A study which investigated bacterial community composition of lab-scale

M

silage from different crops using NGS and traditional denaturing gradient gel electrophoresis, reported data on 259 and 28 genera, respectively (Ni et al., 2017). However, no study has used

D

NGS for bacterial community assessment in commercial silages.

TE

Objectives were to assess effects of the pile management factors feed-out speed, days after

EP

opening and surface density on degree of spoilage of silage at the periphery of the exposed silage face. The hypothesis was that higher feed-out speed and surface density would suppress the degree

CC

of spoilage, as would fewer days after opening, by reducing the time that surface silage was

A

exposed to air that slowly ingresses under plastic covers at the exposed silage face after opening.

2. Materials and methods 2.1. Terms used The terms ‘front ramp’, ‘pile body’, ‘back ramp’, ‘exposed face’ and ‘height’ indicate aspects of a silage pile (Figure 1). In the silage pile building process, fresh crop is piled from the back to

front ramps with tractors using large blades moving back and forth over the pile along its longitudinal axis (Figure 1) in order to achieve desirable pack density. Piles are usually opened from the front ramp and fed-out by daily removal of silage from the exposed face.

SC RI PT

2.2. Pile selection Since the numerous known and unknown factors that impact the quality of silage at an exposed silo face cannot be balanced in a study where the number of silage piles that can be used is limited, several criteria were fixed among the piles selected. Thus, of 15 initially identified corn silage piles located on dairy farms in the SJV, only nine were selected. The criteria used were: 1) the pile was

U

made from a corn crop harvested in August/September 2016, 2) ensiled using a silage inoculant,

N

3) the pile had an ordinary straight shape such as those in Figure 1, 3) the pile had to have good

A

silage face management with a smooth vertical surface to minimize exposure of silage particles to

M

air 4) the pile had a double plastic cover of 5 mm outer with a thin inner underlay, 5) the pile had good fermentation quality (i.e., no substantive increase in pH and temperature in the outer core in

D

a preliminary test core while all piles were unopened), 6) the exposed face had to be anticipated to

TE

be past the front ramp and into the pile body at the time of sample collection, 7) face orientation

EP

could not be south, 8) all piles were to be unloaded using side-to-side actions of a bucket loader, 9) the pile had to be anticipated to have at least 7.5 m between the exposed face and the back ramp

CC

at the time of sample collection, 10) silage samples could be collected between 8 and 12 h, 11) the pile had to be anticipated to have at least 30% of the silage in the body of the pile at sample

A

collection, 12) the expected time after pile opening had to be anticipated to be between 1 and 4 months after opening, and 13) removal of spoiled silage from the surface could not occur during the 14 d prior to pile sampling. 2.3. Silage sample collection

After storage periods of 30 to 120 d from pile building to opening, the plastic covers were cut open at one end of the pile and silage was removed daily with a large front-end loader. Each pile was visited regularly during feed-out to calculate feed-out speed, defined as the speed during the

SC RI PT

14 d immediately before sample collection. Silage samples were collected in 3 ‘lines’ on the surface differentiated by their distance from the exposed face (Figure 2) with Line 1 being 0.3 m, Line 2 being 3.0 m and Line 3 being 6.1 m from the face. Line 1 silage had no plastic cover (removed to facilitate feed-out) while Lines 2 and 3 were plastic covered. In each line, there were 6 coring locations: locations 1 and 6 were 1.5 to

U

1.8 m from the base of the piles (Bottom-side), locations 2 and 5 were in the middle of the side of

N

the piles (Mid-side), and locations 3 and 4 were on top of the pile (Top). Samples were collected

A

from two locations on the exposed face (locations 7 and 8 in Figure 2), both located 1.5 m above

M

grade and ~ 1⁄3 and 2/3 across the width of the exposed face. Inner core silage at these locations is referred to as ‘deep mass silage’, and is accepted to best represent the largest proportion of silage

D

in the pile. Prior to sample collection, the length of the bottom, top and sides of each pile were

TE

measured to determine exposed face area during feed-out using standard geometric calculations.

EP

Each coring event consisted of collecting samples from 2 depths, being 0 to 25 cm (‘outer core’) and 25 to 50 cm (‘inner core’) into the pile surface. All coring used a 4.76 cm (inner

CC

diameter) by 36 cm (for outer core) and 61 cm (for inner core) long stainless steel core tube driven by an 18 volt Ridgid Drill (Model R86008; Ridgid Tool Co., Elyria, OH, USA). Samples were

A

collected through short slits in the plastic covers. After measurements (Section 2.4), silage samples from each line were pooled by core location and depth to create 6 samples/line (i.e., 3 outer core, 3 inner core). Exposed face samples were also pooled to create 1 outer, and 1 inner, core sample.

Pooled samples were subsampled into three, and used for analysis of (1) DM, volatile compounds, yeast and mold counts (125 g), (2) DNA-based bacterial community analysis (15 g) and (3) reserve sample (balance of sample). Samples 1 were kept on ice until analysis, samples 2

were kept on ice during transportation and stored at -20°C. 2.4. On-site measurements

SC RI PT

were kept on ice during transportation and stored at -20°C until DNA extraction, and samples 3

Temperature of the exposed end of outer and inner core samples was measured immediately after removal of each sample by pointing an IR thermometer (Model 561; Fluke, Everett, WA,

U

USA) at the end center silage in the core tube. A small portion, ~4 g, of silage from the core tip

N

was placed in a 150 mL plastic cup to which 80 mL of distilled water was added and mixed. After

A

2 min, pH was measured with an ‘Oyster 15’ pH meter (Extech Instruments, Nashua, NH, USA).

in the steel core by core volume.

M

Cored silage samples were weighed to calculate sample density by dividing silage sample weight

D

Ambient temperature and relative humidity in the area of each pile was recorded every 30 min

TE

during the 2 weeks prior to sample collection with a ONSET Hobo portable weather station (Onset

EP

Computer Corporation, Bourne, MA, USA). 2.5. Analytical procedures (DM, volatile compounds, yeast and mold counts)

CC

Silage samples, dried at 55°C for 48 h and air equilibrated for 24 h, were ground to pass a 1 mm screen on a model 4 Wiley Mill (Thomas Scientific, Swedesboro, NJ, USA). The air

A

equilibrated ground samples were dried for 4 h at 105oC and analytical DM, reported in all tables, was calculated by multiplying air equilibrated DM by the 105oC DM. Silage samples were analyzed for acids (i.e., lactic, acetic, propionic, butyric, succinic, formic), and alcohols (i.e., ethanol, 1 propanol, 2 propanol, 1,2-propanediol, 2,3-butanediol, 2 butanol)

using high performance liquid chromatography (Muck and Dickerson, 1988) in a system with a refractive index detector (model 2414; Waters Corporation, Milford, CN, USA) and a BioRad Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA, USA) at 35°C. Yeast and mold

2.6. Analytical procedures (Bacterial community composition) 2.6.1. DNA extraction

SC RI PT

counts were determined by pour plating (Adesogan et al., 2004).

Silage samples (10 g) were ground prior to DNA extraction with liquid N using a mortar and pestle. Total genomic DNA was extracted from 500 mg of sample with a FastDNA SPIN Kit for

U

Soil (MP Biomedical, Solon, OH, USA) according to manufacturer’s instructions. Quantity and

N

purity of extracted DNA was measured with a NanoDrop ND-2000 UV-Vis spectrophotometer

A

(Thermo Fisher Scientific, Waltham, MA, USA) and stored at -20°C. Only outer and inner samples

M

from the ‘Top’ part of the piles in Line 1 and the deep mass samples were sequenced. 2.6.2. Library preparation and Illumina MiSeq sequencing

D

DNA amplification and sequencing of bacterial 16S rRNA genes were completed at RTL

TE

genomics (Lubbock, TX, USA). The V4 region of the 16S rRNA gene was amplified in a 2-step

made

EP

polymerase chain reaction (PCR) process. Target DNA was amplified using the forward primer, with

the

Illumina

i5

sequencing

primer

CC

(5’TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG3’) and the 515F primer, and the reverse

primer,

made

with

the

Illumina

i7

sequencing

primer

A

(5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG3’) and the 806R primer (Caporaso et al. 2011). Each reaction contained Qiagen HotStar Taq master mix (Qiagen, Inc. Valencia, CA, USA), primers and template DNA. Reactions were run on ABI Veriti thermocyclers (Applied Biosytems, Carlsbad, CA, USA) with cycling conditions: 95°C for 5 min, 25 cycles of 94°C for

30 sec, 54°C for 40 sec, 72°C for 1 min, followed by one cycle of 72°C for 10 min. Amplified products were added to a 2nd PCR for a 2nd amplification by the forward primer (5’AATGATACGGCGACCACCGAGATCTACAC

[Nextera

i5

index]

SC RI PT

TCGTCGGCAGCGTC3’) and reverse primer (5’CAAGCAGAAGACGGCATACGAGAT [Nextera i7 index] GTCTCGTGGGCTCGG3’) based on Illumina Nextera PCR primers (Illumina, Inc. San Diego, CA, USA). Cycling conditions for the 2nd stage amplification were the same as the first stage except for 10 rather than 25 cycles.

The PCR products were confirmed by electrophoresis using eGels (Life Technologies, Grand

U

Island, NY, USA). Products were pooled and purified using Agencourt AMPure XP beads

N

(BeckmanCoulter, Indianapolis, IN, USA). After size selection, a Qubit 2.0 fluorometer (Life

A

Technologies, Carlsbad, CA, USA) was used to quantify the PCR products and the pools were

M

loaded on an Illumina MiSeq (Illumina, Inc. San Diego, CA, USA) 2 x 250 flow cell. 2.6.3. Data analysis

version

1.39.5

following

TE

mothur

D

Obtained sequence reads were de-multiplexed, quality filtered and further analyzed using the

mothur

standard

operation

procedure

EP

(mothur.org/wiki/MiSeq_SOP). Paired-end sequences from each sample were merged and reads with ambiguous base pairs or lengths longer than expected were removed. Unique sequences were

CC

aligned to the SILVA 16S rRNA reference database (release 128) with a threshold of 0.5. The database was customized to the region of interest using the pcr.seqs command prior to alignment.

A

After removing reads that did not align with the target region using the filter.seqs command, the chimera.vsearch command was used to identify and remove chimeric sequences based on the VSEARCH algorithm (Rognes et al., 2016). Reads were taxonomically classified by the ribosomal database project classifier based on Greengenes bacterial 16S rRNA database 13_8 release. After

sequences belonging to nonbacterial domains (i.e., chloroplasts, mitochondria, Archaea, eukaryotes) were removed as a final quality control, sequences were clustered into operational taxonomic units (OTUs) at 97% identity using the dist.seqs and cluster command of mothur.

SC RI PT

Coverage, numbers of OTUs and the inverse simpson index (a parameter of alpha-diversity) were calculated after subsampling to normalize the sequence data by rarefaction to the lowest number of reads in the dataset. Relative abundance of main genera (abundance >0.1%) were calculated and the abundance of LAB, AAB, Enterobacteriaceae, Bacillus and Clostridium were specifically calculated because they are the groups of bacteria commonly found in silage and

U

reported to impact fermentation and/or the spoilage process. The 16S rRNA amplicon sequence

N

data was submitted to the NCBI under Bioproject ID PRJNA484848.

A

2.7. Statistical analysis

M

In spite of this study being the largest to date that examined so many response parameters in silage piles, the number of piles sampled (i.e., 9) was insufficient to simultaneously evaluate

D

impacts of pile feed-out speed, days after pile opening and surface density. Even if more piles had

TE

been used it is not possible to pre-plan levels of these factors prior to pile opening. Thus, piles

EP

were divided into upper and lower groups (i.e., high- and low- for feed-out speed, short- and longfor days after opening, high- and low- for surface density) for each characteristic in order to

CC

independently analyze impacts of each. Significance was accepted if P<0.05 and a tendency if P<0.20. The latter high P value was used due to the low statistical power due to use of 9 piles.

A

2.7.1. Effects of feed-out speed on silage spoilage Temperature, pH, acids, alcohols, DM, ammonia, yeast and mold counts, and bacterial

abundance in Line 1 (i.e., exposed face peripheral silage) were analysed using the GLM procedure of SAS (2016) with pile feed-out speed (i.e., high speed and low speed) and core depth (i.e., outer

and inner) as fixed factors. The interaction of feed-out speed*depth was included. Parameters affected by feed-out speed were further analysed, but with line (i.e., distance from the exposed face) as a fixed factor.

SC RI PT

2.7.2. Effects of days after opening on silage spoilage The same parameters as described in Section 2.7.1. were analyzed using the GLM procedure of SAS with days after opening (i.e., short and long days open) and core depth (i.e., outer and inner) as fixed factors. The interaction of days after opening*depth was included. Parameters affected by days after opening were further analyzed using the GLM procedure, but with line (i.e.,

U

distance from the exposed face) as a fixed factor.

N

2.7.3. Effects of surface density on silage spoilage

A

The same parameters as described in Section 2.7.1. were analyzed using the GLM procedure

M

of SAS with surface density (i.e., high and low density) and core depth (i.e., outer and inner) as

2.8. Degree of spoilage

D

fixed factors. The interaction of surface density*depth was included.

TE

All parameters including temperature, pH, acids, alcohols, yeast and mold counts and bacteria

EP

abundance were used to determine degree of spoilage (Table 1), and to evaluate impacts of each

CC

of feed-out speed, days after opening and surface density on spoilage progression.

3. Results

A

3.1. General characteristics of all the corn silage piles used in study Physical and deep mass fermentation characteristics of all piles (Table 2) show an average

feed-out speed of 0.42 m/day, faster than that of 0.18 m/day recommended by Jones et al. (2004) for a horizontal silo. This speed was also faster than the 0.25 m/day recommended by Borreani

and Tabacco (2012) for bunkers. Average days after opening at sample collection was 92, with a range of 55 to 120, and the average face area was 112 m2 with high variation. The average temperature and pH in deep mass silage was 36.1°C, and 3.66, respectively, with

SC RI PT

relatively low variation. Both temperature and pH are in the range of well-preserved silage as reported in previous studies (e.g., Kung, 2011). Average deep mass pack density was 801 kg/m3 wet weight, which is higher than the minimum set by the San Joaquin Valley Air Pollution Control District (Rule 4570, SJVAPCD, Fresno, CA, USA) of 641 kg/m3 wet weight for corn silage, and similar to the deep mass density of 7 corn silage piles in the SJV (Robinson and Swanepoel, 2016).

U

Acids detected in deep mass silage included lactic, acetic, propionic and butyric, with all values

N

within ranges reported by Weissbach and Strubelt (2008). Succinic and formic acid occurred at

A

similarly low levels to those reported by Robinson and Swanepoel (2016). Alcohols detected in

M

deep mass included 1,2-propanediol, 2,3-butanediol and ethanol, and their average levels were within ranges reported by Weissbach and Strubelt (2008). Average yeast and mold counts in the

D

deep mass silage was 1400 and 7400 cfu/g respectively, with a bacterial population dominated by

TE

LAB, which indicates no substantive spoilage (Table 1) in deep mass silage.

EP

3.2. Effects of feed-out speed on surface silage spoilage Temperature, and the inner/deep mass temperature ratios, were higher (P<0.05) in low-speed

CC

piles (Table 3), which also tended (P<0.20) to have higher pH, although the Speed*Depth interaction (P=0.06) suggested higher pH in low-speed pile outer cores, but no difference in the

A

inner cores (Figure 3). Ammonia tended (P<0.20) to be lower, lactic acid levels were lower (P<0.05), and propionic acid was higher (P<0.05) in low-speed piles. Butyric and succinic acids tended (P<0.20) to be higher, 2,3-butanediol was lower (P<0.05) and ethanol levels were higher (P<0.05) in low-speed piles.

The LAB abundance was much lower (P<0.05) in low-speed piles, and the genus Bacillus tended (P<0.20) to be higher in low-speed piles. 3.3. Effects of days after opening on surface silage spoilage

SC RI PT

Ammonia tended (P<0.20) to be higher, but lactic and acetic acids tended (P<0.20) to be lower in long-open piles (Table 4). The 1,2-propanediol level was lower (P<0.05), and mold counts tended (P<0.20) to be higher, in long-open piles. 3.4. Effects of surface density on surface silage spoilage

Temperature tended (P<0.20) to be higher (Table 5), and ammonia tended (P<0.20) to be

U

lower, in low-density piles. The inner/deep mass temperature ratio tended (P=0.20) to be higher in

N

low-density piles, which also tended (P<0.20) to lower LAB, and increase AAB abundance.

A

3.5. Effects of surface sample depth on surface silage spoilage

M

Impacts of sample depth on surface silage fermentation and degree of spoilage (Tables 4, 5 and 6) show that lactic and acetic acids, and DM, were higher (P<0.05) in inner cores, whereas

D

mold and yeast counts were lower (P<0.05). The speed*depth and density*depth interactions

EP

TE

(P=0.06) indicate different pH due to core depth in low density and low speed piles (Figure 3).

4. Discussion

CC

During feed-out of silage from piles, the plastic covers are progressively cut away as silage is removed for feeding, resulting in the silage on the periphery of the face being directly exposed to

A

air. As a result, acid-tolerant and facultative or obligate aerobes such as Enterobacteriaceae and AAB proliferate by consuming sugars, lactic acid, acetic acid and ethanol. In the presence of O2, these bacteria can metabolize substrate into water and CO2 via the tricarboxylic acid cycle which enables them to obtain energy more efficiently than through fermentation (Pahlow et al., 2003). Although LAB are aerotolerant anaerobes, they cannot utilize O2 for biological activities. Thus,

facultative and obligate aerobes can outcompete LAB in an aerobic environment by depriving them of nutrients. During this oxidation, the heat released increases silage temperature. As acids are degraded by microbes, the silage pH increases, which opens the way for a wider range of

SC RI PT

microbes including Bacillus and acid-tolerant yeasts, which results in further degradation of acids. Once pH has increased, yeasts will start to grow followed by an increase in molds, which are less tolerant to low O2 and low pH than are yeasts. 4.1. Effects of feed-out speed on surface silage spoilage

Rapid feed-out speed is considered an effective way to prevent spoilage of silage in piles (e.g.,

U

Wilkinson and Davies, 2013) because it minimizes the time that silage particles are exposed to

N

ambient air, and prevents microbes from being activated by O2 to cause silage spoilage.

A

The higher temperature and pH, as well as lower lactic acid level, in low- versus high-speed

M

piles suggests that the slower feed-out speed allowed aerobic microbes to proliferate and initiate spoilage to a greater extent in the exposed face peripheral area of low-speed piles. In piles with no

D

spoilage, temperature will be higher in deep mass versus inner core versus outer core as heat which

TE

continues to be produced in the deep mass dissipates to, and then from, the surface. Thus the higher

EP

inner core/deep mass temperature ratio in low-speed piles suggests that its sub-surface silage hosted spoilage microbes that created heat. The lower LAB, and higher Bacillus abundance, in the

CC

low-speed pile bacterial community also supports spoilage initiation being more progressed. In addition, the speed*depth interaction of pH (Figure 3) shows that pH was higher in low-speed

A

piles, but only in the outer 25 cm of the surface, suggesting that this surface silage had progressed further the spoilage process than silage from the sub-surface 25 to 50 cm depth, likely due to more exposure of silage to ambient air in the outer 25 cm. Higher levels of propionic and butyric acids in the low-speed piles may indicate activity of Clostridium and yeasts, although their levels are

too low for it to be certain that they were associated with spoilage. Nevertheless, the similarity in yeast and mold counts between low and high-speed piles suggests that the spoilage process in lowspeed piles was still in transition from the initiation to mid-stage as indicated by increases in

SC RI PT

temperature and pH, and decreases in lactic and acetic acids, and LAB. It is clear that there was no difference in temperature between outer and inner cores in both low and high-speed piles at Line 3 (i.e., the furthest from the exposed face; Figure 4). However, in Line 2, 3.0 m closer to the exposed face, inner core silage had a higher temperature than outer core in high-speed piles. Even though the temperature difference between core depths is small in

U

Line 2, it suggests initiation of spoilage had begun in Line 2, likely due to ingress of air between

N

surface silage and the plastic cover due to pile opening. That temperature differences between the

A

core depths of low-speed piles became bigger, and the temperature of the low-speed pile outer core

M

was higher, suggesting that at both core depths of low-speed piles the spoilage had progressed further into the piles (i.e., farther back of the exposed face). Indeed the higher temperature increase

D

from Line 3 to 1 in low- versus high-speed piles suggests that slower feed-out speed allowed

TE

aerobic microbes to proliferate faster by consuming sugars and acids. This is supported by the

EP

lactic acid level decreasing more in low-speed piles from Line 3 to 1. This increase in temperature, and more gradual decrease in lactic acid levels from Line 3 to 1 in inner core silage, was probably

CC

because inner core silage was less exposed to air. In contrast, that pH did not change between Line 3 and 1, and there was no difference between high and low-speed piles in any line, suggests that

A

pH does not respond to progressive spoilage stages as quickly as temperature and lactic acid levels. The feed-out speeds of the slow- and high-speed piles, 0.29 and 0.59 m/day respectively, are

both faster than previously recommended: 0.28 m/day by Borreani and Tabacco (2012), 0.10 to 0.30 m/day by Vissers et al. (2007) and 0.15 m/day by Pitt and Muck (1993). While comparison

of our results with previous studies is difficult due to difference in definition of spoilage, our results suggest that low-speed piles had a generally low degree of spoilage and that increasing feed-out speed to 0.59 m/day did not substantively impact it. This finding contrasts with Robinson and

SC RI PT

Swanepoel (2016), who reported substantial levels of spoilage associated with face movement in surface silage (i.e., 0 to 50 cm depth) as far as 7 m back of the exposed face in a corn silage pile with a feed-out speed of 0.24 m/day. In that study the surface silage temperature increase closer to the exposed face, with pH not changing regardless of distance from the exposed face, supports our view that pH is a less sensitive indicator of spoilage progression in corn silage than is temperature.

U

Succinic acid is usually found in aerated silage as it is produced by LAB from organic acids at

N

higher pH (McDonald, 1981). It has also been reported to be associated with Enterobacteriaceae

A

(Muck, 2010). Even though there was no clear pattern in levels of succinic acid from Line 3 to 1

M

(Figure 5), its seemingly higher levels in low-speed piles might be due to increased activity of Enterobacteriaceae caused by longer air exposure. As 2,3-butanediol is produced from sugars by

D

a wide range of microbes including Bacillus, Enterobacteriaceae and yeasts (McDonald, 1981),

TE

as well as from citric and malic acid by LAB, it is not possible to identify which microbe(s)

EP

accounted for the 2,3-butanediol in our piles. Nishino and Shinde (2007) reported a decrease in 2,3-butanediol concentrations in whole crop rice silage inoculated with LAB, suggesting that its

CC

presence may be related to acid-intolerant microbes such as Bacillus and Enterobacteriaceae. However, the level of 2,3-butanediol did not change from Line 3 to 1 (Figure 5), probably due to

A

the range of microbes associated with its production. Why its level was higher in high-, versus low-speed, piles, inconsistent with Nishino and Shinde (2007), is unclear. Studies are needed to understand microbial activities which account for 2,3-butanediol production in corn silage.

Overall, low-speed piles were judged to be in transition from initiation to the mid-stage of spoilage, as indicated by increases in temperature and pH, and decreases in lactic and acetic acids, as well as LAB. Even though the surface silage lactic acid level of high-speed piles was lower than

SC RI PT

in deep mass silage, the surface silage in high-speed piles was clearly in the early part of the initiation stage of spoilage as indicated by its low temperature, low pH and high abundance of LAB. Overall, results suggest that feed-out speed impacted degree of spoilage in face area peripheral silage, but that spoilage had not progressed past initiation (high-speed piles) or midstage (low-speed piles) of spoilage in our range of feed-out speeds.

U

4.2. Effects of days after opening on surface silage spoilage

N

Theoretically, silage should remain stable well back of the exposed face during feed-out if it

A

was well covered and an anaerobic environment is maintained. The problem is that even when a pile

M

is well covered, the passing of time inevitably allows holes and tears in the plastic due to rodent/bird/human actions. Thus, over a period of months, oxygen gets under the plastic and allows spoilage to start so that

D

when the pile is uncovered the spoilage process can advance more rapidly. Robinson and Swanepoel

TE

(2016) reported surface silage 7 m back of the exposed face had a substantial level of spoilage,

EP

which was likely caused by air ingress between plastic covers and exposed face of silage piles. If spoilage in surface silage is caused by air ingress between plastic covers and silage, piles with

CC

fewer days after opening should have a lower degree of spoilage than piles that have been open longer. However, there is no study which has determined if days after silo opening impacts the

A

degree of spoilage in surface silage. The decrease in lactic and acetic acid levels in long-open piles indicates that these acids were

likely degraded by facultative or obligate aerobic microbes to a greater extent in these piles due to longer exposure time to air. Longer time of exposure to air appeared to allow microbes to degrade acids, which let molds proliferate in long-open piles. That these piles tended to have higher

Clostridium abundance suggests co-existence of an anaerobic and aerobic environment during spoilage (Pahlow et al., 2003). When silage is exposed to air during feed-out, aerobic microbes outcompete LAB by rapidly consuming sugars and acids. In this process, the O2 between silage

SC RI PT

particles could be consumed by aerobic microbes if their speed of using O2 is faster than the speed of air penetrating the silage mass. Thus the environment can become anaerobic again, albeit temporarily, suggesting that higher abundance of Clostridium in long-open piles might have been caused by this temporal anaerobic environment even though the Clostridium population was small. 1,2-Propanediol is produced by a wide range of bacteria including Escherichia coli (Cabiscol

U

et al., 1992) and Clostridium sphenoides (Tran-Din and Gottschalk, 1985). However, a novel

N

pathway of L. buchneri, which ferments 1 mole of lactic acid to 0.5 mole of acetic acid, 0.5 mole

A

of 1,2-butanediol and a trace of ethanol, was reported by Oude Elferink et al. (2001). Indeed many

M

studies have shown increased 1,2-butanediol, and aerobic stability, in silage inoculated with L. buchneri (Filya et al., 2000, Adesogan et al., 2004). That the level of 1,2-propanediol was stable

D

from Line 3 to 1 in both groups and core sample depths (Figure 6), and inner core silage of short-

TE

open piles had the highest level suggests the possibility that these piles had more 1,2- propanediol

EP

production from L. buchneri due to a shorter time of air exposure, while long-open piles allowed other microbes to grow and outcompete L. buchneri. However, phylogenic identification of LAB

CC

to the species level is difficult by 16S rRNA gene sequencing and it is not clear why short-open piles had generally higher levels of 1,2-propanediol than did long-open piles.

A

In general, long-open piles were judged to be in transition from mid- to advanced-stage of

spoilage as indicated by increases in mold counts and pH, and decreases in lactic and acetic acid levels. In contrast, short-open piles seemed to be between initiation and mid-stage of spoilage as indicated by lower mold counts and pH, and higher lactic and acetic acid levels, relative to long-

open piles. Longer days after opening had a generally detrimental impact on degree of spoilage, as suggested by its effects on mold counts, lactic acid, acetic acid and bacterial community composition. This is consistent with Robinson and Swanepoel (2016) who measured the degree of

SC RI PT

surface silage spoilage at 90 and 160 d after pile opening, and found that mold counts in exposed face peripheral area silage 160 d after opening was higher than that at 90 d. 4.3. Effects of surface density on surface silage spoilage

Silage density is considered to be an important factor in silage spoilage (Ruppel et al., 1995) because lower density silage has more space between particles, which lets air penetrate the silage

U

mass to enable acid-tolerant, facultative or obligate aerobic microbes to outcompete LAB. The

N

higher temperature in exposed face peripheral silage of low, versus high, density piles indicates

A

that lower surface density allowed more air penetration between silage particles thereby allowing

M

more heat to be produced from metabolic activities of acid-tolerant, facultative/obligate aerobic microbes during early stages of spoilage initiation. The slightly lower LAB, and higher AAB

D

(aerobic bacteria reported to be associated with spoilage initiation) in low-density piles also

TE

suggests that high surface density had a suppressive impact on progression of spoilage.

EP

Both low and high-density piles were judged to be in transition from initiation to mid-stage spoilage because both had high pH, low lactic acid and LAB, and high AAB. But low-density piles

CC

were judged to be slightly more progressed as they had higher temperature and lower LAB abundance. However, because there were few meaningful differences in response parameters

A

between low and high-density piles, it appears that surface density impacted the progress of spoilage to a low extent. While Griswold et al. (2009) and Sucu et al. (2016) reported lower DM loss associated with an increase in density, these studies focused on deep mass silage. In addition to its small effect on spoilage progression, pile surface density was not related to deep mass

density, probably due to lack of self-compaction of surface silage, an impact reported in a study that examined a range in silage densities from different bunker silo locations (Oelberg et al., 2006).

SC RI PT

5. Conclusions Impacts of feed-out speed, days after opening and surface density on degree of silage spoilage at the periphery of the exposed face of corn silage piles was assessed. As potential interactions among these factors could not be examined due to the limited number of piles used, their overall impacts should be considered in this context.

U

Feed-out speed of corn silage piles impacted spoilage progression in exposed face peripheral

N

silage as low-speed pile silages were judged to be transitioning from initiation to mid-stage

A

spoilage based on increases in temperature and pH, and decreases in lactic and acetic acids as well

M

as LAB. In contrast, high-speed piles were still in the initiation stage as indicated by a low temperature and pH, and a high abundance of LAB. Nevertheless, as feed-out speed did not have

D

a substantive impact on the degree of spoilage, the average feed-out speed of 0.29 m/day was

TE

judged to be sufficient to prevent substantive surface silage spoilage in the exposed face periphery.

EP

Days after pile opening impacted degree of spoilage as suggested by increases in mold counts in long- versus short-open piles. Long-open piles were judged to be in transition from the mid- to

CC

advanced-stage of spoilage. In contrast, short-open piles were judged to be between initiation and mid-stage as indicated by lower mold counts and pH, and higher lactic and acetic acid levels. As

A

surface spoilage in long-open piles was more progressed than in short-open piles, 110 days after silo opening was too long to prevent substantive surface silage spoilage in the exposed face silage. Surface density impacted the progression of surface silage spoilage because silage in piles with low surface density was judged to be in transition from the initiation to mid-stage of spoilage,

while high density piles were in the initiation stage, as indicated by lower temperature and higher LAB abundance. That there were few substantive differences in response parameters between low and high density piles, suggests that an average surface density of 312 kg/m3 wet weight was

SC RI PT

sufficient to prevent substantive surface silage spoilage in exposed face peripheral silage.

Conflict of Interest

The authors declare that none of them have a conflict of interest regarding this manuscript.

U

Acknowledgements

N

We thank the dairy farmers for their interest, assistance and patience during the study. The

A

CC

EP

TE

D

M

A

senior author also thanks Hannah and Tamar for help during sample collection and processing.

References Adesogan, A.T., Krueger, N., Salawu, M.B., Dean, D.B., Staples, C.R., 2004. The influence of

SC RI PT

treatment with dual purpose bacterial inoculants or soluble carbohydrates on the fermentation and aerobic stability of Bermudagrass. J. Dairy Sci. 87, 3407-16.

Borreani, G., Tabacco, E., 2012. Effect of silo management factors on aerobic stability and extent of spoilage in farm maize silages. In: Proc. XVI Int. Sil. Conf., Helsinki, Finland, pp. 71-2.

Cabiscol, E., Badia, J., Baldoma, L., Hidalgo, E., Aguilar, J., Ros, J., 1992. Inactivation of

U

propanediol oxidoreductase of Escherichia coli by metal-catalyzed oxidation. Biochimica et

N

Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1118, 155-60.

A

Caporaso, J.G., Lauber, C.L., Walters, W.A., 2011. Global patterns of 16S rRNA diversity at a

M

depth of millions of sequences per sample. In: Proc. Natl Acad Sci, USA, 108 Suppl. 1, pp 4516-22.

D

Eikmeyer, F.G., Köfinger, P., Poschenel, A., Jünemann, S., Zakrzewski, M., Heinl, S., Mayrhuber,

TE

E., Grabherr, R., Pühler, A., Schwab, H., Schlüter, A., 2013. Metagenome analyses reveal the

EP

influence of the inoculant Lactobacillus buchneri CD034 on the microbial community involved in grass ensiling. J. Biotechnol. 167, 334-43.

CC

Filya, I., Ashbell, G., Hen, Y., Weinberg, Z., 2000. The effect of bacterial inoculants on the fermentation and aerobic stability of whole crop wheat silage. J. Anim. Feed Sci. 88, 39-46.

A

Griswold, K.E., Craig, P.H., Dinh, S.K., 2009. Relating dry matter density to dry matter loss in corn silage bunker silos in Southeastern Pennsylvania. In: Proc. XV Int. Silage Conf., Madison, WI, USA. pp. 223-24. Harris, C., Raymond, W., Wilson, R., 1966. The voluntary intake of silage. In: Proc. 10th Int. Grassland Congress, Helsinki, Finland. p. 4.

Henderson, N., 1993. Silage additives. J. Anim. Feed Sci. 45, 35-56. Holmes, B.J., 1998. Choosing forage storage facilities. Dairy Feeding Systems Management, Components and Nutrients, 38-59.

SC RI PT

Honig, H., 1991. Reducing losses during storage and unloading of silage. Landbauforschung Voelkenrode. Sonderheft, Germany.

Jones, C., Heinrichs, A., Roth, G., Ishler, V., 2004. From harvest to feed: understanding silage management. The Pennsylvania State University Extension, University Park, PA, USA.

Kung, L., 2011. Silage Temperatures: How hot is too hot? University of Delaware, Dairy Research,

U

Teaching and Extension, Newark, DE, USA.

N

McDonald, P., 1981. The Biochemistry of Silage. John Wiley & Sons Ltd., San Francisco, CA.

A

USA.

M

Muck, R.E., 2011. The art and science of making silage. In: Proc. Western Alfalfa & Forage Conf., Las Vegas, NV, USA.

D

Muck, R.E., 2010. Silage microbiology and its control through additives. R. Bras. Zootec. 39, 183-

TE

91.

EP

Muck R. E., and Dickerson J. T., 1988. Storage temperature effects on proteolysis in alfalfa silage. Trans. Am. Soc. Agric. Biol. Eng. 31, 1005–9.

CC

Ni, K., Minh, T.T., Tu, T.T.M., Tsuruta, T., Pang, H., Nishino, N., 2017. Comparative microbiota assessment of wilted Italian ryegrass, whole crop corn, and wilted alfalfa silage using

A

denaturing gradient gel electrophoresis and next-generation sequencing. Appl. Microbiol. and Biotechnol. 101, 1385-94.

Nishino, N., Shinde, S., 2007. Ethanol and 2,3-butanediol production in whole-crop rice silage. Grassl. Sci. 53, 196-8.

Oelberg, T., Harms, C., Ohman, D., Hinen, J., Defrain, J., 2006. Silage density - survey shows more packing of bunkers and piles is needed. In: Proc. High Plains Dairy Conf., Albuquerque, NM, USA. pp. 47-54.

SC RI PT

Oude Elferink, S.J.W.H., Krooneman, J., Gottschal, J.C., Spoelstra, S.F., Faber F., Driehuis F., 2001. Anaerobic conversion of lactic acid to acetic acid and 1,2-propanediol by Lactobacillus buchneri. Appl. Environ. Microbiol. 67, 125-32.

Pahlow, G., Muck, R.E., Driehuis, F., Oude Elferink, S.J.W.H., Spoelstra, S.F., 2003. Microbiology of Ensiling. In: D. R. Buxton, R. E. Muck, J. H. Harrison, editors, Silage Science

U

and Technology, Agron. Monogr. 42. ASA, CSSA, Madison, WI, USA. pp. 31-93.

N

Pitt, R., Muck, R., 1993. A diffusion model of aerobic deterioration at the exposed face of bunker

A

silos. J. Agr Eng Res. 55, 11-26.

M

Robinson, P.H., Swanepoel, N., 2016. Impacts of a polyethylene silage pile underlay plastic with or without enhanced oxygen barrier (EOB) characteristics on preservation of whole crop maize

D

silage, as well as a short investigation of peripheral deterioration on exposed silage faces. J.

TE

Anim. Feed Sci. 215, 13-24.

EP

Rognes, T., Flouri, T., Nichols, B., Quince, C., Mahé, F., 2016. VSEARCH: a versatile open source tool for metagenomics. PeerJ. 4, e2584.

CC

Ruppel, K.A., Pitt R.E., Chase L.E., Galton D.M., 1995. Bunker silo management and its relationship to forage preservation on dairy farms. J. Dairy Sci. 78, 141-53.

A

SAS Institute Inc., SAS/STAR® Software: Changes and enhancements, Release 9.4. 2016. SAS Institute Inc., Cary, NC, USA.

Spoelstra, S., Courtin, M., Beers, J., 1988. Acetic acid bacteria can initiate aerobic deterioration of whole crop maize silage. J Agric Sci, 111, 127-32.

Stewart, E.J., 2012. Growing unculturable bacteria. J. Bacteriol. 194, 4151-60. Sucu, E., Kalkan, H., Canbolat, O., Filya, I., 2016. Effects of ensiling density on nutritive value of maize and sorghum silages. R. Bras. Zootec. 45, 596-603.

SC RI PT

Tran-Din, K., Gottschalk, G., 1985. Formation of d(-)-1,2-propanediol and d(-)-lactate from glucose by Clostridium sphenoides under phosphate limitation. Arch. Microbiol. 142, 87-92. Vissers, M.M.M., Driehuis, F., Te Giffel, M.C., De Jong, P., Lankveld J.M.G., 2007. Concentrations of butyric acid bacteria spores in silage and relationships with aerobic deterioration. J. Dairy Sci. 90, 928-36.

U

Weissbach, F., Strubelt, C., 2008. Correcting the dry matter content of maize silages as a substrate

N

for biogas production. Landtechnik 63, 82-3.

A

CC

EP

TE

D

M

developments. Grass Forage Sci. 68, 1-19

A

Wilkinson, J.M., Davies, D.R., 2013. The aerobic stability of silage: key findings and recent

A

CC

EP

TE

D

M

A

N

U

SC RI PT

Figure 1. Upper photo: A closed corn silage pile. [The ‘front ramp’ is the part of the pile surrounded by red (left) lines; the ‘pile body’ is the part of the pile surrounded by yellow (middle) lines; the ‘back ramp’ is the part of the pile surrounded by blue (right) lines; The arrow shows the direction of tractors moving during the packing process.] Lower photo: A corn silage pile during feed-out. [The ‘exposed face’ is surrounded by purple (trapezoid) lines; the ‘height’ of the pile is the length of red-dotted (vertical) arrow; the direction of feedout is indicated by the green (horizontal) arrow.]

A

CC

EP

TE

D

M

A

N

U

SC RI PT

Figure 2. Schematic of the sampling locations in the piles. [Dots indicate sample locations from the Bottom-side (locations 1 and 6 are 1.5 to 1.8 m from the base); Mid-side (locations 2 and 5 are middle of the side; Top (locations 3 and 4 are the flat top of the pile; Deep Mass (locations 7 and 8 are in the lower exposed face. Locations 1 to 6 occurred in all 3 lines.]

A

CC

EP

TE

D

M

A

N

U

SC RI PT

Figure 3. The Speed*Depth interaction for pH (upper), and Density*Depth interaction for pH (lower).

A

CC

EP

TE

D

M

A

N

U

SC RI PT

Figure 4. Temperature and lactic acid level from Line 3 to Line 1 in high- and low-speed piles at two depths of sampling. [HS: High-speed piles, LS: Low-speed piles; A,B – values differ at P<0.05 within line; Line 1: 0.3 m from the exposed face; Line 2: 3.5 m from the exposed face; Line 3: 6.5 m from the exposed face; Outer: 0 to 25 cm from the surface; Inner: 25 to 50 cm from the surface.]

A

CC

EP

TE

D

M

A

N

U

SC RI PT

Figure 5. Changes in succinic acid and 2,3 butanediol from Line 3 to 1 of high- and low-speed piles at two depths of sampling. [HS: High-speed piles, LS: Low-speed piles; A,B – values differ at P<0.05 within line; Line 1: 0.3 m from the exposed face; Line 2: 3.5 m from the exposed face; Line 3: 6.5 m from the exposed face; Outer: 0 to 25 cm from the surface; Inner: 25 to 50 cm from the surface.]

A

CC

EP

TE

D

M

A

N

U

SC RI PT

Figure 6. Changes in 1,2 propanediol levels from Line 3 to 1 of long- and short-open piles at two depths of sampling. [LO: Long-open piles, SO: Short-open piles; A,B – values differ at P<0.05 within line; Line 1: 0.3 m from the exposed face; Line 2: 3.5 m from the exposed face; Line 3: 6.5 m from the exposed face; Outer: 0 to 25 m from the surface; Inner: 25 to 50 cm from the surface.]

Table 1 Description of spoilage terms used in the text. Description

None

No proliferation of undesirable bacteria, yeasts or molds. No increases in temperature and pH. Yeasts <100,000 cfu/g, Molds < 500,000 cfu/g.

Initiation

Acid tolerant aerobic bacteria (e.g., AAB) and yeasts start to grow due to exposure to ambient air and their metabolism (i.e., degradation of acids) causes increased silage temperature and pH.

Mid

Wider range of bacteria (e.g., Bacillaceae, Enterococcaceae) start to proliferate as acids are degraded and silage pH increases. These bacteria outcompete LAB by consuming sugars and acids, which also causes higher increases in temperature and pH.

Advanced

Molds start to increase as acids are degraded by bacteria and yeasts. Severely increased temperature and pH.

M

A

N

U

SC RI PT

Degree of spoilage

A

CC

EP

TE

D

Adapted from Pahlow et al. (2003), Muck (2010) and the authors’ experience.

Table 2 Characteristics of the nine piles and their deep massa silage. Mean

SE

0.42

0.075

91.7

8.17

112

17.2

6.5

0.71

11.6

3.58

85.1

4.71

36.1

0.74

3.66

0.036

801

39.6

351

7.1

1.1

0.05

9.93

2.79

45.4

4.47

19.7

1.74

01.4

0.58

1,2-Propanediol

4.7

1.35

2,3-Butanediol

4.8

2.54

2.1

0.81

Yeasts

1.4

0.61

Molds

7.0

5.58

Days after pile opening at sample collection Exposed face area (m2) Maximum face height (m) Ambient temperature during feed-out (°C)c Ambient humidity during feed-out (%)d

U

General Characteristics of the Deep Massa Silage of the Nine Piles Temperature (°C)

N

pH

A

Wet weight density (kg/m3)

M

Dry matter (g/kg) Ammonia (g/kg DM)

Lactic

EP

Acetic

TE

Acids (g/kg DM) e

D

Crude protein (g/kg DM)

Succinic

SC RI PT

General Characteristics of the Nine Piles Pile feed-out speed (m/day)b

A

CC

Alcohols (g/kg DM)

Ethanol

Yeasts and molds (*1000 cfu/g)

Bacterial abundance (% in total population)

76.4

6.79

AABg

3.1

0.97

Enterobacteriaceae

0.4

0.22

Clostridium

0.34

0.148

Bacillus Others

0.8

0.59

SC RI PT

LABf

18.9

A

CC

EP

TE

D

M

A

N

U

a – Deep Mass silage is silage collected from the center bottom of the exposed face (see Figure 2). b – The speed that the exposed face moved back due to silage removal. c – Average ambient temperature in the area of the piles during the 14 days prior to sample collection. d – Average ambient humidity in the area of the piles during the 14 days prior to sample collection. e – There were no detectable levels of propionic, butyric and formic acids in any sample. f – Lactic acid producing bacteria in the families Acetoanaerobium, Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae and Leuconostocaceae. g – Acetic acid producing bacteria in the family Acetobacteriaceae.

5.97

Table 3 Impacts of feed-out speed and depth on surface silage fermentation and degree of spoilage. Feed-out speeda (m/d)

Depth (cm)b (Outer) (Inner) 0-25 25-50

Temperature (°C) Outer: Innerc Inner:Deep massd

38.8 1.07 0.97

30.2 0.93 0.83

35.26 -

33.75 -

pH

4.17

3.89

4.21

3.86

DM (g/kg)

341

320

314

348

Ammonia (g/kg DM)

1.0

2.7

2.0

1.7

Lactic

19.8

26.7

15.1

Acetic

15.9

16.9

12.2

Propionic

0.4

<0.0

Butyric

0.6

<0.0

Succinic

1.0

1,2-Propanediol

CC

Ethanol

Depth Spd*Depth 0.64 -

0.51 -

0.122

0.17

0.09

0.06

16.0

0.13

0.02

0.34

1.24

0.13

0.74

0.82

3.59

0.03

<0.01

0.38

20.6

4.03

0.79

0.02

0.88

0.2

0.2

0.20

0.02

0.88

0.88

0.1

0.4

0.34

0.07

0.26

0.26

0.3

0.3

1.0

0.49

0.09

0.10

0.79

N

U

0.02 0.14 0.03

D

A

31.4

3.3

3.6

1.9

5.0

1.50

0.90

0.22

0.76

3.1

15.7

10.3

8.5

2.73

0.01

0.67

0.99

1.7

<0.0

0.4

1.3

0.97

0.05

0.28

0.28

EP

2,3-Butanediol

Spd

2.02 0.05 0.08

Acids (g/kg DM) e

Alcohols (% DM)

SEM

SC RI PT

(High) 0.59

TE

(Low) 0.29

M

P

Yeasts and molds (*1000 cfu/g) 1089

1060

1934

214

822

0.97

0.02

0.90

Molds

685

588

1064

209

462

0.80

0.04

0.62

A

Yeasts

Bacterial abundance (% in total population) f LABg

19.7

70.3

42.2

47.8

10.8

<0.01

0.69

0.37

AABh

17.7

12.2

16.2

13.7

11.3

0.57

0.79

0.52

Enterobacteriaceae

1.9

3.0

2.8

2.1

2.55

0.60

0.75

0.56

Clostridium Bacillus Others

0.7 11.1 39.9

0.5 1.3 13.6

0.3 4.4 34.0

0.9 7.9 27.6

0.55 6.70 13.9

0.74 0.11 <0.01

0.23 0.56 0.41

A

CC

EP

TE

D

M

A

N

U

SC RI PT

a – The speed that the exposed face moved back due to silage removal. b – ‘Outer’ is 0 to 25 cm from surface and ‘Inner’ is 25 to 50 cm from surface. c – Ratio of temperature in outer core over inner core. d – Ratio of temperature in inner core over deep mass silage. e– There were no detectable levels of formic acid in any sample. f – Average abundance of bacteria in piles of each group. g – Lactic acid producing bacteria in the families Acetoanaerobium, Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae and Leuconostocaceae. h – Acetic acid producing bacteria in the family Acetobacteriaceae.

0.64 0.46 0.35

Table 4 Impacts of days after opening and depth on surface silage fermentation and degree of spoilage. Days after pile openinga

(Outer) (Inner) 0-25 25-50

35.6 1.02 0.92

34.2 0.98 0.88

35.77 -

34.02 -

pH

4.15

3.93

4.24

3.84

DM (g/kg)

331

333

317

347

Ammonia (g/kg DM)

2.4

0.9

1.8

1.5

Lactic

20.9

25.4

15.0

Acetic

13.9

19.4

12.3

Propionic

0.3

0.2

Butyric

0.1

0.5

Succinic

0.8

0.72 0.37 0.55

0.66 -

0.98 -

0.143

0.35

0.09

0.90

17.0

0.86

0.05

0.25

1.25

0.15

0.79

0.69

3.91

0.18

<0.01

0.29

21.1

3.50

0.08

<0.01

0.33

0.2

0.3

0.24

0.81

0.83

0.55

0.1

0.5

0.38

0.24

0.24

0.73

1.0

0.54

0.70

0.13

0.93

M

D

Ethanol

1.0

6.5

2.1

5.4

1.13

<0.01

0.08

0.25

8.3

9.1

9.6

7.8

3.46

0.89

0.75

0.89

1.2

0.5

0.4

1.4

1.13

0.46

0.29

0.94

EP

2,3-Butanediol

31.2

0.3

0.6

TE

1,2-Propanediol

Days*Depth

2.51 0.058 0.096

Acids (g/kg DM) e

Alcohols (g/kg DM)

Depth

U

Temperature (°C) Outer: Innerc Inner: Deep massd

Days

SC RI PT

SEM

N

(Short) 69

P

A

(Long) 110

Depth (cm)b

CC

Yeasts and molds (*1000 cfu/g) 1195

927

1913

209

748

0.69

0.02

0.72

Molds

887

336

1037

186

425

0.13

0.03

0.54

A

Yeasts

Bacterial abundance (% in total population) f LABg

41.9

42.6

39.9

44.6

23.9

0.97

0.82

0.82

AABd

17.3

12.6

16.2

13.7

11.40

0.62

0.80

0.55

Enterobacteriaceae

3.3

1.2

2.5

2.0

2.49

0.32

0.82

0.83

Clostridium Bacillus Others

0.9

0.3

0.3

0.8

4.5 32.1

9.5 33.8

5.0 36.0

9.0 29.8

0.53 7.74 19.30

0.16

0.23

0.45 0.91

0.54 0.70

A

CC

EP

TE

D

M

A

N

U

SC RI PT

a – The number of days between pile opening and sample collection. b – ‘Outer’ is 0 to 25 cm from surface and ‘Inner’ is 25 to 50 cm from surface. c – Ratio of temperature in outer core over inner core. d – Ratio of temperature in inner core over deep mass silage. e – There were no detectable levels of formic acid in any sample. f – Average abundance of bacteria in piles of each group. g – Lactic acid producing bacteria in the families Acetoanaerobium, Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae and Leuconostocaceae. h – Acetic acid producing bacteria in the family Acetobacteriaceae.

0.83 0.97 0.97

Table 5 Impacts of surface density and depth on surface silage fermentation and degree of spoilage. Depth (cm)b

P

(Inner) 25-50

Temperature (°C) Outer: Innerc Inner: Deep massd

38.3 0.99 0.94

32.3 0.99 0.85

36.29 -

34.33 -

pH

4.18

3.94

4.28

3.84

DM (g/kg)

334

330

317

348

Ammonia (g/kg DM)

0.9

2.4

1.8

1.5

Lactic

20.6

24.7

14.3

Acetic

15.6

17.0

11.9

Propionic

0.2

0.3

Butyric

0.5

0.1

Succinic

0.6

0.59 -

0.61 -

0.125

0.24

0.04

0.06

17.5

0.79

0.05

0.51

1.26

0.17

0.78

0.78

4.09

0.24

<0.01

0.64

20.7

3.86

0.65

0.01

0.31

0.2

0.24

0.81

0.83

0.55

0.1

0.5

0.38

0.24

0.24

0.73

0.8

0.54

0.70

0.13

0.93

U

0.12 0.93 0.20

M

Ethanol

31.0

0.3

0.3

1.0

3.5

3.4

1.8

5.0

1.51

0.98

0.20

0.84

9.2

8.3

9.7

7.8

3.46

0.87

0.73

0.90

0.5

1.2

0.4

1.4

1.13

0.46

0.29

0.94

EP

2,3-Butanediol

Depth Den*Depth

D

1,2-Propanediol

Den

2.28 0.05 0.08

Acids (g/kg DM) e

Alcohols (g/kg DM)

SEM

N

(Outer) 0-25

A

(High) 360

TE

(Low) 312

SC RI PT

Density (kg/m3)a

CC

Yeasts and molds (*1000 cfu/g) 1506

732

2023

214

748

0.23

0.01

0.27

Molds

722

578

1099

201

460

0.71

0.03

0.61

A

Yeasts

Bacterial abundance (% in total population) f LABg

26.3

54.9

38.1

43.1

20.9

0.13

0.78

0.65

AABh

22.6

9.3

17.2

14.8

10.70

0.15

0.79

0.57

Enterobacteriaceae

1.6

3.0

2.6

2.0

2.54

0.49

0.80

0.96

Clostridium

0.4

0.8

0.3

0.9

0.54

0.44

0.27

0.46

Bacillus Others

9.5 39.6

4.5 27.4

5.0 36.8

9.0 30.2

7.74 18.87

0.44 0.44

0.54 0.68

0.97 0.85

A

CC

EP

TE

D

M

A

N

U

SC RI PT

a – The surface density (i.e., average wet weight density of samples collected from Line 2 and 3 (0 to 50 cm depth). b – ‘Outer’ is 0 to 25 cm from surface and ‘Inner’ is 25 to 50 cm from surface. c – Ratio of temperature in outer core over inner core. d – Ratio of temperature in inner core over deep mass silage. e – There were no detectable levels of formic acid in any sample. f – Average abundance of bacteria in piles of each group. g – Lactic acid producing bacteria in the families Acetoanaerobium, Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae and Leuconostocaceae. h – Acetic acid producing bacteria in the family Acetobacteriaceae.