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EFFECTS OF MICROBIAL CULTURES

AND VARIOUS OTHER ADDITIVES ON THE

FEEDING QUALITY, FERMENTATION PATTERN,

DRY MATTER RECOVERY AND AEROBIC

STABILITY OF HIGH MOISTURE CORN

By

Frank Arthur Wardynski

A Thesis

Major Professor
Steven R. Rust

Submitted to Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Animal Science

1991

ABSTRACT

EFFECTS OF MICROBIAL CULTURES AND VARIOUS OTHER ADDTTIVES ON
THE FEEDING QUALITY, FERMENTATION PATTERN, DRY MATTER
RECOVERY AND AEROBIC STABILITY OF HIGH MOISTURE CORN

By

Frank Arthur Wardynslci

Three experiments were conducted to evaluate the effects of lactic acid producing
bacteria inoculation (LABI) on the preservation, nutritive quality and aerobic stability of
high moisture corn. In experiment one, large concrete upright silos were utilized to.
evaluate control (CON) vs LABI. Experiment two used small laboratory silos to evaluate
CON vs LABI and whole vs ground corn in trial 1 and CON, LABI, reconstitution with
water, nitrogen addition and acid addition in trial 2. Experiment three evaluated corn
stored at four dry matter (DM) contents (78; 74; 66; 63 % DM), three storage
temperatures (4; 12.5; 20 °C), and treated with CON or LABI. Dry matter recovery was
decreased with LABI in two trials, similar in two trials and increased in one trial. Corn
treated with LABI was less stable after aerobic exposure as compared to CON (P <
.05). Feeding quality was similar between LABI and CON (P > .10). Experiment

results indicate inoculant use is not consistently beneficial or cost effective.

ACKNOWLEDGEMENTS

. The reported research could not have been finished without the help of many
people. I greatly appreciate everyone for their efforts.

I offer my warmest thanks to Dr. Steven Rust, who served as my advisor and
friend. I appreciate Dr. Rust’s uncompromising principles and work ethic. Dr. Rust
was supportive and offered encouragement when needed. I also would like to thank.
Steve and his family for their care and closeness.

Special thanks goes to Dr. Harlan Ritchie and Dr. David Hawkins for serving on
my committee. Their shared expertise and experience in the beef industry and their
friendship will be treasured indefinitely.

Dr. Melvin Yokoyama deserves thanks for serving on my committee, his expertise
in microbial fermentation, the use of his microbiology lab and technical assistance.

Thanks also goes to Dr. Oran Hesterman for serving on my committee and his
special knowledge from the agronomic viewpoint.

I owe a great debt to many faculty, staff and students. Sincere thanks goes to all
who assisted in the many hours of microbiological work. I also appreciate the assistance
of technicians Sue Hengemuehle and Elaine Fink. Special thanks are also offered to
Kaye Hillock for typing the manuscript and to Tadd Dawson in preparing the graphics.

I also need to offer great thanks to David Main, David Lust and Peter Anderson for their

iii

unselfish help and fiiendship.

I can never express the thanks which my parents deserve. Their love, support
and guidance will always be appreciated. My values, morals and accomplishments are
a direct result of them. I am truly grateful and I love you. Other members of my
family, my sister and grandparents also are recipients of my love and gratitude.

I also would like to thank Kathy, my wife. She has been a special inspiration and

worked diligently on the manuscript.

iv

TABLE OF CONTENTS

PAGE

List of Tables ......................................... vii
List of Figures ......................................... x
Introduction ........................................... 1‘
Literature Review

Physical factors which effect the efficiency

of silage fermentation ................................. 3

Effects of chemical constituents of the plant

on silage fermentation ................................ 6

Harvest and storage losses associated with

high moisture grains ................................. ll

Microbiology of silage ................................ 15

Additives and inoculants ............................... 19

Feeding value of high moisture corn ........................ 26
Experiment 1. The effects of microbial inoculation

for the preservation of high moisture corn

on cattle performance, fermentation

characteristics and aerobic stability ......................... 31

Experiment 2. The effect of inoculation, mechanical
processing, reconstitution and chemical -
addition on the fermentation of high moisture corn ............... 54

Experiment 3. Effects of dry matter content, storage
temperature and microbial inoculation on the
fermentation and aerobic stability of high

moisture corn ...................................... 77
Conclusion ........................................... 124
Appendix ............................................ 130
Literature Cited ........................................ 140

vi

Experiment 1

Table 1.

Table 2.

Table 3.

Experiment 2

Table 1.

Table 2.

Experiment 3

Table 1.

LIST OF TABLES

Composition of diet fed to cattle . . .

Effects of inoculation of high moisture
corn on efficiency of utilization for

growing and finishing eattle ......

Effects of inoculation on chemical
changes in ensiled high moisture corn

Effects of inoculation and
mechanical processing on the chemical
characteristics of corn after 40d

ensilement at 25 % moisture ......

Effects of inoculation, reconstitution
and chemieal addition on fermentation
characteristics of high moisture corn
after 40d ensilement at 20% moisture

Initial (d-O) chemical characteristics
of corn harvested at various dry

matter contents ..............

PAGE

.............. 38

.............. 43‘

.............. 45

.............. 65

.............. 72

.............. 84

Table 2.

Table 3.

Table 4.

Table 5 .

Table 6.

Table 7.

Table 8.

Table 9.

Table 10.

Table 11.

Table 12.

Table 13.

Effect of inoculation on dry matter

recovery after 67d ensilement ................... 86
Correlation of means after 67d of

fermentation ............................. 87

Calculation of substrate conversion
efficiency ................ 104

Effects of dry matter content and
inoculation on fermentation ................... 107

Effects of storage temperature and
inoculation on fermentation
characteristics after 67d ensilement ............... 109

Effects of dry matter content on
dry matter recovery after 48 hours
of aerobic exposure ........................ 111

Correlation of means after 48 hours
of aerobic exposure ........................ 112

Temperature rise above ambient
during aerobic exposure ..................... 114

Effects of dry matter content by storage

temperature interaction on high

moisture corn after 48 hours of

aerobic exposure .......................... 115

Effects of dry matter content by inoculation
interaction on high moisture corn
after 48 hours of aerobic exposure ............... 117

Effects of storage temperature by

inoculation interaction on high

moisture corn after 48 hours of

aerobic exposure .......................... 118

Recovery of dry matter and crude protein

after 67d of ensilement and 48 hours of aerobic
exposure as affected by inoculation .............. 120

viii

Table 14.

Table 15.

Recovery of dry matter and crude
protein after 67d of ensilement and
48 hours of aerobic exposure as

affected by dry matter content .................. 121
Calculated savings by the use of
an inoculant ............................. 122

Experiment 1

Figure 1.

Figure 2.

Figure 3.

Experiment 2

Figure 1.

Figure 2.

Figure 3.

LIST OF FIGURES

Change of temperature in silos
during fermentation due to inocu-

lation treatment ....................

Change in microbial population of
high moisture corn during aerobic

exposure due to inoculation ............

Change in temperature of high moisture
corn during aerobic exposure due to

inoculation ......................

Effect of inoculation rate and
mechanical processing on pH of high

moisture corn during fermentation ........

Effect of inoculation rate and
mechanical processing on the lactic
acid content of high moisture corn

during fermentation .................

Effect of inoculation rate and
mechanical processing on the acetic
acid content of high moisture corn

during fermentation .................

PAGE

........ 50

........ 51

........ 52

........ 64

........ 66

........ 67

Figure 4. Effect of inoculation rate and
mechanical processing on the ethanol
content of high moisture corn during
fermentation ............................. 69

Figure 5 . Effect of inoculation rate and
mechanical processing on the soluble
earbohydrate content of high moisture
corn during fermentation ...................... 70

Experiment 3

Figure 1. Effects of storage temperature and
dry matter content on pH decline ................. 89

Figure 2. Effects of storage temperature and
dry matter content on lactic acid
concentration ............................. 90

Figure 3. Effects of storage temperature and
dry matter content on acetic acid
concentration ............................. 91

Figure 4. Effects of storage temperature and
dry matter content on ethanol
concentration ............................. 92

Figure 5. Effects of storage temperature and dry
matter content on water soluble
carbohydrate concentration ..................... 93

Figure 6. Effects of storage temperature and dry
matter content on crude protein concentration ......... 94

Figure 7. Effects of storage temperature and dry matter content
on water soluble nitrogen concentration ............. 95

Figure 8. Effects of storage temperature and dry matter content
on ammonia nitrogen concentration ............... 96

Figure 9. Effects of storage temperature and dry matter content
on lactic acid bacteria counts ................... 97

xi

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Effects of storage temperature and dry matter content
on enterobacterioceae counts ............... ’ . . . . 98

Effects of storage temperature and dry matter content
on Bacillus counts .......................... 99

Effects of storage temperature and dry matter content
on yeast counts ........................... 100

Effects of storage temperature and dry matter content
on mold counts ........................... 101

Effects of storage temperature and dry matter content
on dry matter recovery ...................... 102

xii

INTRODUCTION

Corn is the most common grain fed to livestock in Michigan. The popularity of
corn grain can largely be attributed to the energy density and cost of production of other
energy sources. Livestock producers are accustomed to fading corn, have a good
knowledge base of its feeding characteristics and usually have on-farm storage
capabilities.

Corn, for all practical purposes, is harvested for storage as dry or high moisture
corn. Corn is often harvested wet and dried to 15 96 moisture. This allows the product
to be stored in a relatively stable state, allowing minimal mold and microbial growth to
occur. Dry storage of corn has advantages over high moisture corn. Some of the
advantages include: 1) can be moved through existing marketing channels; 2) can be used
by the milling industry; 3) easily transported; 4) is an excellent energy source for cattle
and maintains condition for extended periods of time in the feedbunk.

. However, certain disadvantages associated with dry corn can be overcome by
harvest as an ensiled feedstuff. Fermentation removes the problems associated with
insects and mold contamination and may lessen the negative impact of mycotoxins.
Other advantages of high moisture corn storage include: 1) an earlier and extended
harvest season allowing for more efficient labor utilization; 2) less harvest loss as
opposed to harvesting dry corn; 3) improved digestibility. Likewise, drying corn to an
acceptable moisture content is expensive due to high energy costs.

Unfortunately, storage of high moisture corn as ensilage has certain draw backs

2

as well. Ten to twenty percent of the dry matter in high moisture corn can be lost during
storage and feeding. This problem can be minimized by proper harvest, storage and
feeding practices.

Filling the silo or storage structure should be rapid. to minimize respiration losses.
Feeding rate should be rapid to reduce aerobic deterioration on exposed surfaces.
Storage structures must be air tight or well-consolidated to ensure an anaerobic
environment.

To minimize losses in the silo structure, several amendments have been added to
the crop material before ensilement. One of these amendments has been the addition of,
starter cultures to redirect the fermentation pattern toward less carbon dioxide production
and hence more carbon retention in the silo.

This thesis project was designed to evaluate the ability of starter cultures to
enhance the fermentation process in a low-moisture silage. Trials one and two were
designed to evaluate the benefit of a microbial inoculum to high moisture corn in field-
scale silos. Once it became evident the commercially available inoculants had little
benefit in high moisture corn, experiment three was designed to characterize the effects
of environmental factors on the fermentation process. It was expected this
characterization would provide the necessary background to initiate studies to develop a

more efficacious inoculant for high moisture com.

LITERATURE REVIEW

Physical Factors Which Affect The Efficiency

Of Silage Fermentation

Oxygen exclusion is an important factor in the efficient fermentation of feedstuffs.
Oxygen may be excluded by compaction during silo filling, enzymatic respiration or
through bacterial fermentation. Compaction is the firmness and tightness with which
feedstuffs are packed into the silo. A well compacted silo creates the environment for
rapid lactic acid accumulation. Silo type, particle size and moisture content influence
consolidation, compaction and oxygen depletion in silages.

Feedstuffs in upright silos are firmly packed by the weight of feed mass, whereas
compaction is achieved mechanically in bunker silos. Dry matter (DM) losses are
greater from bunker than upright silos (Voelker et al., 1985). Typically, the dry matter
recovery from bunker and oxygen limiting upright silos is 85 96 and 98.6% respectively
(Williams et al., 1979).

Losses during feedout and from surface exposure due to aerobic deterioration
varies among silo structure type. Bunker silos have greater surface exposure, allowing
more time for air to penetrate the feed mass than in upright silos. Typieal energy losses

attributed to aerobic deterioration during storage and after unloading range from 0-1095

3

4
and 0—1596, respectively (McDonald, 1981).

Particle size reduction will enhance compaction and reduce trapped air during silo
filling which allows fermentation to develop more rapidly. Decreasing chop length or
particle size increases fermentation rate, which results in lower pH values and greater
lactic acid accumulation (Kroqu et a1. , 1955; Gordon et al. , 1959). Increased
fermentation rate due to smaller particle size results from the release of juices,
consolidation and air exclusion (Woolford, 19840).

Stage of maturity and moisture content can influence degree of compaction and
fermentation rate. The optimum moisture content for fermentation of high moisture.
grains is between 25 and 30% (Fox, 1976; Aguirre et al., 1983). Grains ensiled with
adequate moisture compact more completely. Moisture content is more critical in grains
stored in bunker silos as the opportunity for air exposure is greater. Low moisture,
ensiled grains are difficult to consolidate adequately which enhances the risk of an
extended aerobic exposure period. During ensilement of low moisture grains, mold,
yeast and aerobic bacteria dominate the fermentation which increases fermentation losses
and may reduce aerobic stability.

Likewise, high moisture grains can be ensiled too wet which results in excessive
fermentation (Goodrich and Meiske, 1976). An extended fermentation increases dry
matter and energy losses and reduces palatability. Dry matter consumption of wet
ensiled grains is reduced which coincides with elevated levels of soluble nitrogen (Prigge
et al., 1976) and acids (Wilkinson et al., 1976; Shaver et al., 1984).

Varietal differences (genetics) and external factors (environment) affect the

5

maturity, yield, nutrient accumulation and epiphytic microbial populations present. A
readily available source of carbohydrate is needed for an adequate fermentation to occur.
Variety differences in soluble carbohydrate composition and response to stressful
environmental conditions can influence the efficiency of the fermentation.

Corn reaches physiological maturity at 30-44% moisture (Goodrich and Meiske,
1976). Maturity can be determined by presence of a black layer at the tip of the kernel.
Once the black layer has developed, nutrient deposition is near completion. Immature
seeds are high in cell wall, crude fiber, ash, protein and water (Copeland and McDonald,
1985). Starch, sugar and lipids are the last chemical constituents to be deposited .
(Daynard and Duncan, 1969). Later maturing corn varieties generally produce greater
yields, however, these varieties contain higher moisture levels later into the harvest
season. Consequently, harvest of corn at higher moisture allows the use of later
maturing and higher yielding varieties. Another advantage of harvesting corn at higher
moisture levels is 10% less field loss (Goodrich and Meiske, 1976).

Genetics and environment have been shown to alter nutrient composition of grains
(Jones et al., 1981; Kofoid et al., 1982). Protein content can be increased by nitrogen
fertilization, low plant density and dry weather. High protein content during dry weather
is a function of less carbohydrate deposition in the kernel. In other crops such as
soybeans, elevated temperatures have increased lipid content. Lipids are minor
constituents of cereal grains (Copeland and McDonald, 1985) and of small importance
during fermentation.

Environmental stresses ean greatly influence the number and species of

6

microorganisms present on field crops. Stresses which limit lactic acid bacteria are
ultraviolet light, temperature and available nutrients (McDonald, 1981). Lactic acid
bacteria accumulate on areas of the plant which have available sap and increase in

numbers as plants become more mature (Daeschel et al., 1987).

Effects Of Chemical Constituents Of The Plant On Silage Fermentation

Preservation of corn through fermentation has been described as a four stage
process (Barnett, 1954). The first stage involves continued plant respiration through the
action of enzymes. During this stage carbon dioxide and heat are produced. Plant.
respiration involves enzymatic oxidation of soluble carbohydrates to carbon dioxide and
proteolysis of protein to less complex forms of nitrogen. During stage 2, aerobic
metabolism is initiated which yields primarily acetic acid. Management techniques which
minimize the duration of stages 1 and 2 should increase the efficiency of nutrient
conservation in the ensilage process. Stage 3 involves the initiation of lactic acid
production and anaerobiosis. The final phase involves a rather quiescent period where
lactic acid production reaches its peak and pH remains stable at 4.0.

A simplified chemical equation of respiration is Col-1,20. + 60, = 6C0; + 6H,O
+ 2870 Kilojoules. Cells from intact and freshly harvested plants respire. During
growth, plants capture and use respiration energy as ATP (adenosine triphosphate) for
biosynthesis of new plant material. A portion of captured energy is lost as heat to the
environment (Salisbury and Ross, 1969). Respiration continues in harvested plant

material which liberates larger amounts of energy from oxidized glucose as heat with

7
virtually no biosynthesis (McDonald, 1981).

Plant respiration is accomplished primarily by three sequential pathways
(McDonald, 1981). First, simple sugars are converted to pyruvate by glycolysis.
Secondly, pyruvate is oxidized to form acetyl Co—A which is oxidized in the Krebs cycle
to produce water, carbon dioxide, FADHZ and NADHz. The FADH, and NADH, are
used in the electron transport system as electron donors to phosphorylate ADP to ATP.

The primary concern of plant respiration during ensiling involves substrate
depletion and proteolysis. Several factors influence the extent of plant respiration during
the early stages of ensilement. Ruptured plant cells and elevated ambient temperature .
have been shown to increase the rate and velocity of respiration (McDonald, 1981).
Duration of plant respiration depends upon oxygen supply, cellular pH and temperature.
Firmly and rapidly consolidated silage undergoes less plant respiration beeause of less
available oxygen (McDonald et al. , 1966). In inadequately consolidated silos, silage
temperature can increase and reduce nutritional value (Henderson et al. , 1982).

Fermentation is a process in which microorganisms metabolize substrates for
energy and produce organic acids as endproducts (Salisbury and Ross, 1969).
Fermentation accelerates as plant respiration ceases. Available substrate is metabolized
by aerobic coliforms and heterolactic microorganisms. As oxygen is depleted and pH
decreases, homolactic fermentation dominates which establishes a relatively quiescent
forage mass. During the aerobic phase, the primary end products are acetic and lactic
acids and ethanol. The homolactic phase yields primarily lactic acid (Woolford, 1984c).

Plant carbohydrates can be classified into 3 types; consisting of soluble, Storage

8
and structural. Water soluble carbohydrates or simple sugars are readily available and

fermented by microorganisms. The chemical linkages in storage and structural
carbohydrates limits the availability of these compounds as substrates for microbial
growth. Starch, which is the most abundant storage carbohydrate in grains, can be
utilized by lactic acid bacteria (Lingren and Refai, 1984) and more readily than structural
carbohydrates; however, many of the desirable microorganisms lack an amylase enzyme
(McDonald, 1981). Structural carbohydrates are of minor importance in silage
fermentation because of the low cellulase activity in Lactobacillus species. Enzymes such
as cellulase (McHan, 1986) and hemicellulase (Dewar et al., 1963) may increase soluble .
sugar content of grass and legume silages.

Hexoses are fermented by either homolactic or heterolactic fermentation.
Fermentation type is determined by type of sugar fermented and microorganisms present.
Homolactic fermentation is a more efficient pathway for acid production (McDonald et
al. , 1987). Homoferrnentative organisms convert one mole of 6—carbon sugar into 2
moles of lactic acid.

Heterolactic fermentation has several different pathways in which 1 mole of
hexose is converted to 1 mole of lactate and other end products (McDonald, 1981).
During heterofermentation, 1 mole of glucose or fructose can be fermented to 1 mole
each of lactate, ethanol or acetate and earbon dioxide. Other microorganisms, in the
presence of fructose, a hydrogen acceptor, convert 2 moles of fructose to 1 mole of
mannitol and 1 mole of glucose. The glucose is further metabolized to lactate, acetate

and carbon dioxide. Heterofermentative bacteria may use oxygen as a hydrogen acceptor

9
to convert acetyl phosphate to acetate which yields an additional mole of ATP. Final end

products produced are lactate, acetate, water and carbon dioxide.

Pentoses can be fermented by both homolactic and heterolactic microorganisms
with similar efficiencies as hexoses. One mole of pentose is converted to 1 mole each
of lactate and acetate. Clostridia] bacteria ean metabolize sugars to butyrate, carbon
dioxide and heat (Gibson et al. , 195 8). Clostidial fermentation is inefficient in terms of
energy conservation and is, therefore, undesirable.

Lactic acid bacteria have a small role in the fermentation of organic acids such
as malic, succinic and citric acids (Woolford and Sawezyc, 1984 a,b; Grazia and Suzzi,
1984). Organic acids can be substrates for lactic acid bacteria but are used readily by
clostridia (Leibenspergen and Pitt, 1987). Clostridia ean ferment 2 moles of lactate to
produce butyrate and carbon dioxide.

Organic acids serve as buffers in silage. Organic acids tend to buffer between pH
4 and 6. Buffering capacity is commonly expressed as milliequivalents of alkali needed
to increase the pH of 1 kg of silage from 4 to 6. During fermentation, organic acids are
replaced with fermentation acids. Fermentation acids, such as lactic or acetic acid, have
stronger buffering capacities than organic acids originally present such as malic and citric
acid (Woolford, 1984). Accumulation of fermentation acids lower the pH and tend to
buffer at a lower pH.

Structural relationships and composition of nitrogenous compounds change during
fermentation (Bergen et al. , 1974). Proteolysis involves the solubilization and cleavage

of proteins into polypeptides. Much of the proteolytic activity in forage crops occurs

10
in the first few hours and is caused by enzymatic activity (Bergen et al., 1974). After

the initial enzymatic proteolysis, microorganisms may continue the proteolytic process.
Soluble nitrogen content has been shown to increase between day 28 and 56 in ensiled
high moisture corn (Prigge et al. , 1976) while microbial populations remained constant.
Baron et al. (1986) showed little proteolysis occurred in high moisture corn during the
early phases of ensilement but demonstrated a significant increase in the later
fermentation period. The authors concluded the low acidity may have contributed to the
increase in soluble nitrogen.

Lactic acid bacteria have limited proteolytic capabilities, however, clostridia] .
organisms do possess proteolytic fermentation pathways (Leibensperger and Pitt, 1987).
Clostridia can deaminate and deearboxylate amino acids to form ammonia, amines,
amides and organic acids (Edwards and McDonald, 1978). Another proteolytic pathway
used by Clostridia is the Stickland reaction (Woolford, 1984) which is an oxidation-
reduction reaction. One amino acid is oxidized and another is reduced to form organic
acids.

Many factors affect the velocity and extent of proteolysis. Moisture content has
a large effect on proteolysis. Proteolysis is positively correlated with moisture and
negatively with rate of acidification in forages (Brady, 1965). However, it has been
shown that com ensiled at high moisture content undergoes extensive fermentation with
greater amounts of acid and soluble nitrogen produced (Thornton et al. , 1977a). High
sugar to protein ratio has been shown to improve protein stability. High moisture corn

is relatively low in sugar, protein and moisture. Proteolysis activity is lower in high

ll

moisture corn than forages, as indicated by lower soluble nitrogen contents (Baron et a1. ,
1986). Proteolytic activity increases the proportion of soluble nitrogen to 60% of the
total nitrogen in grass silage (Oshima and McDonald, 1978), 50% in whole plant corn
silage or ear corn silage (Buchanan-Smith, 1982; Aumaitre and Zelter, 1975), and 40%
in high moisture corn (McKnight et al., 1973). The limited fermentation that high
moisture corn undergoes ean lessen the stability upon exposure to air as compared to
other silages. Yeast, molds and aerobic bacteria begin to respire as oxygen is introduced
into the silage mass. Oxygen can enter the silage mass by infiltration through silo walls,
exposed surface areas or during feedout. As aerobic organisms begin rapid growth, heat .
is generated which increases the temperature of the silage mass. An elevated temperature
is positively correlated with dry matter lost as carbon dioxide (Woolford, 1984b). These
organisms primarily utilize fermentation acids and sugars as substrates to produce earbon
dioxide, ammonia and water. Aerobic activity results in dry matter loss and

concentration of crude fiber (Woolford, 1984).

Harvest And Storage Losses Associated With High Moisture Grains
Storage of grain in high moisture form results in greater DM and nutrient losses.
Management systems which minimize the extent of these losses would be beneficial.
Nutrient losses can occur during several stages of preservation which include harvest,
storage or feedout. The primary focus of this review involves losses encountered during

storage and feedout.

12

During storage or the ensiling process, nutrients may be lost in effluent, through
fermentation by microorganisms and losses due to oxygen infiltration (Pitt, 1986).
Microorganisms and enzymes degrade soluble nutrients such as carbohydrates and
proteins to simpler chemieal compounds. Fermentation losses as earbon dioxide are
believed to contribute 1-2 % of the total dry matter loss (McDonald, 1981). Fifty percent
of the DM and 18% of the energy losses occur as earbon dioxide (Woolford, 1984c).

Effluent losses from silages can range from 0% in grains and wilted forages to
7% in unwilted forages (Pitt, 1986). Total effluent produced is determined by moisture
content and force applied to the forage mass. Rate of effluent production is a function -
of time required for plant cell membrane breakdown and rate of cell contents to be forced
out of the cell (Pitt and Parlange, 1987).

Oxygen can migrate more readily into silage from exposed surfaces and
improperly consolidated silage. Oxygen can move through silo walls and into the silage,
however, greatest air movement into ensiled feeds occurs with uncovered or unsealed
surfaces (Ruxton et al. , 1975). Unsealed bunker or trench silos filled with high moisture
fodder (50% DM) resulted in 70% less usable DM as compared to sealed silos (64 vs
90% DM recovery; Brown and Kerr, 1965). Losses due to oxygen infiltration during
the fermentation process are similar to losses associated with air exposure during feedout.
Maintenance of an anaerobic environment in ensilage is critical to minimize storage
losses. Dormant aerobic organisms become active and utilize a vast array of readily
available substrates upon exposure to air.

During feedout, soluble carbohydrates and fermentation acids provide substrates

13

for aerobic microorganisms to proliferate. Yeast organisms are primary contributors to
aerobic deterioration (Woolford and Wilkie, 1984), however, bacteria also contribute to
the process (Woolford and Cook, 1978). Reviews of aerobic deterioration in research
trials have shown DM losses of 16.1% (Watson and Nash, 1960) as cited by Ashbell and
Lisker (1988), whereas on farm losses due to aerobic deterioration range from 8-71 %
(Zimmer, 1967) as cited by Ashbell and Lisker (1988).

Yeasts have been shown to metabolize both sugars and acids (Middelhoven and
Franzen, 1986). Metabolism of lactic acid to earbon dioxide by yeast elevates the pH
and allows bacterial growth to begin (Lindgren et al., 1985b).

Dry matter content influences retention during fermentation and feedout. Lower
DM content at ensiling will cause more rapid and extensive fermentation (Goodrich et
al. , 1975). Higher acid, ethanol and soluble nitrogen content, greater gas production and
lower pH result from enhanced fermentation (Goodrich and Meiske, 1976; Prigge et al. ,
1976). The amount of DM lost increases as gas production increases. Soluble nitrogen
content is greater in low DM silage. Consequently, moisture content and nutrient losses
are positively correlated. Silages ensiled at less than 30% DM result in excessive
effluent production, however, high moisture grains are not ensiled at these levels.
Therefore, effluent loss is not a problem with high moisture grains. Ensiling feedstuffs
with high DM content may have deleterious effects on recovery, since the limited
fermentation may increase aerobic instability.

Amount of DM loss is influenced by the type of silo structure. Oxygen-limiting,

concrete tower and horizontal silos are commonly used to store silages. Oxygen limiting

l4

silos do not allow oxygen exposure as readily as other silo types; consequently moisture
content is less critical (Fox, 1976) for adequate preservation. Concrete tower silos
consolidate more thoroughly than horizontal silos (Pitt and Parlange, 1987) causing
greater oxygen depletion, less oxygen infiltration and more effluent production in
excessively wet forages. Horizontal silos have relatively large surface areas as compared
to upright silos. Large surface areas allow greater oxygen penetration into the silage
mass and greater DM losses.

Different methods have been used to evaluate the losses and changes which occur
during the ensiling process. Ensiled feeds are characterized by the conversion of soluble.
carbohydrates to organic acids and the solublization of protein to soluble nonprotein
nitrogen (Bergen et al. , 1974). Under favorable conditions, pH will decrease and lactic
acid content will be high relative to other fermentation acids produced (Thornton, 1976).
An unfavorable fermentation is characterized by high butyric acid content and excessive
proteolysis.

Large silos are frequently used to measure nutrient losses during fermentation.
Large silos give an accurate assessment of fermentation losses, however, cost and size
of scale limit replications. Multiple dacron bags filled with plant material and buried
within each silo can be used to estimate DM recovery. Unfortunately, DM recovery
estimates from buried bags have low correlations with large silo DM recovery estimates
(McDonald, 1981). Location of buried bags within the silo will influence the DM
recovery estimates as well.

Small seale or laboratory silos have been used to evaluate the fermentation

15
process. Small silos are relatively inexpensive and easily replicated. Laboratory silos

have been criticized (Mayne and Gordon, 1986) for lack of similarity with types of
fermentations in field seale silos. These silos are frequently more anaerobic than farm
scale silos. However, laboratory silos have been used frequently to evaluate fermentation
(Lindgren et al., 1985a; Baron et al., 1986). Aerobic stability can be evaluated by
exposure of silage to air in insulated containers. Temperature rise, microbial growth and

dry matter loss are commonly reported parameters to quantify the extent of deterioration.

Microbiology Of Silage

Epiphytic microorganisms present on plants before ensiling are extremely variable
(Daeschel et al. , 1987). Facultative anaerobes living in an aerobic environment are
important for adequate fermentation. Numbers of epiphytic microorganisms are
dependent upon several factors. Bacterial populations vary according to loeation on the
plant. Microorganisms are more numerous on damaged or decayed foliage with few
organisms found on intact inflorescence or seeds (McDonald, 1981). Bacteria are found
on damaged areas because nutrients are readily available at these points. Since the lower
portions of the plants are more likely to contain higher numbers of bacteria, the amount
of stubble left in the field ean influence the resulting fermentation. Secondly, plant
maturity and climatic conditions can influence the number of organisms present on
growing plants (Woolford, 1984c). As plant maturity and season progresses, the number
of lactic acid producing bacteria (LAB) increase (Daeschel et al., 1987). Warm, humid

weather and cloudy skies are generally associated with elevated populations of epiphytic

16

bacteria. In addition, ealm days with little air movement reduce evaporation and
consequently, increase bacteria numbers. Clostridial species are not normal inhabitants
on growing plants but are present in soil. These organisms are influenced little by
weather conditions (Woolford, 1984c).

Numbers of LAB species may be small on the growing plant, but larger
populations have been routinely reported at the silo (Muck and Specherd, 1984; Muck
and O’Connor, 1985). This increase may be explained by extended time periods
between cutting and placement in the silo which allows the populations to multiply.
Secondly, the harvest machinery may accumulate plant material which allows epiphytic,
populations to multiply and serve to inoculate plant material. It is likely that the
increased population at the silo is a combination of both (Fenton, 1987).

Clostridial and Bacillus species are present in low numbers on fresh crops. These
organisms are usually of soil origin and high plant levels pre—ensiling are usually due to
contamination during harvest (Woolford, 1984c). Tractor and implement tires may be
a source of these organisms during the consolidation of bunk or trench silos.

Lactic acid producing microorganisms are gram positive, microaerophilic, non-
spore forming and usually non-motile organisms (Beck, 1978). The genera of
microorganisms comprised in LAB are Lactobacillus, Streptococcus, Pediococcus and
Leuconostoc. Lactobacillus belong to the family Lactobacillaceae. These organisms are
rod shaped and possess species of both heterofermentative and homofermentative
characteristics. The other three genera belong to the family Streptococcaceae and are

spherieal in shape. Leuconostoc species are heterofermentative while Streptococcus and

17
Pediococcus species are homofermentative (McDonald, 1981). Domination of the

fermentation by lactic acid bacteria is important to ensure sufficient amounts of lactic
acid production and a rapid pH decline. .

Sugar is utilized by LAB as the main substrate, but organic acids can also be
used. Lactic acid producing microorganisms have been shown to ferment lactate (Beck,
1978) under adverse conditions. Proteolytic activity on plant proteins is small in LAB.

Lactobacillus can survive in pH from 4.0—6.8 with 6.0 being optimum. Although,
certain strains may have lower pH optimums. Optimum temperature for Lactobacillus
is 30C. These microorganisms grow slowly above 45C. Pediococci organisms are ,
thought to grow best at pH 5.5 (Woolford, 1984c). Clostridia are more heat tolerant
than LAB with optimum temperature at 37C. Excessive respiration activity increases
silage mass temperature and favors Clostridial fermentation. Clostridia cannot tolerate
high acid levels and have an optimum pH range of 7.0-7 .4. This emphasizes the
importance of lactic acid production and pH reduction.

Clostridia are members of the family Bacillaceae. Most clostridia are obligate
anaerobes with some aerotolerant species. Additionally, most clostridia are gram positive
bacteria but may switch cell wall structure to become gram negative in older cultures.
There are two types of clostridia involved in fermentation; l) saccharolytic bacteria
which utilize sugars and lactic acid with weak proteolytic activity and 2) proteolytic
bacteria which degrade protein with weak saccharolytic activity (Leibensperger and Pitt,
1987). W have both saccharolytic and proteolytic capabilities. Silage
dominated by clostridia is characterized by elevated levels of butyric acid, ammonia and

18
pH (Leibensperger and Pitt, 1987). Clostridia ferment lactic acid to produce butyric acid.

Butyric acid is weakly acidic which hampers pH decline.

Another bacterial species which is commonly present in silages are
Enterobacteriaceae. The organisms are rod shaped facultative anaerobic bacteria. Many
enterobacteria are pathogenic, however, those found in silage are thought to be non—
pathogenic (McDonald, 1981). The role of enterobacteria in silage fermentation is minor
provided LAB growth is sufficiently rapid and pH is lower than 5 .5 . Since the pH
optimum for enterobacteria is 7.0, activity is greatest early in the fermentation process.
Enterobacteria, commonly referred to as coliforrn bacteria, are inefficient ferrnenters as -
evidenced by the end-products of metabolism. Sugars are fermented to lactate, acetate,
succinate, ethanol, 2,3-butanediol and formate. Formic acid is reduced to hydrogen ions
and carbon dioxide under acid conditions. Coliforrns have proteolytic activity and are
responsible for a portion of protein degradation in silage.

Other microorganisms present include: yeasts, molds, and other aerobic bacteria.
Under normal silage fermentation conditions, these organisms contribute only a minor
role during anaerobic fermentation. However, these microorganisms can be extremely
prolific upon exposure to oxygen. In general, these microorganisms have a detrimental
effect on the nutritional value of silages.

Yeasts are oval or elliptical in shape and reproduce by budding or binary fission,
often forming chains of elongated cells. Certain species of yeasts grow in anaerobic
conditions and are acid tolerant. Under anaerobic conditions, yeast ferment sugars to

ethanol and compete with LAB for soluble sugars.

l9

Yeasts are more active in ensilage after oxygen exposure. Two physiological
types of yeasts are present which attribute to silage instability. Bottom or ground
growing yeast ferment sugars to carbon dioxide and water. Another type, top growing
yeast, consume lactic acid resulting in decreased energy content and preservation of the
feedstuffs (Woolford, 1984c).

Filamentous fungi or molds contribute little to the fermentation process under
anaerobic conditions but are associated with decomposition of fermented feedstuffs and
mycotoxin production (Lee et al. , 1986). Molds and fungi are more prevalent in silage
which has undergone an inadequate fermentation. Molds ean utilize cellulose, cell wall,
material, sugars or lactic acid as substrates. The most prominent by-product of mold and
fungi fermentation is carbon dioxide.

Aerobic bacteria such as Bacillus species are involved with aerobic instability as
well. Some are facultative anaerobes. Little is known about Bacillus, but are thought
to be significant contributors to aerobic deterioration in well preserved silages (Lindgren
et al., 1985b).

Additives and Inoculants

There are several management techniques to improve silage fermentation and
minimize nutrient losses. Previous studies of the chemistry and microbiology of silage
have suggested that methods to enhance the rate and extent of lactic acid production
would be beneficial. Several strategies have been utilized to enhance lactic acid
production such as microbial cultures, enzymes, nutrient amendments, fermentation

inhibitors and reconstitution. Current technology allows the production of

20

microorganisms to add as starter cultures to fermenting biomass to facilitate lactic acid
production. The array of microorganisms present in the unfermented crop material has
a major impact on the efficiency of the fermentation (Muck and O’Connor, 1985).
Beneficial microorganisms, whether epiphytic or added as an inoculant, must be abundant
and dominate over deleterious organisms for a favorable fermentation to occur.

Addition of microbial cultures can be beneficial when epiphytic LAB populations
are low (Muck and O’Connor, 1985). Addition of microbial cultures containing LAB
have been shown to favorably influence fermentation patterns in ensiled crops (Ely et al. ,
1981; Lindgren et al., 1983; Lindgren et al., 1985a; Kung et al., 1987).

The criteria established for a successful microbial inoculant (Whittenburg, 1961 ,
as cited by Woolford, 1984) are: 1) high growth rate, competitive and must be able to
dominate the fermentation; 2) homofermentative; 3) acid tolerant with activity to pH
4.0; 4) ferment glucose, fructose, sucrose and also preferably fructosans and pentosans
as well; 5) inability to convert dextrose to sucrose or fructose to mannitol; 6) limited
ability to ferment organic acid; 7) survive at temperatures exceeding 40C; and 8) no
proteolytic activities.

Two Lactobacillus species, Lam and Winn, meet the majority
of these criteria (Seale et al., 1986). W, SJaecium and W
acidflagtia possess many of the beneficial characteristics as well (Lindgren et al. , 1985a).
Wm has been identified as possessing most of the qualities identified
by Whittenburg for an ideal inoculant, however, this organism tends to grow slowly

during the early stages of fermentation (Bryan-Jones, 1969 as cited by McDonald, 1981).

21

Therefore, the addition of inoculants containing multiple species or strains may be
advantageous. Streptococcus may be a candidate as a second organism to an inoculant
containing Lactobacillus plantarum. Streptococcus grows rapidly under aerobic
conditions and endures a wide temperature range. Secondly this organism is less acid
tolerant and tends to disappear at pH 5.0 which is a condition optimal for Mm
(Bryan—J ones, 1969 as cited by McDonald, 1981). Another possible companion organism
is mm. It is acid sensitive and has an optimum temperature of 40C
(Buchanan and Gibbons, 1974).

Addition of enzymes may increase substrate for fermentation, but the enzymes are,
active shortly after ensiling but are inactivated by anaerobic conditions and low pH
(Ruxton et al. , 1975). Mineral acids added to ensiled feeds can prevent enzymatic
activity, however consumption by livestock may be reduced (Thomas, 1978).

Since “many Lactobacillus organisms lack amylase enzymes and crop materials
such as corn and alfalfa contain starches, the addition of amylase has been evaluated
(McDonald, 1981). Amylase addition breaks down polysaccharides which are otherwise
unavailable to lactic acid bacteria. Likewise, cellulose is a major carbohydrate fraction
in forages and under certain climatic conditions (McDonald, 1981), soluble carbohydrate
may be limiting. Therefore cellulase addition has been evaluated (Huhtanen et al. ,
1985). Cellulose is degraded by cellulase addition which provides more substrate for
growth of lactic acid bacteria (McHan, 1986). Hemicellulose is another carbohydrate
reservoir which can be hydrolyzed by addition of enzymes to fermenting plant material

(Dewar et al. , 1963). These enzymes have shown some benefit, however, the magnitude

22
has rarely been large enough to justify the cost.

Nutritional amendments added to ensilage may contribute nutrients or provide
substrate to allow a more efficient fermentation. Nutritional amendments evaluated
previously include grains (Jones, 1988), molasses (Singh et al. , 1985), limestone
(McDonald, 1981) and whey (Thomas, 1978). Increased supply of available sugars to
substrate—deficient forages has shown an improvement in silage characteristics especially
in high protein feeds (Svensson and Treit, 1964; Seale et al., 1986; Jones, 1988).

Grain has been added to alfalfa and grass silages as a nutritional amendment.
These amendments were effective in enhancing the energy value of the resulting silage,
but did not affect efficiency of fermentation (Jones, 1988). McDonald (1981) suggested
malt extract would be a more suitable amendment because the carbohydrate fractions
present would have less complex structures. Another source of soluble earbohydrate
added to silage has been molasses or molasses by-products. Under certain conditions,
favorable responses have been recorded (Woolford, 1984c). Clostridial-type fermenta-
tions have been reported in molasses treated silage which may result fi'om excess
substrate and reduced competition between LAB and clostridial organisms.

Whey, a major by-product of cheese production, has been used to increase soluble
carbohydrate content in ensiled feedstuffs. Lactose, the primary sugar component in
whey, is unavailable to LAB organisms (Thomas, 1978). Whey contains greater than
90% water which can be useful with forages lacking adequate moisture for fermentation.

Nitrogenous compounds have been added to silages to enhance the crude protein

content and as a preservative (Alaspaa, 1986). Most nitrogenous amendments are

23

comprised of non-protein nitrogen such as urea, ammonium hydroxide and anhydrous
ammonia. Ammonia compounds increase nitrogen content, buffer ensilage and exhibit
fungicide properties (Russell et al. , 1988). Ammonia buffers the pH decline and thereby
extends the fermentation period which increases lactic acid accumulation. On a wet
matter or as is basis, current recommendations suggest application of 0.208%
anhydrous ammonium hydrate or 0.5 %-1.5% urea to low protein feedstuffs (Owens et
a1. , 1981).

Limestone (Ely, 1978) has been used as a nutritional amendment to corn silage
and high moisture corn. Limestone and other ealciurn salts tend to buffer the pH decline,
and extend the fermentation. Corn silage and high moisture corn contain minimal
amounts of calcium. Addition of calcium salts fortify the calcium density in the
feedstuffs. Since corn silage has low concentrations of crude protein and calcium,
addition of NPN and calcium salts provides a more complete feedstuff in addition to the
fermentation benefits (Ely, 1978). _

Several researchers (Siddons et al., 1984;1Woolford, 1984c; Murphy, 1986;
Voelker et al. , 1989) have attempted to improve the fermentation process by addition of
fermentation inhibitors such as: acids, esters, salts, sterilants and antibiotics. Mineral
and organic acids have been used to inhibit fermentation by maintenance of high
hydrogen ion concentration. Sulfuric acid has strong acidifying potential and is effective
as a preservative, however, feed consumption may be hindered (Murphy, 1986). In
addition, corrosive properties make it an unattractive alternative.

Organic acids inhibit fermentation by reduction in the hydrogen ion concentration

24
and antimicrobial activity (Woolford, 1984). As earbon chain length increases, acidic

properties decrease and antimicrobial characteristics increase. Formic acid is widely
used in the United Kingdom as a silage preservative. It is a strong acid (pk, = 3.75 ;
Waldo, 1978), has bactericidal properties, inhibits coliform bacterial growth and
enhances lactic acid fermentation (McDonald, 1981). However, yeast organisms are
tolerant of formic acid (Thomas, 1978). Combinations of formic acid and formaldehyde
have been used to preserve direct cut grass silages (Haigh et al., 1987).

Acetic acid is commonly used for preservation of foods for human consumption.
Acetic acid is classified as a weak acid; has a pk. of 4.64 (McDonald, 1981) and inhibits.
enterobacteria and yeast. Acetic acid has been shown to increase residual soluble
carbohydrates and decrease soluble nitrogen content and dry matter loss (Mann and
McDonald, 1976). Acetic acid has been shown to improve aerobic stability (Moon,
1983). Improved stability may be attributed to antimicrobial properties against yeast.

Propionic acid and esters of propionic acid have antifungal characteristics and
have been benefical for low dry matter silages (Voelker et al., 1989). Silage pH
influences the antimicrobial effects of propionic acid. Aerobic stability is improved in
silages with high propionate levels and Driedger (1976) suggests this effect is due to the
antimicrobial aspects of propionic acid. Attempts to utilize propionic acid producing
bacteria as a fermentation enhancer have been unsuccessful (McDonald, 1981).

Long chain volatile fatty acids (valeric, caproic and heptanoic acid) have
inhibitory action against molds, yeast and enterobacteria (Thomas, 1978). Some other

acids which have received investigation are acrylic, sulfamic, benzoic, sorbic, citric and

25
glycolic acid (McDonald, 1981). The effects of these acids on microorganisms have

been variable (Thomas, 1978).

Another form of additive used to restrict or inhibit fermentation is sterilants.
Extent of fermentation restriction is highly dependent upon application rate of the
sterilant. Formaldehyde is the most commonly used sterilant and is usually applied as
formalin (40% formaldehyde in aqueous solution). Another form, paraformaldehyde, is
a crystalline material which contains 82-92% formaldehyde. Formaldehyde is a
bacteriostat that suppresses fermentation and results in less acid and ammonia production
(Henderson et al. , 1982). Formaldehyde treated silage can become less stable than,
untreated silage as formaldehyde is lost through volatilization (Kung et al. , 1986).

Many antibiotics have been used as fermentation inhibitors. Bacitracin has been
used to inhibit gram positive microorganisms (McDonald, 1981). Antibiotics such as
clindamycin, tylosin and pimaricin (Woolford et al. , 1975; Woolford and Wilkins, 1975
as cited by Thomas, 197 8) have been shown to reduce fermentation through antibacterial,
antiprotozoa and antifungal properties, however, it has not proven to be cost effective.

Fermentation of dry feedstuffs can be enhanced by addition of water (Hinders,
1976). Crop materials with less than 40% moisture undergo limited fermentation because
of low epiphytic bacteria, limited substrate and inadequate moisture for anaerobic
bacterial growth (Kung et al., 1987). Crop material ensiled too dry may undergo
excessive heating, carmelization and nutrient loss. Reconstitution of dry corn has been
shown to enhance fermentation (Goodrich and Meiske, 1976). Grains harvested at 24%

moisture reconstituted to 30% have similar fermentation characteristics as corn harvested

26
and ensiled at 30% moisture (Aguirre et al., 1983).

Feeding Value of High Moisture Corn

Preservation and storage of HMC has several advantages. Harvesting and storing
of corn in the high moisture form allows an earlier and extended harvest season.
Secondly, less field loss occurs (Fox, 1976; Goodrich and Meiske, 1976) with harvest
of high moisture versus dry corn. Another benefit of high moisture as compared to dry
corn involves the increased digestibility (Buchanan-Smith, 1976) and feed conversion
efficiency (Perry, 1976). The fourth advantage of HMC involves the elimination of ‘
drying costs. Currently, the cost to dry one bushel is $.02 with a 1.4% shrink per
percentage unit of moisture removed. In addition, transportation cost is minimized as
thecornisusedonthefarminstmdoftransportedtoaterrninalmarket. Several
disadvantages are associated with high moisture harvest and storage. Grain stored as
high moisture cannot be sold through commercial channels. Secondly, harvest and filling
rate must be rapid to minimize respiration, thus storage facilities should be relatively
close to harvest site. Rate of removal once feeding begins must also be rapid to
minimize aerobic deterioration and high moisture feeds have a shorter bunk life as
compared to dry feeds.

Previous research has suggested corn must be ground before feeding (Baker,
1973). However, weight gains are similar for whole and ground corn in high concentrate
diets with less than 15% roughage (Dexheimer, 1973). Prigge (1976) showed that

soluble nitrogen concentrations are higher in ground versus whole ensiled corn. Soluble

27

nitrogen concentration is commonly associated with depressed animal performance
(Sprague and Bremiman, 1969) Studies from 1971-1973 reported by Guyer and Farlin
(1976) indicate efficiency and rate of gain are improved by 5 and 3 % respectively for
cattle fed ensiled whole corn versus ground HMC. Gill et al. (1982) showed a 15%
improvement in feed conversion efficiency with ground ensiled corn as compared to a
mixture of whole and ground blended corn prior to ensilement.

Dry matter content has a direct impact on preservability and feeding quality.
Acceptable preservation has been observed with dry matter content as high as 79% dry
matter in oxygen-limiting silos (Goodrich et al. , 1975), however, earmelization ean occur,
in excess of 76% dry matter (Fox, 1976). Corn stored at low dry matter content will
have elevated acid and soluble nitrogen content (Fox, 1976) which ean decrease dry
matter consumption (Goodrich and Meiske, 1976). Dry matter intake has been shown
to be reduced with ensiled corn as compared to dry corn (Wilkinson et al., 1976).
Palmquist and Conrad (1970) demonstrated that intake, milk fat percentage and acetate
to propionate ratio within the rumen were decreased with high moisture corn diets for
dairy cows. In growing-finishing cattle fed high moisture corn, conversion of
metabolizable energy into tissue gain was more efficient than for cattle fed dry corn
(Goodrich and Meiske, 1976). Henderson and Bergen (1970) and Klosterrnan et al.
(1960) suggested the improved efficiency was due to the high availability of acids in high
moisture corn. Burroughs et al. ( 1970) reported decreased dry matter intake and
improved feed conversion efficiency as high moisture corn replaced dry corn in the diet.

Tonroy et al. (1974) reported a 3-13% reduction in dry matter intake with high moisture

28

corn diets, however, weight gains were similar.

Buchanan-Smith (1976) reviewed 17 studies which compared high moisture and
dry corn in diets of growing-finishing cattle. Cattle fed HMC gained faster or equal to
cattle fed dry corn in 11 of the trials. In addition, in 16 of the 17 trials, feed conversion
efficiency was improved with HMC as compared to dry corn. Digestibility of HMC
tended to be greater than dry corn as well. Aguirre et al. (1984) demonstrated that total
tract starch digestion increased as moisture content increased, whereas ruminal fiber
digestion decreased. A quadratic effect was seen in organic matter (OM) and dry matter
(DM) digestion. Digestion of OM and DM was highest at 65 % DM and lowest at
intermediate levels (75 %). Thornton et al. (197 8) showed that digestibility was enhanced
in corn stored at 30% moisture as compared to 23 % moisture corn and suggested the
difference was due to greater starch availability. Aguirre et al. (1984) showed total
nitrogen digestibility was greater at the highest moisture content (35 %).

The type of protein fed with HMC is a concern. Martin et al. (1980) summarized
10 trials conducted in Oklahoma that compared soybean meal (SBM) and urea as
supplemental protein sources for HMC diets and reported an advantage in intake, rate
of gain and feed conversion efficiency with SBM. However, these trials indieated an
advantage for urea over soybean meal with dry corn, though these results were more
variable. The benefit of urea may be less with dry whole corn than with dry processed
corn.

Elevated levels of soluble nitrogen can adversely affect animal performance

primarily through depressed intakes (Bergen, 1971). Several factors can affect soluble

29
nitrogen content of the diet as listed by Sprague (1976): grain type, level and type of

roughage in the diet and type of supplemental protein. Each of these factors may
influence the rate of proteolysis in the rumen.

High dry matter content of ensiled corn has been associated with inadequate
oxygen expulsion which allows mold growth to occur. Elevated levels of mold in the
diet may depress animal performance (Goodrich et al. , 1974).

Microbial inoculants have been added to HMC prior to ensilement (Bolsen et al. ,
1984) to enhance the fermentation. Dry matter recovery was improved by more than 5 %
with the addition of lactic acid producing microorganisms. Cattle fed the inoculated corn,
gained weight more efficiently than cattle fed untreated corn. However, cattle fed the
control corn had greater dry matter intake and weight gain.

Deterioration that occurs after oxygen exposure results in DM loss; increased pH;
reduced lactic acid; increased yeast, mold and aerobic bacteria populations (Stevenson,
1976). Yeast may eause the initial microbial deterioration in forage silages and aerobic
bacteria in corn silage (Woolford, 1984b). Well preserved silages, as defined by low pH
and minimum residual soluble carbohydrate, (Woolford, 1984c) tend to be stable upon
air exposure. Certain researchers have suggested rapid fermentation with high lactic acid
levels are necessary for a stable silage. However, Ohyama et al. (1975) states the only
two chemieal constituents which constantly create stable silages are elevated ammonia
and butyric acid content. In addition, Ohyama et al. ( 1980) suggests acetic acid has a
higher antimycotic activity than lactic acid. Inoculation with rapidly growing lactic acid

producing bacteria has been shown to decrease stability of silages even though pH was

30
below 4.5 and lactic acid levels were high (Rust et al. , 1989). All silages that are stable

upon air exposure are not desirable. For instance, silages which have undergone a
clostridial type fermentation are stable beeause of the high ammonia and butyric acid
levels. Aerobic deterioration may also be critieal in the feedbunk. Feedbunks must be
managed to prevent accumulation of stale and moldy feed. The longer the stability
period of HMC, the easier bunk management becomes. Grains which are fed within 24
hours of initial oxygen exposure should have minimal loss and be of high nutritional
quality (Stevenson, 1976). Ensiled grains exposed to oxygen for relatively long periods
of time before feeding will have increased microorganisms present, elevated temperatures,

and decreased nutritive quality (Henderson et al. , 1979).

THE EFFECTS OF MICROBIAL INOCULATION
FOR THE PRESERVATION OF HIGH MOISTURE CORN
ON CATTLE PERFORMANCE, FERMENTATION

CHARACTERISTICS AND AEROBIC STABILITY

31

32
ABSTRACT

Two trials were conducted to evaluate the effects of microbial inoculation for
preservation of high moisture corn (HMC). In trial 1, three concrete upright silos were
filled with 83 t of HMC that was treated with either a dry microbial inoculant (D1),
preferrnented liquid microbial inoculant (PFLI) or an unheated control (CON).
Prefermented liquid microbial inoculant or control untreated HMC were placed in two
silos in trial 2. Inoculants were applied to supply 2X106 cfu/ g of wet corn. Cattle
feeding trials were performed to evaluate nutritive value of the HMC in trials 1 and 2.
Probe samples were collected from the bottom two doors of each silo and during feedout
to evaluate fermentation changes over time. Buried dacron bags at 4 elevations within
each silo were recovered during feedout to determine dry matter losses and chemical
composition changes during the fermentation period. In both trials, concentrations of
soluble nitrogen (SN), lactate, acetate and lactic acid producing bacteria (LAB) increased
over time (P < .05). Soluble carbohydrates and pH decreased through d 21 but
increased (P < .05) by time of feedout in trial 1. Acetate content was lower with PFLI
as compared to CON or DI (trial 1) indicating a more homolactic fermentation. In trial
2, SN and LAB concentrations were greater while pH was lower with PFLI treated corn
(P < .05). Dry matter recovery tended to be greater with CON (P < .05) than PFLI
treated HMC in trial 2. Aerobic stability was evaluated on corn from silos in trial 11 by
placement in styrofoam containers. Temperature and microbial numbers increased (P <

.05) over the 5 d incubation period for both HMC treatments. Microbial enumeration

33
was greater for PFLI on d 3 as compared to CON. Average daily gains were similar for

all treatments in both trials, however, the DI treated HMC was consumed to a greater

extent(P < .05)thanPFLIcornintrial 1.

Key words: High Moisture Corn, Lactic Acid Bacteria, Inoculant

34
INTRODUCTION

Moisture conditions during harvest season encourages livestock producers to store
corn in a high moisture form. However, losses during storage and feedout can be
substantial (McDonald, 1981). Fox (1976) and Goodrich and Meiske (1976) have
previously reported 5-20% of ensiled dry matter may not be recovered from the silo.
In addition, the low moisture content of high moisture corn (HMC) limits the extent and
type of fermentation. Consequently, most ensiled HMC begins deterioration in less than
24 h post-exposure to oxygen (Stevenson, 1976).

Microbial inoculants have been shown to successfully shift fermentation patterns
toward more efficient metabolic pathways resulting in less dry matter loss during the
fermentation process with whole corn plant and sorghum silages (Bolsen et al. , 1984) and
grass silages (McDonald, 1981). Microbial inoculation to stimulate a homolactic
fermentation has been shown to decrease aerobic stability in whole corn plant silage (Rust
et al., 1989). Since the use of microbial inoculants in high moisture corn has not been
extensively investigated, this study was conducted to evaluate the potential for microbial
inoculants to enhance preservation of the nutritional value of the fresh crop and reduce
deterioration upon air exposure. A secondary objective was to evaluate the effects of

inoculation of HMC on dry matter recovery from upright silos.

MATERIAL AND METHODS
Trial 1

High moisture corn (HMC) was harvested at approximately 72 % dry matter and

35
ensiled in one of three upright concrete silos (3.6 m x 13.5 m). An alternate load

sequence was used to fill the three silos by placement of two loads in a silo
(approximately 12.6 t) before rotation to the next silo. A separate silage blower was
used for each silo. High moisture corn was placed through a New Holland 355 portable
grinder-mixer fitted with a 1.6 cm screen. Silo filling was completed in 6 d and
approximately 83 t (72% DM) were placed in each silo. The three treatments alloeated
to silos were: control (no inocula, CON); dry granular inocula (DI); and prefermented
liquid inocula (PFLI). The inoculantsl (DI and PFLI) were comprised of 1W1“:
mm.8mwcusfaecinmmd2eiiomnsacidilactici. ThePFLIwas
reconstituted by mixing 280 g of commercial soluble inoculant (565 x 109 cfu/package)
in 9.5 L of distilled water and allowing the mixture to set at room temperature for 24 h
prior to use. The application rate of D1 and PFLI was 1 kg and 2.1 L per t of
harvested corn (as is basis), respectively. The respective inoculation rates were used
to supply 2 x 10‘ cfu/ g of corn. Inoculants were added at the silage blower. A Gandy
applieator2 was used to apply DI while PFLI was applied with a sprayer apparatus
equipped with two nozzles using 1.76-2.11 kg/cm’ of pressure. A thermocouple and two
dacron bags were placed at each of four elevations (2.4, 4.8, 7.2 and 9.6 m) in each silo.
Temperatures were recorded daily for the first 32 d post-ensiling. Dacron bags were

filled with a known weight of HMC and buried. Strips of colored plastic were placed

 

‘ Medipharrn, USA Des Moines, IA

2 Gandy Company Manufacturers, Owatonna, MN

36
45-60 cm above the buried bags to identify bag loeation during feedout. On appearance

of plastic strips, bags were uncovered, weighed and samples frozen for chemieal
analysis. Differences in DM weights of buried bags between placement and removal
from silos were utilized to estimate dry matter losses during ensilement. In addition, dry
matter recovery from each of the upright silos was determined from the difference in
weight of dry matter placed and dry matter fed. .

Each load of HMC placed into each silo was subsampled and stored at 4 °C until
the end of each day. Samples were composited to provide a single sample for each silo
per day and frozen for laboratory analysis. Two 5 cm holes were bored in the bottom
two doors of each silo and fitted with rubber stoppers. A 500 g sample was obtained
from a single door from each silo on 0, 1, 2, 3, 4, 5, 6, 11, and 21 d post-ensilement.
A Penn State forage samplerJ was utilized to collect samples through bore holes. One
hundred fifty g of each bore sample were taken to the laboratory for microbial analysis
and the remainder frozen at -19 °C. During the feed out period for each silo, samples
were collected every two weeks and analyzed for DM content to ealculate DM intake.

Thirty pens of cattle consisting of 12 pens of steers (382 kg) and 18 pens of
heifers (342 kg) were randomly assigned to the three treatments to evaluate dry matter
intake, weight gain and feed conversion efficiency of the three corn treatments. One
hundred eighteen steers were placed in the twelve pens and 104 heifers in 18 pens.

Cattle were fed the respective treatments for 56 d.

 

3 Nasco Agricultural Sciences, Fort Atkinson, WI.

37

Two initial and final weights were obtained on consecutive days. Cattle were
previously adapted to HMC diets and allowed a 5 (1 period to adapt to the addition of
treated HMC in the diets before the trial was initiated. Diets consisted of 85 % HMC
10% corn silage and 5 % supplement (Table 1).

Trial 2

Two upright concrete silos were filled with approximately 90 t of HMC (75 %
DM). Corn was ground through a 1.6 cm screen on a New Holland 390 tub grinder
prior to ensilement. Each silo was assigned one of two treatments, either CON or PFLI.
Silos were filled in a similar load sequence and PFLI applied as previously described.
A thermocouple wire and two dacron bags filled with HMC were placed at four
elevations within each silo as in Trial 1. Temperatures were recorded from
thermocouples during the first 28 d post—ensilement. Samples were collected from the
bore holes in the bottom doors on O, 1, 2, 4, 8, 16 and 40 d and stored in a similar
manner as described in Trial 1. Samples were collected every 2 wk from each silo
during the feedout period for dry matter analysis to determine dry matter intake.

Twenty styrofoam containers, ten per treatment, were filled with 4.8 kg of
ensiled HMC and maintained at 20 °C after exposure to air. Cooking thermometers were
placed into the HMC in each container to record daily temperature changes. Duplicate
containers for each treatment were evacuated on 1, 3, 5, 8, or 14 d and weighed for
calculation of DM recovery. Subsamples were collected as containers were evacuated
and microbial and chemical analyses performed.

One hundred seventeen crossbred steers (328 kg) were allotted to 18 pens and fed

38

Table 1. Composition of Diet Fed To Cattle
(Expressed as a percent of dry matter)

IriaLl IliaLZ
High Moisture Corn 85 I 85
Corn Silage 10 -
Alfalfa-Grass Hay - 10
Supplement‘ 5 5

' Supplements were balanced to provide 11% crude
protein, .5 % calcium, .35% phosphorous, .7% potassium,
.25 % sodium chloride and 250 ppm monensin.

39

one of the two HMC treatments for 126 (1. Two weights obtained on consecutive d were
recorded for initial and final weight determination. Steers were adjusted from an all corn
silage diet to the treated corn diets over a 14 d period. The diet consisted of 85 % HMC,
10% chopped hay and 5 % supplement (Table 1).
Laboratory Analysis

Frozen samples were allowed to thaw at 20 °C and chopped in a Hobart
Macerator‘. A 100 g sample of chopped wet material was placed in a 60 °C forced air
oven for DM determination (AOAC, 1984). Total nitrogen concentration was determined
on wet tissue with a micro—kjeldahl procedure (AOAC, 1984) using a Technicon
Autoanalyzer‘. A 10% homogenate of the chopped wet material and distilled water was
diluted 50-fold and analyzed for soluble earbohydrate content (Dubois et al. , 1956).
Soluble nitrogen was measured by placing 20 g of chopped wet material into 80 ml of
Ohio buffer (Johnson, 1969) and 5 ml of the homogenate was analyzed for nitrogen using
micro-kjeldahl analysis with a Technicon Autoanalyzer (AOAC, 1984). Fifty ml of the
homogenate were strained through cheesecloth and pH determined with a combination
electrode (Baertsche et al. , 1986). Volatile fatty acid analysis in trial 1 was performed
by gas chromatography. Twenty ml of strained homogenate was acidified with 4 ml of
25 % metaphosphoric acid solution (w/v). After mixing, the extract was centrifuged at

27000 x g for 15 min. Two ul of supernatant were injected into a Hewlett Packard

 

‘ The Hobart Manufacturing Company, Troy, OH

5 Technicon Instruments Corporation, Tarrytown, NY

40
Gas Chromatograph (5 840A)‘ equipped with a flame ionization detector and

microprocessor. The column (2 ml x 2 mm id) was packed with 10% SP - 1200 and 1%
mm, on chromosorb WAW (80/ 100). Lactic acid concentration (Trial 1) of the
strained homogenate was determined by the procedures of Barker and Summerson
( 1941). In trial 2, lactic acid, volatile fatty acids and ethanol concentrations were
analyzed with high pressure liquid chromatography (HPLC) using a modified procedure
from Siegfried et al., (1984). One ml of 20.92% (w/w) ealcium hydroxide and .5 ml
of 10% (w/w) cupric sulfate were added to 1 ml of the acidified homogenate. Samples
were vortexed after addition of each reagent. Cupric sulfate reagent contained .4 %
(w/w) crotonic acid which served as an internal standard. After reagent addition,
samples were allowed to stand at 5 °C for 30 min followed by centrifugation at
10,000 x g. The supernatant was acidified with 25 ul of concentrated sulfuric acid and
frozen and thawed twice to remove any remaining protein. Samples were centrifuged at
10,000 x g and filtered through .22 um nylon filters" Standards were prepared in an
identical procedure as samples. Thirty-five ul were injected into the I-IPLC system with
a 30 min run time. A Biorad ion exchange column HPX-87H (Cat no. 125-0140)’ was
heated to 45 °C for succinate, lactate, forrnate, acetate, propionate and butanediol

separation and 30 °C for ethanol and butyrate separation. A Biorad guard column (cat.

 

° Hewlett Packard, Palo Alto, CA

7 Millipore Corporation, Bedford, MA.

41
no. 125-0129)“ was utilized to protect the ion exchange column. Mobile phase was

prepared by diluting 1.66 ml of concentrated sulfuric acid and .41 g of EDTA to a
volume of 4 L with double distilled millipore filtered water. The mobile phase was
boiled to dissolve EDTA, allowed to cool and filtered through .22 um nylon filters.
Mobile phase flow rate was .7 ml/ min. The HPLC system’ was comprised of a Waters
6000A pump, 712 WISP, 730 data module, 720 system controller and 410 refractive
index detector. The detector was set at 8 x 10‘ refractive index units full scale.
Ammonia-N was determined on the Technicon Autoanalyzer by injection of strained

homogenate.

Microbial Analysis

A 100 g sample of HMC was added to 900 ml of sterile distilled water. The
mixture was vigorously stirred and filtered through two layers of cheesecloth. The
filterate was serially diluted in lO-fold increments to 10”, using sterile .1% peptone
solution. Enumeration of lactic acid producing microorganisms (LAB) was by the spread
plate method (Hungate and Fletcher, 1962). A propipettor was utilized to apply 0.2 ml
on LBS (BBL Microbiological Systems, MD) agar plates of each dilution and incubated
at 39 °C for 72 h. Colonies were grown, and counted and presumptively identified as

lactic acid producing microorganisms. Samples from the aerobic stability study were

 

3 Bio Rad Chemical Division, Richmond, CA.

9 Waters Associates Inc. , Milford, MA

42

prepared and counted in a similar manner, except the agar plates contained starch.
Statistical Analys's

Cattle performance, fermentation characteristics and aerobic stability were
analyzed as a completely randomized design using the analysis of variance procedure
(SAS Institute, 1987). Nonorthogonal designed contrasts were made to separate means

of cattle performance using Bonferronni-t statistics (Gill, 1978).

RESULTS AND DISCUSSION

Cattle Performance

In trial 1, dry matter intakes were greater (P < .05) for eattle fed DI as compared
to PFLI (Table 2). Weight gain and fed conversion efficiency were similar. Likewise,
weight gain, dry matter intake and feed efficiency were similar for cattle fed the two
treatments in trial 2. Results from these two trials suggest the nutritive value of HMC
is not significantly influenced by inoculation prior to ensilement. The results of these
trials support the conclusion drawn from studies which evaluate the effects of inoculation
on the nutritive value of corn silage (Luther, 1986; Rust et al., 1989) or alfalfa (Mader
et al. , 1985) that microbial inoculation does not enhance nutrient value. Since the
nutritive content and acceptability is not altered, the beneficial effects of an inoculant for
HMC preservation would have to be manifest in improved bunk stability or enhanced

nutrient preservation.

43

Table 2 Effects of Inoculation of High Moisture Corn On
Efficiency of Utilization for Growing-Finishing

 

 

 

 

 

 

Cattle
Treatments
Trial 1
0 DP PFLI‘ SEM'
No. of Pens 10 10 10
ADG,kg 1.24 1.26 1.21 .045
DMI,kg 9.75° 10.05c 9.03" .185
Gain/feed .127 .126 .135 .004
Trial 2
No. of Pens 9 9
ADG,kg 1.41 1.41 .027
DMI,kg 8.72 8.68 .155
Gain/feed .163 .162 .003

‘ SEM=standard error of the mean; C=control; DI=dry inoculant;

PFL1=prefermented liquid inoculant

“ Means in a row with unlike superscripts differ (P < .05)

Fermentation Characteristics

Barnett (1954) subdivided silage fermentation into four phases: 1) cellular
respiration; (2) aerobic bacteria producing acetic acid; (3) LAB producing lactic and
acetic acids; and (4) a quiescent period providing sufficient fermentation has occurred.
The concept of phases was utilized by Rust et a1. (1989) to study changes over time in
bunker silos. In the study, phase 4 was separated into two components, (1 5-21
representing the beginning and fwd out representing the end (d 151-242). The concept
of phases established by Barnett as modified by Rust et al. was incorporated into the
analysis of chemical changes over time in the upright silos. The probe samples collected
through the bored holes were composited to provide three time periods; phase 1-3 (1-4
(1), beginning of phase 4 (5-21 d) and the end of phase 4 during feedout (from buried
bags). Greater soluble nitrogen content was exhibited at the beginning of phase 4 over
phases 1-3 and continued to increase until the end of phase 4 in trial 1 (Table 3). As in
trial 1, soluble nitrogen content did not change in trial 2 until the beginning of phase 4
and continued to increase until the end of phase 4 (P < .05). Ammonia nitrogen
concentration increased (P < .0001) throughout the period of ensilement in trial 2.
Further calculation indicates 20.7 and 18.2% of the soluble nitrogen were in the form
of ammonia at feed out time for CON and PFLI, respectively. In support of the trend
observed in this study, Prigge et a1. (1976) demonstrated solubilization of nitrogen
occurred through 56 d. This finding suggests solubilization and deamination continue
through the fermentation period which in turn suggests the true protein content of high

moisture corn decreases the longer it is stored. Consequently, the type of supplemental

45

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47

protein may influence the efficiency of ruminal organic matter fermentation and ultimately, the
efficiency of utilization of metabolizable energy. Microbial counts were not evaluated at the end
of phase 4, but the elevated soluble nitrogen content may result from acid solubilization (Prigge
et al., 1976) or proteolysis by microbial enzymes (Bergen et al., 1974). In trial 1, soluble
carbohydrate content and pH decreased until phase 4 began and then increased during phase 4
(P < .01) for all treatments. Soluble carbohydrates and pH values were lowest during the
beginning portion of phase 4 and increased during the remainder of the ensilement period. The
pH rise suggests microbial activity occurred throughout the period of ensilement and sufficient
fermentation endproducts did not accumulate to suppress microbial metabolism. A significant
interaction (P < .02) between inoculation treatment and time was observed for pH changes in
trial 2. Acidity decreased at a faster rate for the inoculated HMC as compared to CON.
Soluble carbohydrate content was unchanged by treatment. The discrepancy between the two
trials is not readily explained, however, PFLI in trial 1 had a very high pH at the end of phase
4 which is difficult to explain unless extensive deterioration occurred prior to the removal from
the silo. Lactic acid content increased over time from the early phases until the end of phase
4 (P < .05) and was not affected by inoculation treatment in trials 1 and 2. Acetic acid content
was greater in phase 4 than phases 1-3 (P < .01) in trials 1 and 2. In trial 1, PFLI exhibited
lower acetic acid concentrations (P < .01) than D1 or CON. However, in trial 2, inoculation
treatment did not influence acetic acid production (P > .2). lactic acid concentrations
continued to increase after the initiation of phase 4, while acetic acid accumulation was not
evident. The elevated lactic acid content without acetic acid increase would suggest a more

homolactic fermentation occurred after the early portion of phase 4 as opposed to early

48
fermentation (phases 1-3). In contrast to these results, Woolford (1984) reported results which

indicate the homolactic species are predominant during the early stages of fermentation while
heterolactic bacteria become more prevalent as fermentation progresses to the later stages.
lactic acid content was considerably lower in trial 2 than trial 1 and values previously reported
by Bolsen et al. (1984). A portion of the difference in lactic acid content between the two trials
can be explained by the lower dry matter content in trial 2 and consequently the extent of
fermentation may have been less. The trend of lactic acid accumulation in trial 2 follows a more
typical fermentation pattern than trial 1. Another potential explanation for discrepancy between
the two trials may involve the methodology. Trial one results were measured by a gas
chromatography procedure whereas trial two endproducts were quantified by HPLC analysis.

In trial 1, propionic acid content increased (P < .004) during phase 4. A significant
phase x inoculation effect was detected for butyric acid (P < .10) and isovaleric acid (P < .03)
. in trial 1. The control corn exhibited greater butyric acid content than PFLI (P < .10) while
DI had an extremely high butyric acid value at the end of phase 4. Corn treated with PFLI
showed a greater isovaleric content than CON or DI (P < .02) at the end of phase 4. Propionic
acid (P < .01) and butyric acid (P < .05) content were greater with DI over CON or PFLI.
The elevated concentrations of branched chain fatty acids may partially explain the pH rise
during phase 4. The source of the branched chain acids may have resulted from deamination
of amino acids which would produce ammonia and buffer the pH upward. Propionic acid and
longer chain fatty acids were not detected in trial 2. Ethanol content was evaluated in trial 2 and
was found to increase (P < .0001) continuously until the end of phase 4.

In both trials, LAB were found to be greater (P < .02) at the beginning of phase 4 as

49

compared to phases 1-3 . Lactic acid bacteria were not measured in the buried bags, however,
lactic acid content was increased which would suggest LAB levels may have been increased.
A phase x inoculation interaction trend was observed in trial 2 (P < .10) for LAB. Lactic acid
producing bacteria were greater with PFLI over CON (P < .0031) during phases 1-3.
However, LAB populations increased after phase 1-3 with CON to similar populations as PFLI
during phase 4.

Dry matter recovery as measured from the buried bags was decreased (P < .08) with
PFLI as compared to CON in trial 2, however, DMR was similar in trial 1. Temperature of
corn in silos did not appear to be affected by inoculation treatment in either study (Figure 1).
In contrast, Rust et al. (1989) showed that temperature was lower in inoculated whole plant corn
silage as compared to untreated silage.

Microbial counts and temperature of the corn treatments during aerobic exposure in trial
2 are shown in Figures 2 and 3. Microbial enumeration after 3 d of air exposure were greater
for the inoculated corn (P < .05) as compared to CON. lBy d - 5, enumeration estimates for
both treatments were similar. Temperature was greater on 3 and 5 d with PFLI treated corn as
opposed to control corn. Control corn exhibited an accelerated heat production between 5 and
8 d exceeding the PFLI corn by 8 d. The elevated microbial population and temperature as seen
with PFLI indicate a less stable product. Rust et al. (1989) showed that corn silage treated with
a microbial inoculant adversely affected stability as measured by elevated temperature.

Previous research efforts with crops containing higher moisture levels were directed
toward the control of fermentation by addition of microorganisms capable of early, rapid growth

(Ely et a1. , 1981). Given the inability of accelerated early growth from microbial starter cultures

50

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Figure 2. Change in microbial population of high moisture corn during aerobic
exposure due to inoculation. Bars represent standard deviation for each
mean.

52

 

 

 

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Figure 3. Change in temperature of high moisture corn during aerobic exposure
due to inoculation. Bars represent standard deviation for each mean.

53

to control microbial activity after 4-7 d, alternative strategies to control fermentation

beyond 7 d may be beneficial for high moisture corn.

THE EFFECTS OF INOCULATION, LIECHANICAL
PROCESSING, RECONSTITUTION, AND CHEMICAL
ADDITION ON THE FERMENTATION OF

HIGH MOISTURE CORN

54

55
ABSTRACT

Two trials were conducted with laboratory silos to evaluate the effectiveness of
various methods of ensiling high moisture corn (HMC). In trial one, a 2X3X6 factorial
arrangement of treatments was utilized to evaluate two particle sizes of HMC stored at
25 96 moisture (whole versus ground); three inoculation rates of lactic acid producing
bacteria (LAB; 0, l x 10‘ and 2 x 10‘ cfng of wet corn); and six time periods of
ensilement (1, 2, 4, 8, 16 and 40 d). Lactate, acetate, ammonia-N content and LAB
enumeration were greater with ensiled ground HMC as compared to whole corn (P <
.05); however, dry matter recovery (DMR) was greater with whole corn treatment (P <
.05). Ground HMC had lower ethanol content and pH, but greater soluble nitrogen
content than whole corn within both LAB inoculation rates (P < .05). Higher
inoculation rates improved DMR in whole corn and depressed recovery with ground corn
(P < .05). In trial 2, fermentation characteristics of ground corn ensiled at low moisture
content (20%) was evaluated. A 9 x 4 factorial arrangement of treatments was used.
The nine treatments applied to the corn prior to ensilement included no treatment or
control; LAB inoculation (2 x 106 cfu/g); water to reconstitute corn to 75 96 DM; LAB
plus reconstitution, 1% ammonium hydroxide; .8996 urea; 1% propionic acid; . 15%
sorbic acid or a combination of .3 % propionic and .1% sorbic acid. Duplicate silos were
evacuated at four time endpoints for each treatment (1, 3, 7 or 40 d). Crude protein,
ammonia-N, soluble nitrogen and pH were greater while microbial enumeration was

reduced (P < .05) with addition of urea or ammonium hydroxide. A portion of the

56
added urea was not hydrolyzed in the low moisture corn, as evidenced by increased

soluble N but lower ammonia N (P < .05) as compared to the ammonium hydroxide
treated corn. Treatment with organic acid reduced the number of bacteria and pH (P <
.05) as compared to control. Sorbic acid was a more potent inhibitor of fermentation
than propionic acid (P < .05). Reconstitution of corn increased bacterial growth and
lowered pH (P < .05) as compared to untreated corn. Fermentation was more extensive
with water addition and restricted with nitrogen compounds or organic acids. Dry
matter recovery was similar after 40 d ensiling between treatments in trial 2 (P >
.10). As indicated by DMR, inoculation was beneficial with whole corn and deleterious
with ground corn stored at 25 % moisture; however, DMR was not improved by addition

of amendments or additives to 20% moisture corn.

Key Words
High Moisture Corn, Fermentation, Additives, Inoculation, Particle Size,

Reconstitution.

57
INTRODUCTION

High moisture corn (HMC) has been widely used as a feedstock for ruminant
animals. Due to diversified farm enterprises, labor availability and adverse climatic
conditions may limit the timeliness and efficiency of harvest. Consequently, HMC is
often harvested at moisture contents less than 25 % moisture which can create an
environment for an unfavorable fermentation (Fox, 1976). The rate and extent of
acidification through fermentation are limited by the low moisture content and limited
bacterial activity. The restricted fermentation reduces the nutritive value (Owens and
Thornton, 1976) and resistance to deterioration upon air exposure (Stevenson, 1976).
The addition of chemical or microbial additives prior to ensiling may lessen the
detrimental effects of the restricted fermentation.

Various treatments have been used during the ensilement of forage and grain
silages (Bolsen et al., 1984; Russell et al., 1988; and Rust et al., 1989). Microbial
inoculants have been shown to favorably influence fermentation and improve dry matter
recovery (Bolsen et al., 1984). Goodrich et al. (1975) has shown a more rapid
fermentation occurred with water addition to low moisture corn to a final moisture
content of 33.1%. Organic acids have also been used to restrict microbial growth.
Propionic acid is routinely utilized to preserve HMC stored at low moisture contents
(Voelker et al., 1985). Propionic acid has caustic properties and can be hazardous.
Sorbic acid, a less corrosive material, has been shown to inhibit microbial growth (Lee
et al. , 1986). Furthermore, combinations of organic acids may inhibit microbial

activities more effectively than a single acid (Moon, 1983). Decreasing particle size has

58

been shown to increase consolidation and improve fermentation ( Woolford, 1984c).

The purpose of this study was to determine the effects of microbial inoculation
rate and mechanical treatment on fermentation characteristics and dry matter recovery
(DMR) in corn ensiled at 75 % DM, and secondly, to evaluate chemical and microbial
additives to enhance preservation of low moisture corn.

MATERIALS AND METHODS

Trial 1

High moisture corn was harvested at approximately 75 % DM and ensiled in 108
laboratory silos (45.7 x 10.2 cm dia.) constructed of PVC pipe and fitted with rubber
caps. A bunsen valve was inserted in the top of each silo to allow gas escape. The 36
treatments were in a 2x3x6 factorial arrangement of treatments with three replications.
Treatments included two physical forms (ground vs whole), three levels of microbial
inoculation‘ (0, l x 10‘ or 2 x 10‘ cfu/g of wet corn) and six periods of ensilement (1,
2, 4, 8, 16 or 40 d). High moisture com (75% DM) was ground through a New Holland
390 tub grinder fitted with 1.6 cm screen. Three kg of corn were placed and
consolidated in each silo. Inoculants contained My: mm, W
faggjum and W W organisms. The freeze dried inoculant was '
reconstituted by mixing 280 g of commercial inoculant (565 x 10’ cfu per 280 g
package) in 9.5 L of distilled water. The mixture was allowed to ferment at room

temperature for 24 h prior to use. Prefermented inoculant was applied at the rate of 204

 

‘ Medipharm USA, DesMoines, IA

59
ml per t of wet corn. The number of epiphytic lactic acid producing microorganisms on

the fresh corn crop was 6.8 x 103 cfu/g and the prefermented inoculant contained 1 x 10‘
cfu/ ml at time of application. Inoculum was manually dispersed on the corn prior to
ensilement. Three silos from each inoculant treatment within a physical form of corn
were opened on 1, 2, 4, 8,16 and 40 (1. Dry matter recovery from each silo was
determined from differences in initial and final dry weight. Two sub—samples from each
silo were stored at either -19 °C for future laboratory analysis or placed on ice for less

than 5 h at 4 °C before microbial enumeration.

Trial 2

Seventy-two PVC laboratory silos (45.7 x 10.2 cm dia.) were filled and
consolidated with 3 kg of corn containing 80% DM. A 9x4 factorial arrangement of
treatments included nine treatment amendments and four silo evacuation dates post-
ensilement. The nine additive treatments were: 1) control, no treatment; 2) reconstitution
with water to 75% DM; 3) inoculation with LAB (2 x 10‘ cfu/g of wet corn; 4)
reconstitution to 75 % DM and inoculation (2x10‘ cfu/g of wet corn); 5). 1% ammonium
hydroxide; 6) .89% urea; 7) 1% propionic acid; 8) .15% sorbic acid and; 9) .3%
propionic and .1% sorbic acids. Inoculant was prepared as described in trial 1.
Prefermented inoculant was applied at a rate of 47.3 ml per 22.7 kg of fresh corn to
supply 2x106 cfu/g of fresh corn. Sixty-seven ml of water per kg of corn was added to
reconstitute 50 kg of corn to 25% DM. Duplieate silos of each treatment were evacuated

on 1, 3, 7, and 40 d post-ensiling. A 150 g sub-sample was taken, placed on ice and

60
microbial enumeration was performed within 5 h. Three hundred g were stored at -19

°C for laboratory analysis.
Microbial Analysis

A 100 g sample was mixed with 900 ml of sterilized water, blended for 5 min
with a magnetic stirrer and filtered through four layers of cheesecloth. The water extract
was serially diluted in lO-fold increments in sterile .1 % peptone solution. Enumeration
of lactic acid producing bacteria (LAB) was by the spread plate method (Hungate and
Fletcher, 1962) using selective LBS agar (BBL Microbiological Systems, MD). Each
dilution was plated in triplicate. Two hundred ul were placed on each plate, incubated
aerobically at 39 °C and colonies counted after 72 h. Colonies were presumptively
identified as lactic acid producing microorganisms.
Laboratory Analysis

Samples were allowed to thaw at room temperature and chopped in a Hobart
Macerator’. A 100 g sample was dried at 60 °C in a forced air oven for dry matter
determination (AOAC, 1984). Samples were analyzed for total nitrogen using a micro-
kjeldahl technique (AOAC, 1984) with a Technician Autoanalyzer“. A 20 g sample of
chopped corn was placed in 80 ml of Ohio buffer for soluble nitrogen content
determination (Johnson, 1969) by analyzing five ml of the homogenate was analyzed for

nitrogen using micro-kjeldahl technique with a Technicon Autoanalyzer (AOAC, 1984).

 

2 The Hobart Manufacturing Company, Troy, OH

3 Technicon Instruments Corporation, Tarrytown, NY

61
A 20% homogenate was prepared with wet corn and distilled water and strained through

four layers of cheesecloth for pH, ammonia nitrogen, soluble carbohydrate, volatile fatty
acid, lactic acid and ethanol content. Determination of pH was performed on the
homogenate with a combination electrode (Baertsche et al. , 1986). The homogenate was
diluted (1:50) and analyzed for soluble carbohydrate content (Dubois et al. , 1956). The
strained homogenate was centrifuged at 27,000 x g for 15 min and injected into the
Technicon Autoanalyzer for ammonia-N determination. Twenty ml of the strained
homogenate was acidified and deproteinized with 4 ml of 25 % metaphosphoric acid
solution. After mixing, the solution was centrifuged 27,000 x g for 15 min. Supernatant
was prepared for lactic acid, volatile fatty acid and ethanol analysis by HPLC procedure
of Siegfried et al. (1984) with modifications. One ml of 20.92% (w/w) calcium
hydroxide reagent and .5 ml of 10% (w/w) cupric sulfate reagent was added to 1 ml of
the corn extract. Samples were vortexed after the addition of each reagent. The cupric
sulfate reagent contained crotonic acid (.4 %) which served as an internal standard.
Samples were stored at 5 °C for 30 min after reagent addition and centrifuged 10,000 x
g for 15 min. The supernatant was acidified with 25 ul of concentrated sulfuric acid,
frozen and thawed twice followed by centrifugation at 10,000 x g for 15 min to remove
the remaining protein. The supernatant was filtered using a .22 um nylon filter
(Millipore Products Division, MA). Standards were prepared by adding individual acids
together and diluting with water. Standards were prepared in an identical procedure to
samples. Thirty-five ml of prepared samples and standards were injected into the HPLC

system with a 30 min run time. A Biorad ion exchange column (I-IPX-87H) was encased

62

in a water bath at 45 °C for succinate, lactate, forrnate, acetate, propionate and
butanediol separation and 30 °C for ethanol and butyrate separation. A Biorad guard
column (catalogue number 125—0129) was utilized to protect the ion exchange column.
Mobile phase was prepared by diluting 1.66 ml of concentrated sulfuric acid and .40 g
of EDTA to a volume of 4 L with double distilled millipore filtered water. The mixture
was boiled to dissolve the EDTA, cooled and filtered through a .22 um nylon filter.
Mobile phase flow rate was .7 ml per min. The HPLC system was comprised of a
Waters 6000A pump, 712 WISP, 730 data module, 720 system controller and 410
refractive index detector‘. The detector was set at 8 x 106 refractive index units full

scale.

Statistical Analyst

Fermentation characteristics were analyzed using the analysis of variance
procedure (SAS Institute, 1987). Nonorthoganal designed contrasts were made among
d 40 means using Bonferroni - t statistic (Gill, 1978). Contrasts of interest, in trial 1,
were whole versus ground corn within each inoculation treatment; and no inoculant
versus low rate of inoculation and low versus high inoculation rate within whole and
ground corn. In trial 2, contrasts included: Control versus inoculation, control versus
reconstitution, reconstitution alone versus reconstitution and inoculation, control versus

nitrogen compounds, ammonium hydroxide versus urea, control versus organic acid

 

‘ Waters Associates Inc., Milford, MA.

63

treatment and propionic versus sorbic acid.
RESULTS

Trial 1

Across both inoculation rates, the rate (Figure 1) and extent (Table 1) of
fermentation, as evidenced by pH changes, were reduced with whole corn as compared
to ground corn (P < .05). Ground corn reached a lower pH by 4 d post-ensiling than
whole corn (4.4 versus 5 .5), however, the low pH with ground corn was maintained
only with inoculated corn. The pH increased in the untreated ground corn after d-8.
Lactate (Figure 2) and acetate (Figure 3) concentrations were greater with ground corn
than whole corn (P < .05) across all inoculation treatments. Within the ground corn
treatment, lactate concentration was greater with the low inoculation rate (P < .05) as
compared to control or high inoculation rate. Acetate was greater at the highest
inoculation rate as compared to the other treatments. Inoculation treatments did not
effect the acid profile with ensiled whole shelled corn. Lactate and acetate concentrations
appear low in the study; however; previously reported studies utilized HMC containing
more moisture (Young et al., 1984; Bolsen et al., 1984; Goodrich et al., 1975). The
occurrence of lower lactate and higher acetate with the higher inoculation rate with
ground corn may result from metabolism of lactate to acetate by yeast or clostridia
(W oolford, 1984c). However, LAB have been shown to convert lactate to acetate in
silages as well (Woolford, 1984c) which may indicate excess inoculation. Inoculated
ground corn showed lactic acid accumulation by 4 and 8 d post-ensilment, however,

acetic acid was not detected until 40 d post-ensilement. Whole corn contained greater

6.5 -- Whole Corn

 

 

 

 

 

6.0-
5.5-
I
O.
5.0‘
4.5a?
4,0t::+:::::4%::::::4::
O 10 20 30 40
O—OControl 0—01x106cfu/g 0—02x106cfu/g
S'ST Ground Corn
1
6.0T ‘f
5.5--
I
Q- 50
. I /§
4.5-h >‘ /
4.0cc:::%+::{e::::;:;:§
O 10 20 30 40

Day of Fermentation

Figure 1. Effect of inoculation rate and mechanical processing on pH of high
moisture corn during fermentation. Bars represent standard deviation for
each mean.

Table 1. Effects ofInoculation and Mechanical Processing on the Chemical Characteristics

65
Of Corn After 40d Emilanertt at 25% Moisture

Dry
Soluble Ammonia Soluble Matter

pH Carbohydrate Lactate Acetate Ethanol Nitrogen Nitrogen Recovery LAB‘
Whole Corn
Control" 6.03 1.58 0 0 .588 .069 4.54 100.4 5.11
1 x 10“ 6.07 1.41 .085 0 .588 .060 4.43 99.5 6.25
2 x 10" 6.00 1.49 .112 0 .624 .069 4.36 99.8 6.0
Ground Corn
Control” 6.06 1.91 .156 .071 .449 .120 5.37 99.0 6.69
l x 10“ 4.45 1.63 1.181 .065 .409 .196 8.77 99.2 7.63
2 x 10" 4.47 1.71 .994 .133 .291 .191 7.93 98.9 7.92
SEM" .135 .283 .026 .006 .037 .01 .606 .008 .172

Contrasts

W" Vs. G" Control * * '1' '1'
W‘Vs.G"lx10‘ * * '1' '1' '1' '1' '1' .*
W” Vs. G" 2 x 10‘ "' * * * '1' * * *
Within Whole Corn
Control vs. 1 x 10‘ * *
1 x 10‘ vs. 2 x 10‘ * '1'
Within Ground Corn
Control vs. 1 x 10‘ "' * * * *
1 x 10‘ vs. 2 x 10‘ * '1' * *-

‘ LAB - lactic acid producing bacteria, log cfulg.

" Control - no inoculation treatment; 1*10‘ cfu/g - inoculation rate; 2*10‘ cfu/g - inoculation rate, cfu/g;

SEM-standarderrorofthemean;W-wholecorn;G—groundcorn.

' Asterisks identify treatment differences (P < .05).

 

 

 

      

 

 

 

Whole Com
1.2--
A
2 1.0»
O
33 0.8--
E
o ..
< 0.6
.9
45 0.4--
3
0.2» -
0.0‘Wtc : ::;:::Q-,
0 10 20 30 4O
0—0 Control 0—01x1060fu/g o—o 2x106cfu/g
Ground Corn
1.2-- Q
’5‘ -
o 1.0-- 9
3‘; 0.8--
:9
0 ..
< 0.6
.9
*5 0.4--
3 -
0.2" 6/9
-/=/,
0.0““43‘3_1~ jifvclsécefi
O 10 20 30 40

Day of Fermentation

Figure 2. Effect of inoculation rate and mechanical procesing on the lactic acid
content of high moisture corn during fermentation. Bars represent
standard deviation for each mean.

67

0.14 -- Whole Corn

1

0.12-

0.10-

I

0.08 i

0.06 -

I

0.04 «

Acetic Acid (96 DM)

'7

0.02 -

 

I I
0.00 "-.H—‘—. i 4. i O t 4, c : s 4. g s : 4. 4. .- .
0 10 20 30 40

 

O—OControl O—O1x1060fU/g 0—02x106cfu/g
0.14.. Ground Corn 1

1’

0.12-

0.101

I

I

0.08 -

e-Qe—t

0.06 ..

I

0.04 -

Acetic Acid (96 DM)

0.02 T

 

0.00‘W§11.:§::::%%::1§
O 10 20 .30 40

 

Day of Fermentation

Figure 3. Effect of inoculation rate and mechanical processing on the acetic acid
content of high moisture corn during fermentation. Bars represent
standard deviation of each mean.

68
amounts of ethanol on d - 40 (P < .05) than ground corn (Table 1), however, ethanol

accumulation was not evident until 8 d post-ensilment in the whole corn (Figure 4).
Since ethanol is produced under aerobic conditions primarily by yeast organisms,
(McDonald, 1981), oxygen may have been trapped between corn kemals. Soluble
carbohydrates (Figure 5) were similar for all treatments.

Ensiled ground corn exhibited greater amounts of ammonia N (P < .05) than
ensiled whole corn (Table 1). The enhanced level of deamination in ensiled ground corn
may be explained by the increased availability of nitrogenous compounds in the smaller
particles. Inoculation increased ammonia-N in ensiled ground corn as compared to the
control. Solubilization of nitrogen was increased (P < .05) by ensiled ground corn but
no differences were seen with ensiled whole corn. Ensiled ground corn had greater
numbers of LAB than whole corn (Table 1, P < .05). Presumably, the particle size
reduction created more substrate for microbial growth. Microbial numbers increased (P
< .05) as inoculation rate increased with HMC from both particles sizes. It is peculiar
that the population of LAB increases with levels of inoculation but the fermentation
endproducts are similar in both situations. Perhaps the LAB were metabolizing
fermentation endproducts.

Dry matter recovery was greater for ensiled whole (P < .05) as compared to
ground corn (Table 1). In addition, inoculation of HMC tended (P < .05) to reduce dry
matter recovery.

Trial 2

Dry matter content was greater (P < .05) in untreated corn as compared to

0.7 -- Whole Corn

 

 

 

 

w T

2b""3o"' 4o

1
0.6-- ;
g 0.5T
O
18, 0.4.»
g 0.3..
g r
a 0.2-- g
0.1“ /é
ooO‘M{CS++:::%{{::;5:
0 10 20 30 4O
0—o Control o—e 1x1 a5efu/g 0—02x106cfu/g
0.7" Ground Corn
0.6--
2 0.5..
0
£3, 0.4--
'5
0.3L» 1'
5 1
LIJ 0.2+- T/*
0.1 "t' I. ?
0.0M 4. 4. 4. A. . .
10

0

Day of Fermentation

Figure 4. Effect of inoculation rate and mechanical processing on the ethanol
content of high moisture corn during fermentation. Bars represent
standard deviation for each mean.

70

6.0 -- Whole Corn

 

 

 

I
5.0--
\r
4.0--
, i
/
3.0« \f
2.0..-
LO +t:-{<;tt::filc;%ec;;:
O 10 20 30 40

o—o Control e—o 1x106cfu/g 0—02x106cfu/g

Water Soluble Carbohydrates (% DM)

 

 

 

6'07 Ground Corn
5.0--
r
O
4.0"
3,0"?
l
I
2.O«L
1.053%%%fival---cictcc4_
0 10 20 30 40

Day of Fermentation

Figure 5. Effect of inoculation rate and mechanical processing on the soluble carbohydrate content
of high moisture corn during fermentation. Bars represent standard deviation.

reconstituted corn (Table 2). The lower dry matter content of reconstituted corn is due

71

to added water. Corn treated with urea had a greater DM content than ammonium
hydroxide treated corn (P < .05). The addition of urea and ammonium hydroxide
increased crude protein, soluble nitrogen and ammonia nitrogen content (P < .05) over
control corn after 40 d of ensilment. Urea treated corn had greater crude protein and
soluble nitrogen content, but lower ammonia nitrogen content (P < .05) than ammonium
hydroxide treated corn. Lower pH values were detected with water addition (P < .05)
than nitrogen or acid addition. Urea treated corn had a lower pH (P < .05) than
ammonium hydroxide treatment while propionic acid addition exhibited lower pH values
(P < .05) than sorbic acid addition. Reconstituted corn exhibited greater LAB
proliferation over control corn (P < .05). The addition of nitrogenous or acid
compounds restricted LAB growth as compared to control corn (P < .05). No
differences were detected between treatments for DMR or soluble carbohydrate content
(P > .05).
Discussion

Retention of nutritional value during the ensiling process is the primary goal of
silage enhancements. Dry matter recovery or retention has been the most commonly
reported indicator of nutrient preservation. The purpose for ensiling feedstuffs is to
allow harvest of a moist material while surrendering only a small portion of the
nutritional value during fermentation and storage. Since HMC contains limited amounts
of moisture, the challenge to ensure a favorable fermentation is great.

In trial 1, DMR was adversely affected by grinding and inoculation, although all

72

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74
treatments exhibited acceptable DMR ranging from 100.4 to 98.9 %. Bolsen et al.

(1984) showed that DMR was improved with LAB addition to HMC which is in contrast
to the results of this trial. In other silages such as grass, the extent of mechanical
treatment did not greatly influence DMR (Steen, 1985).

In the current study, DMR was reduced under the conditions that stimulate an
extended fermentation (elevated moisture content and inoculation). This is in agreement
with the results of the study reported by Goodrich et al. (1975). In contrast with trial
1, a Kansas study (Bolsen et al., 1984) reported increased DMR with inoculation. In that
study inoculation reduced lactic and acetic acid contents which suggests the inoculated
corn had a less extensive fermentation. Though DMR was not reported, Prigge (1976)
showed elevated soluble nitrogen content and lower pH with ground corn as compared
to whole shelled corn which are indicators of more extensive fermentation and thereby
elevated DM‘ losses. During the ensiling process, microbial organisms and enzymes
utilize plant substrates and produce carbon dioxide, as one of many endproducts
(McDonald, 1981; Woolford, 1984c). In trial 1, extended enzymatic and microbial
activity may have increased the amount of dry matter lost as carbon dioxide. In trial 2,
no differences in DMR were detected. Recoveries were similar to those in trial 1
ranging from 100.4 to 98.7%.

The concept of extended fermentation being associated with elevated DM losses
has been previously discussed (Barnett, 1954; Mcdonald, 1981), however rapid
accumulation of lactic acid and decreased pH have been associated with suppressed

clostridial and mold growth. Consequently, optimum fermentation is a balance between

75

fermentative losses and aerobic instability.

Reconstitution enhanced fermentation over control corn as indicated by elevated
LAB enumeration and lower pH. However, inoculation did not greatly influence
fermentation in the 20% moisture corn. The addition of nitrogen compounds or organic
acids decreased microbial activity and pH.

Besides DMR, animal performance has also been used to evaluate fermentation
treatments. Nitrogenous compounds have increased the crude protein content of the
ensiled feedstuffs which may be beneficial to cattle performance. Young et al. (1984)
reported similar cattle performance between silages treated with urea before and after
ensilement. Russell et al. (1988) demonstrated similar findings with high moisture milo,
however, an advantage was seen with a natural protein source (soybean meal) in the diet
over urea added pre—ensiling or in the diet.

Soluble nitrogen content was greater while ammonia nitrogen content was lower
in urea treated corn as opposed to ammonium hydroxide treatments. This may indicate
that the urea is not fully hydrolyzed. Previous studies have reported that low moisture
feedstuffs may not have adequate moisture to activate the urease activity, thus showing
decreased urea hydrolysis (Ghate and Bilanski, 1979: Ghate et al., 1981; Alhadhrami et
al. , 1989). Ureolytic activity has been shown to come from bacterial origin (Cook,

1976) with limited urease activity being derived from plant tissue (Rode et al., 1986).

Conclusion

In this study, the production of lactic acid was increased and pH decreased with

76
the grinding process and microbial addition to HMC containing 75 96 DM, however, they

proved to be disadvantageous to DMR. At 80% DM various fermentation characteristics
were affected by microbial inoculation, reconstitution, nitrogen compound addition and
organic acid addition with no differences in DMR. The results of both laboratory studies
failed to demonstrate effieacious amendments for the preservation of HMC.
Extrapolation of the results from these oxygen-limiting silos to field seale silos may be
limited. These studies were performed in air-tight laboratory silos and results may differ

in large seale silos.

EFFECTS OF DRY MATTER CONTENT, STORAGE TEMPERATURES ~
AND MICROBIAL INOCULANTS ON THE FERMENTATION AND

AEROBIC STABILITY OF HIGH MOISTURE CORN

77

78
ABSTRACT

High moisture corn (HMC) was ensiled in laboratory silos for 67 d in a 4x3x2
factorial arrangement of treatments to evaluate the effects of initial dry matter (DM)
content, storage temperature and inoculation rate on the pattern of fermentation, dry
matter recovery (DMR) and aerobic stability. Corn was harvested at 4 DM contents (78,
74, 66 or 63%), ensiled at three temperatures post-ensiling (4,12 or 20 °C) and treated
with O or 2x10“ cfu/g of lactic acid producing bacteria before ensiling. Each of the 24
treatments was replicated three times. Corn was sampled for chemical and microbial
analysis and DMR measured at the end of the ensilement period. Remainder of the corn
from each silo was placed into a plastic lined styrofoam container to evaluate
deterioration during aerobic exposure. Dry matter recovery was improved (P < .05)
with the addition of an inoculant and the effect was greatest at the intermediate moisture
levels. Storage temperature did not signifieantly influence DMR during fermentation or
aerobic exposure (P > .10). Inoculation increased lactic acid and decreased acetic acid
content (P < .05) as compared to control. Lactic and acetic acid accumulations were
greatest (P < .05), with low DM corn. During aerobic exposure, DM losses were less
(P < .05) at high DM content. Inoculation did not influence DM losses during aerobic
exposure (P > .10). Temperature rise during aerobic exposure was more rapid with low
DM corn (P < .05). Temperature of corn stored at low dry matter content and low
storage temperature rose above ambient temperature (P < .05) within 48 h of aerobic

exposure. Corn stored at 20 °C showed no change in temperature. These data indicate

79

that corn stored at high DM content improved DMR during fermentation and aerobic
exposure. Inoculation improved DMR during fermentation and did not adversely effect
stability at high DM contents. Maximum DMR ean be obtained with corn stored at 78%

DM and with the use of an inoculant in air tight mini-silos.

INTRODUCTION

The objective of ensilement of high moisture corn (HMC) is to preserve the
nutrients present in the fresh crop as economically as possible. Unfortunately, nutrient
losses occur as a result of the fermentation and subsequent exposure to oxygen may
further reduce digestible nutrients. Management practices such as rapid silo filling, well
sealed silos and rapid feedout have been shown to improve nutrient recovery (Fox,
1976). Several factors such as dry matter (DM) content, storage temperature and the use
of a microbial inoculant or starter culture have been shown to alter fermentation patterns
of whole plant corn (Luther, 1986; Rust et al., 1989) legume silage (Ely et al., 1981)
and grass silages (Lindgren et al., 1985a). Limited information is available on the
effects of these factors on the fermentation pattern of a low moisture crop such as HMC.
Furthermore, information regarding the effects of fermentation enhancers on aerobic
stability is limited in the published literature. The microorganisms involved in the
deterioration process have been classified by Woolford and Wilkie (1984). In addition,
microbial inoculant starter cultures have been reported to positively influence the rate of
fermentation in HMC under certain ensiling conditions (Rust et al. , 1987).

To accurately determine the conditions in which a starter culture would be

80

advantageous for HMC, a trial was designed to evaluate the effect of moisture content,
storage temperature and addition of a starter culture on nutrient recovery and aerobic

stability.

MATERIALS AND METHODS

General

Corn was harvested at four dry matter contents (63.4, 66.4, 74.0 or 78.1% DM)
from the same field. At each moisture content, corn was placed into a roller mill,
inoculated with water or prefermented liquid inocula and 3 kg consolidated in polyvinyl-
chloride laboratory silos (10.2 cmdia x 45.7 cm). The inocula contained a mixture of
Wham. 311mm andRedimagidflacfia. Theinocula
was reconstituted from a dry form by mixing 280 g of commercial soluble inoculant (565
x 10’ cfu/28f) g) in 9.5 L of water and allowing to set for 24 h prior to application.
Inocula was applied at a rate of 47.3 ml/22.5 kg) of fresh corn to supply 2 x 10‘ cfu/g.
Each moisture by inoculation treatment combination was stored at 4, 12.5 or 20 °C. For
each treatment combination, triplicate silos were opened 67 d post-ensiling. Subsamples
were taken for chemical and microbial analysis. The remainderof theensiled corn was
placed into plastic lined styrofoam containers to evaluate stability during oxygen
exposure. Temperature of the corn mass was recorded at 12 h intervals. Com was
evacuated from containers after 48 h of aerobic exposure and subsamples were collected

for chemical and microbial analysis.

81
Chemical Analysis

A lOOgsample ofwetcornwasdriedat60°Cinaforcedairoven fordry
matter determination (AOAC, 1984). Analyses for pH and total nitrogen were
determined by procedures previously described (Baetsche et al. , 1986). A 20%
homogenate was deproteinized with 5-su1fosalicylic acid, diluted ten-fold and analyzed
for soluble earbohydrate content (Dubois et al., 1956). For ammonia nitrogen analysis,
20 ml of the homogenate was acidified with 4 m1 of 25 96 meta-phosphoric acid,
centrifuged and analyzed on Technicon Autoanalyzerl for ammonia nitrogen (AOAC,
1984). Soluble nitrogen content determination (Johnson, 1968) was performed by placing
a 20 g sample of chopped corn in 80 ml of Ohio buffer. Five ml of the homogenate was
analyzed for nitrogen concentration using micro-kjeldahl technique with a Technicon
Autoanalyzer (AOAC, 1984). Volatile fatty acids, lactic acid and ethanol were analyzed
by HPLC analysis according to Siegfried et a1. (1984) with modifications. The HPLC
system was comprised of a Waters 6000A pump’, 712 WISP, 730 data module, 720
system controller and 410 refractive index detector. The detector was set at 8x104
refractive index units full scale. A Biorad3 exchange column HPX-87H was heated to
45 °C for succinate, lactate, acetate, propionate and butanediol separation and 32 °C for

butyrate and ethanol separation. A guard-column (Biorad 125-0129) was inserted to

 

‘ Technicon Instruments Corporation, Tarrytown, NY.
2 Waters Associates Inc. , Milford, MA.

3 Biorad Chemical Division, Richmond, CA.

82
protect the ion exchange column. The mobile phase was prepared by dilution of 1.66

ml H280, and .4 g EDTA to 4 L of double distilled HPLC water, and filtered through
.22 um nylon filter. Flow rate for the mobile phase was .7 mllmin. Injection volume
was 20 ul with a run time of 30 min. A 40% honogenate was prepared with wet corn
and distilled water and strained through four layers of cheesecloth for HPLC analysis.
The homogenate was acidified by adding 5 ml of concentrated sulfuric acid to 20 ml of
homogenate before centrifugation at 27,000 x g. One ml of 20.92 96 (wlw) calcium
hydroxide and .5 ml of 10% (wlw) cupric sulfate were added and the sample was again
centrifuged at 10,000 x g. Twenty five ul of concentrated sulfuric acid were added to
the supernatant prior to a freeze-drawing to deproteinize the sample. Deproteinization
process was repeated twice to ensure that a clean preparation was injected into the
HPLC. Samples were centrifuged and filtered through .22 um nylon filters. Cupric
sulfate solution containing .496 crotonic acid, served as an internal standard.
Microbial Analysis

Samples of high moisture corn were stored in air-tight plastic bags at 4 °C for no
longer than 5 h prior to microbial enumeration. One hundred g of corn were added to
900 ml of sterile distilled water. The mixture was homogenized for l min in a Waring
blendor and filtered through four layers of cheesecloth. The filtrate was serially diluted
in lO-fold increments in sterile LBS broth (BBL Microbiology Systems, MD) for
determination of lactic acid bacteria. Coliform bacteria were enumerated by serially
diluting filtrate in 10-fold increments in sterile E C Medium (Difco Laboratories, Detroit,

MI). The extract was also serially diluted in sterile .1 % peptone solution. Aliquots of

83 ,
.1 ml were dispersed on agar plates, incubated aerobieally at 39 °C for 72 h and colonies

counted for mold and yeast enumeration. Media for mold and yeast determination were
prepared by boiling 10 g malt extract, 10 g potato starch, 1 g proteose peptone, 2 g
bacto—dextrose and 16 g agar in l L of distilled water. After solubilization, the solution
was autoclaved for 20 min.
Statistical Analyst

Fermentation characteristics were analyzed using the analysis of variance and
correlation coefficients were acquired (SAS Institute, 1987). To establish relationship
between specific variables, correlation analyses were performed. Mean separation was

performed using Tukey’s HSD (Gill, 1978).

RESULTS AND DISCUSSION

Microbial and chemical characteristics of the unensiled corn changed as the
harvest season progressed (Fable 1). Corn harvested at higher DM content contained
greater pH and crude protein content while soluble nitrogen ammonia nitrogen and
soluble carbohydrate content were lower (P < .05). The reason for elevated protein
content at later harvest dates is unknown. Copeland and McDonald (1985) discuss the
nutrient deposition into the kernel and indicate that protein and sugar content in pre-
mature com should be greater than mature corn on a DM basis. Protein and sugars are
deposited relatively early during kernel development followed by starch deposition which

occurs before maturity. The kernel contains no less total protein or sugar as maturity

84

 

Table 1. Initial (d-0) Chemical Characteristics Of Corn Harvested

At Various DM Contents
Harvest Date Oct. 3 Oct. 10 Oct. 31 Dec. 2
Dry Matter.’B 63A EA 14.0 18.1 SEM
Laboratory Analyses, 96 DM
pH 6.5‘I 6.3‘ 6.6“ 6.7“ .03
Crude Protein 9.6° 9.3° 10.6‘ 10.4‘ .16
Soluble N .115° .111‘I .104“ .102c .003
Ammonia N .066f .040c .028“l .025° .002
lactate .13 .01 .02 .01 .13
Soluble Carbohydrate 7.6d 5.1° 4.9° 4.7° .33
Microbial Analysis, log cfu/g
Coliform 6.7 6.7 6.3 6.7 .44
LAB” 4.3 5.0 5.0 4.3 .37
Yeast 2.0“ 4.36 0.0° 1.3“I .37
Mold 0.0 0.0 0.0 0.0 0.0

b

SEM - Standard error of the mean.

LAB - lactic acid producing bacteria.

Means within a row with unlike superscripts differ (P < .05).

85
progresses, however, expressed on a percentage basis, protein and sugar contents

decrease as starch accumulates. Soluble carbohydrate content over time reflects a similar
sequence of events. Analysis supports the traditional view of less sugar expressed as a
percent of DM as the plant matures; however, total nitrogen content does not. Microbial
populations of LAB and coliform were similar across harvest dates and DM contents.
Mold organisms were not present in any of the unensiled corn samples. Yeast
populations appeared to be higher during the early harvest dates, although only the
66.4% DM corn had significantly greater yeast (P < .05).

Inoculation treatment increased DMR (P < .05) as compared to control (Table 2).
The .5 % increase of unrecoverable DM with inoculation seems small and differences that
small may be difficult to demonstrate under field conditions. However, non-significant
improvements in DMR of this magnitude due to inoculation of HMC have been reported
in field studies (Wardynski et al. , 1990). Dry matter recovery was positively correlated
(Table 3) with dry matter content (r=.86; P < .0001), pH (r=.73; P < .0001), soluble
carbohydrate (r=.23; P < .052) crude protien content (r = .57; P < .0001) and mold
population (.28; P < . 16). Ensiled corn with higher DM content had greater crude
protein content and more mold colonies. After the 67 d ensilement period, a negative
relationship was observed between DMR and yeast population (-.33; P < .004), soluble
nitrogen (-.68; P < .0001) ammonia nitrogen (-.59; P < .001) lactic acid (-.64; P <
.0001) acetic acid (-.77; P < .0001) and ethanol (-.71; P < .001). A signifieant (P <
.07) DM by storage temperature interaction effect on DMR was evident in this study.

Overall, dry matter recovery increased as DM content increased, however, 74 % DM

86

Table 2. Effect Of Inoculation On Dry Matter Recovery After
67d Ensilement

Central lament SEM‘
Dry Matter Recovery,% 98.2" 98.7‘ .05

‘ Standard error of the mean.

”° Means within a row with unlike superscripts differ (P < .05).

87

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88
ensilage stored at 12.5 °C had similar DM recovery as ensilage stored at 4 °C. The

results of this study suggest storage of ensiled HMC at 12.5 °C may allow preservation
over a wider moisture range with less DM loss.

The trends for improved DMR as moisture level decreases is in agreement with
previous results reported by Goodrich and Meiske (1976). Dry matter losses associated
with the HMC in this study were less than 5 % which is similar to published results for
other fermented feedstuffs (McDonald, 1981; Woolford, 1984c).

Comparison of the mean squares for storage temperature by the DM by storage
temperature interaction mean square, indicates the majority of the variation in DMR is
associated with DM content. In support of this premise, the correlation between storage
temperature and DMR was nearly zero (r= .01; P > .95). The effects of storage
temperature on DMR with HMC in this study are in agreement with previous results
reported by Garcia et al. (1989) with ensiled alfalfa.

A DM x storage temperature interaction (P < .05) was observed for pH, lactic
acid, acetic acid, ethanol, soluble nitrogen, ammonia nitrogen, LAB, coliform, bacillus,
yeast and mold population (Figures 1-14). Across all storage temperatures, there was
a trend for pH to increase (P < .05) as DM content increased (Figure 1). One
discrepancy from this overall trend occurred with the driest corn. Storage at 12.5 °C
yielded a lower pH than similar HMC stored at 4 or 20 °C. The reason for the
discrepancy is unknown. Lactic acid content increased (P < .05) as DM content
decreased with all three storage temperatures, however, the amount of lactic acid

production per unit of DM increased more with highest storage temperature (Figure 1).

 

 

89

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3.5+4¢¢§4=¢¢§5535{+:::4,

50 65 7O 75 80

Dry Matter (%)

Figure 1. Effects of storage temperature and dry matter content on pH
decline. Bars represent standard deviation for each mean.

4.0--
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. . . .7.5. 80

' 65 70
Dry Matter (%)

Figure 2. Effects of storage temperature and dry matter content on lactic acid
content. Bars represent standard deviation for each mean.

91

 

 

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OD : % e : f 4 : : 4 1 1 4. * ,
60 65 7O 75 80

Dry Matter (%)

Figure 3. Effects of storage temperature and dry matter content on acetic acid
content. Bars represent standard deviation for each mean.

92

 

 

 

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60 65 7O 75 80

Dry Matter (%)

Figure 4. Effects of storage temperature and dry matter content on ethanol
content. Bars represent standard deviation for each mean.

93

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60 65 7O 75 80

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Figure 5. Effects of storage temperature and dry matter content on water soluble
carbohydrate content. Bars represent standard deviation for each mean.

 

 

Water Soluble Carbohydrates (96 DM)

94

 

 

11.0» T
A .
c2) 1%?
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50 65 70 75 80

Dry Matter (%)

Figure 6. Effects of storage temperature and dry matter content on crude protein
content. Bars represent standard deviation for each mean.

.0
00
%

 

 

A ARA
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60 65 7O 75 80

Dry Matter (%)

Figure 7. Effects of storage t-perature and dry matter content on soluble
nitrogen content. Bars represent standard deviation for each mean.

96

 

 

 

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60 65 7O 75 80.

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Figure 8. Effects of storage temperature and dry matter content on ammonia
nitrogen content. Bars represent standard deviation for each mean.

97

 

 

10.5"
A 9.5... /
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60 65 7O 75 80

Dry Matter (%)

Figure 9. Effects of storage temperature and dry matter content on lactic acid
producing bacteria (LAB) counts. Bars represent standard deviation for
each mean.

98

 

 

 

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60 65 7O 75 80

Dry Matter (%)

Figure 10. Effects of storage temperature and dry matter content on
enterobacteriaceae counts. Bars represent standard deviation for each
mean.

 

 

 

 

 

9.5-- ..
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5.5 : : c : : t c + : : t : r s t 4 : : c :
60 65 7O 75 80

Dry Matter (%)

Figure 11. Effects of storage temperature and dry matter content on bacillus
counts. Bars represent standard deviation for each mean.

100

 

 

 

7.0«—
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3 I
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50 55 70 75 80

Dry Matter (%)

Figure 12. Effects of storage temperature and dry matter content on yeast counts.
Bars represent standard deviation for each mean.

101

4.5" 0—04 OC
.. o—o12.5 °c
4'0 a—A20.0 °c

.___p_.-

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60 65 7O 75 80

Dry Matter (%)

Figure 13. Effects of storage temperature and dry matter content on mold counts.
Bars represent standard deviation for each mean.

Molds (log cfu/g)

 

 

102

 

 

A _”_ 0—04 0C I
39/ ‘00 o—o12.5°c O
>\ 15—13200 °C ¥
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60 65 7O 75 80

Dry Matter (%)

Figure 14. Effects of storage temperature and dry matter on dry matter recovery.
Bars represent standard deviation for each mean.

103

Closely, with ensilement of low moisture feedstuffs as used in this study, elevated storage
temperature provided for greater lactic acid concentrations.

For HMC with the two lowest dry matter levels stored at 4 °C acetate
concentrations were signifieantly less than HMC stored at the other temperatures (Figure
3). This trend was observed with lactic acid as well, which suggests storage at lower
temperatures significantly altered the extent or pattern of fermentation.

Ethanol content was less for HMC with low DM content ( < 66%) stored at 20
°C as compared to storage at lower temperatures or high dry matter ensilages (Figure 4).
Since ethanol has little preservative capacity (McDonald, 1981) and results in carbon
loss, high levels are undesirable residual soluble carbohydrate content, a measure of
substrate usage by microorganisms, was greater at lower DM content ensilages (Figure
5) and residual soluble carbohydrate was significantly greater for 78 and 63 96 DM HMC
stored at 4 °C as compared to HMC with similar DM stored at higher temperatures.
This suggests factors other than substrate availability may have limited these fermenta-
tions.

Comparison of the degree of homofermentation expressed as moles of lactate acid
per mole of acetic plus ethanol suggests a more efficient fermentation occurs as moisture
level increases to 34 %. Efficiency of fermentation decreases at 63 % DM largely because
the amount of ethanol produced at all storage temperatures increased more than lactic
acid. Utilization of this ratio of endproducts as predictor of homofermentation and
fermentation efficiency is related to dry matter recovery (W oolford, 1984c). Calculation

of stoichimetry balance for moles of substrate (Table 4) suggests less than 2 % of the

104
Table 4. Calculation Of Substrate Conversion Efficiency

 

Dry Matter Content,%

63A 66.5 M 18.1
Soluble Carbohydrate, 96 DM
Beginning 7.57 5.06 4.94 4.70
Residual 3.35 2.21 1.41 2.57
Soluble Carbohydrate, Moles
Residual .050 .028 .027 .026
Initial .019 .012 .008 .014
Loss .031 .016 .019 .012
Total Moles Of Endproducts ‘
Lactic Acid 2.78 2.31 .94 .30
Acetic Acid .73 .52 .35 .05
Ethanol 3.03 1.54 .98 .88
Carbon in Endproducts

15.86 11.05 5.48 2.76
Carbon Lost
From Substrate .192 .096 .114 .072
From Amino Acid .139 .131 .046 .005
Total .331 .227 .160 .077

105
metabolized substrate could be accounted for in soluble carbohydrate in initial samples

or catabolism of amino acids. Therefore, quantifieation of soluble carbohydrate in
unensiled corn may be an inadequate predictor of the resultant fermentation. Crude
protein content was highest for the lowest DM corn ensilage (Figure 6). Although total
nitrogen content remained similar across storage temperatures, the form of nitrogen
changed.

Soluble nitrogen and ammonia nitrogen content increased (P < .05) as DM
content decreased and was consistently higher at elevated storage temperatures (Figures
7 and 8). Clearly, some of the carbon skeletons of the nitrogenous compounds were
utilized as energy substrates. The proportion of total nitrogen in the soluble form was
7.7, 13.8, 36.4 and 35.7% for 78.1, 74.0, 66.4 and 63.4% HMC, respectively.

Populations of lactic acid producing bacteria (LAB) were significantly lower (P
< .05) at the lowest DM content regardless of storage temperature (Figure 9).
However, the effect of storage temperature on LAB growth was similar across DM
content. Coliform bacteria appeared to decrease (P < .05) in HMC as DM content
decreased with the 4 and 12.5 °C storage temperatures. Conversely, at 20 °C; coliform
tended to increase as DM content decreased. Across all ensilages, the correlation
between LAB and coliform is negative (r= -. 15; P < .20) which supports similar trends
reported by McDonald (1981). As ethanol content increased, coliform populations
decreased (r = -.32; P < .006). Bacillus population remained relatively similar across
storage temperature and DM contents (Figure 11). For HMC stored at 12.5 and 20 °C,

yeast populations increased as DM content decreased (Figure 12). This observation

106

would substantiate the larger influence yeast has on fermentation of HMC as compared
to other fermented feedstuffs. Yeast population was negatively related with dry matter
content, (r = -.49; P < .0001) dry matter recovery (r = -.49; P < .0044), crude
protein (r = -.59; P < .0001) and mold population (r = -.46; P < .0001). It would
appear that coliform is a significant contributor to the fermentation of low moisture corn
ensilage stored at low temperatures, whereas yeast significantly contributes to the
fermentation in low moisture ensilage stored at high temperatures. Yeast populations
were positively correlated with soluble nitrogen (r = .66; P < .0001), lactic acid (r =
.57; P < .0001), acetic acid (r = .46; P < .0001) and ethanol (r = .20; P < .084).
Ethanol concentrations are relatively high in these ensilages and yeast is the only
microorganism positively correlated. This observation would suggest yeast is the
originator of the ethanol. Mold organisms were not present in the initial samples (Table
1). Mold growth was not observed in ensilage with a final pH of 4.5 or less. Mold
colonies were identified in HMC stored at low temperatures or in low moisture corn
stored at 20 °C (Figure 13).

Effects of inoculation with various DM contents on pH, lactic acid, acetic acid,
ethanol, soluble nitrogen and yeast are shown in Table 5. Addition of the starter culture
significantly reduced pH with 74 and 78.1% DM ensilage. Populations of LAB on d-67
were increased (P < .05) by inoculation of the lowest moisture ensilage. Above 74 96
DM ensilage, LAB populations were not influenced by moisture level or inoculation.
However, it is possible the strains and species of microorganisms contained in LAB

fraction were altered. Yeast population decreased as DM content increased which

107

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108
follows similar trends observed with LAB.

Lactic acid concentration was increased (P < .05) by inoculation of the higher
DM ensilage but unchanged with the wetter ensilage. Conversely, inoculation reduced
(P < .05) acetate levels with the 74, 66 and 63% DM HMC. This observation supports
the concept that the starter culture did influence the outcome of the fermentation even
though lactic acid levels or LAB enumeration estimates were similar.

Ethanol content was reduced (P < .05) by inoculation of the 63.4% DM ensilage
as compared to control ensilage with similar DM content. Relatively small inconsistent
changes due to inoculation at the different DM levels were seen in the nitrogenous
fractions.

Storage temperature by inoculation interaction was significant (P < .05) for pH,
lactic acid, acetate acid, and ethanol (Table 6). Inoculation reduced pH, ethanol, and
acetate levels but increased lactic acid levels at the 12.5 and 20 °C storage temperatures.
Dry matter recovery was not influenced by the addition of inoculant at the lower
temperature. Consequently, at low ambient temperatures routinely exhibited during
harvest of HMC, microbial starter culture may be ineffective.

Mold growth was positively correlated with DM content (r = .42; P < .0002)
and DMR (r = .28; P < .016). Corn stored at high DM content has been reported to
cause increased mold growth (Fox, 1976). Elevated mold growth in dry corn is often
a result of poor compaction or accumulation of insufficient fermentation endproducts.
The air tight storage structure ued in this study may have prevented oxygen penetration

and thereby, prevented the mold growth seen with the low moisture corn ensilage under

.4

 

 

109

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110

field conditions.

Dry matter recovery after aerobic exposure (DMRE) was greater (P < .0001) in
corn which was stored at 78% DM as compared to 74, 66, or 63% DM (Table 7). The
magnitude of the losses are relatively small ( < 1%), however, more extended exposure
may increase these losses substantially (Rust et al. , 1989). Table 8 depicts the
correlation coefficients between parameters measured during the aerobic stability test.
Positive correlation coefficients were observed between DMRE and DM (r = .48; P <
.0001), mold (r = .40; P < .0005), butyric acid (r = .27; P < .02) and dry matter
recovery during the ensilement phase (r = .22; P < .07). Conversely, a negative
relationship was present between DMRE and coliform (r = -.32; P < .006), yeast (r =
-.47; P , .0001). Bacillus (r = -.30; P < .01), soluble nitrogen (r = -.36; P < .002),
ammonia nitrogen (r = -.37; P < .001), lactic acid (r = -.31; P < .009) and
temperature rise (r = -.24; P < .04). The factors positively correlated with DMRE
were also positively related to DM content. In general, the more extensive the
fermentation during the ensilement period, the less likely an ensilage with aerobic
stability will be produced.

Temperature of the ensilage mass has been used to evaluate the degree of stability
upon air exposure (Rust et al. , 1989). The results from this trial would support that
relationship since temperature rise and DMRE were negatively correlated (r = -.24; P
< .04). However, the correlation coefficient is relatively low which suggests other
factors may be involved. For instance, yeast population is negatively related with DMRE

(r = -47; P < .0001) but is not significantly related to temperature rise (r = .06; P =

111

Table 7. Effects of Dry Matter Content on Dry Matter Recovery During
Aerobic Exposure

Dry Matter Content, %

53.5. 6.6.4. 21.9 18.1
Dry Matter Recovery,% 99.1” 99.4" 99.4” 1006‘

 

‘ Standard error of the mean.

”5 Means with unlike superscripts differ (p < .001).

SEMI
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113

.60). The logical interpretation suggests yeast metabolism results in nutritive loss but does not
contribute to temperature rise.

The amount of time that elapsed before a significant increase ;(P < .056) in temperature
occurred above ambient for the various treatments is shown in Table 9. As storage temperature
increased, temperature rise during aerobic exposure decreased (r = -.64); P < .0001).
Regardless of DM content or inoculation treatment, corn ensilage stored at 20 °C did not exhibit
an increase in temperature. However, the inoculated corn appeared to deteriorate more rapidly
than control corn with the other storage temperatures. With the inoculated corn stored at 12.5
°C, all DM levels within the 48 h period were unstable (P < .05) whereas only the 74 and 78%
DM HMC were unstable at that storage temperature. Temperature stability was similar for all
DM levels within inoculation treatments at the lowest storage temperature. The temperature
rise during the first 48 h was positively associated with coliform (r - .39; P < .0007) and
bacillus (r : .23; P .< .05) bacteria.

The effects of DM and storage temperature on aerobic stability of HMC upon the storage
temperature under which the ensilage was preserved are shown in Table 10. High moisture corn
which contained 74 and 78% DM and stored at 20 °C had a net increase in soluble carbohydrate
(P < .0001) after 48 h of exposure. Fermentation endproducts were greater or unchanged after
exposure for the 55, 74, and 78% DM com. A general trend was evident of bacterial
populations to increase (P < .01) as DM content decreased while trends in fungi populations
appeared more variable. It would appear the bacteria are the principle organisms in the
deterioration process and the role of the fungi is less clear. However, the yeast organisms may

initiate the deterioration process by metabolism of acids and subsequent increase in pH which

l 14
Table 9. Temperature like Above Ambient During Aerobic Exposure

 

 

 

 

 

Inoculation Treatment
Control Inoculant
Storage TemP-. °C 1 12.5 29 1 12.5 2.0
time', 1:
Dry Matter, % 78 >48 48 >48 >48 48 >48
74 36 36 >48 36 36 >48
66 36 >48 >48 24 24 >48
63 36 >48 >48 36 36 >48

Time required for a significant (P > .05) temperature increase above ambient.

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116

allows the bacteria to grow. Mold organisms may become principle organisms beyond
48 h of air exposure.

As would be expected, as endproduct utilization decreased, microbial populations
and temperature rise decreased as well. Storage temperature did not appear to influence
DMR (r = .0008; P < .94) or DMRE (r = .03; P < .77) in this study even though
many of the parameters used to monitor fermentation and deterioration were significantly
altered.

Inoculated HMC containing 63 % DM lost more lactic acid (P < .02) during the
48 h stability test than control corn, however, both control and treated 63 % ‘DM
ensilages lost more lactic acid than the other DM treatments (Table 11). The major
substrate for deterioration in all silages was ethanol. Inoculation of the 63 and 66% DM
HMC had greater temperature rise and ethanol reduction (P < .007) than control silages
of the same DM. Inoculation of low DM HMC appears to lessen the ability of the
resultant ensilage to resist deterioration. Microbial shifts that occurred during the aerobic
stability test were not influenced by inoculation treatments.

The higher the storage temperature, the smaller the temperature rise (Table 12)
and the greater the mold population (P < .007) after 48 h of exposure. With HMC
preserved at the 20 °C temperature, inoculant did increase butyric acid content (P < .07)
as compared to the controls. This coincided with increased mold populations (r = .32;
P < .006).

The magnitude of DM losses from the laboratory silos in this study were less than

5 % which is similar to losses from buried bags in 225 t upright concrete stave silos

117

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1 19
(Wardynski et al. , 1988). Therefore, it would appear that the losses from the laboratory

silos or buried bags represent fermentation losses and the remainder of the 10-15 96 DM
losses from field scale silos represent oxidative losses from the exposed surface and air
penetration. Estimates for aerobic losses in this study are much less (1%) than the 10-
15 96 , however, the length of exposure in field scale silos is undoubtedly longer than 48
h.

Comparison of dry matter and crude protein recovery from ensilement through
48 h of air exposure is shown in Tables 13 and 14, respectively. Main effects of DM
content and inoculation were the only significant variables for DM recovery estimates.
Inoculation significantly increased (P < .001) total recovery, however, another .396
increase in DM loss during aerobic stability would negate this benefit (Table 13).
Overall, crude protein recovery was not influenced by inoculation treatment. Dry matter
and crude protein recovery increased (P < .001) as the DM content increased (Table
14). The consequence of this observation would encourage ensilement of HMC with
22% moisture.

The economic value of inoculation based on the results of this laboratory study
is shown in Table 15. The economic savings from use of an inoculant is 8.3 pounds of
com (72% DM). The savings are .25, .33, .42 and $.50/ton for corn priced at 1.68,
2.24, 2.8 and $3.36/bushel. Currently, corn would need to be valued at $6.75/bushel
to cover the cost of inoculation of $1.00 per ton.

Based on the results of this study producers may benefit from delayed harvest of

corn until the moisture content nears 22% . The ambient temperature during the

120

Table 13. Effect of Inoculation on Recovery of DM and Crude Protein After 67d
of Ensilement and 48 h of Aerobic Exposure.

 

 

DM Crude Protein
Emmi _C_ .1. M _C_ .1. .SEM:
Silo 98.2" 98.7“ .08 99.4“ 99.5“ .02
Exposure Test 99.7 99.5 .01 98.7 98.8 .009
Total 97.9‘ 98.2“ .001 98.1 98.3 .03
' Standard error of the mean.

Means within a row with unlike superscripts differ (P < .001).

Means within a row with unlike superscripts differ (P < .08).

121

Table 14. Effects of Dry Matter Content on Recovery of DM and Crude
Protein After 67d of Emilement and 48 h of Aerobic Exposure.

Dry Matter Content, %

 

53 fi 14 18 SEM:

DM Recovery, %

Silo 97.5” 97.6” 99.0c 99.7‘‘ .12

Exposure Test 99.1" 99.4” 99.4” 100.6° .19

Total 96.6" 97.0” 98.3c 110.3‘ .17
Crude Protein Recovery, %

Silo 99.1b 99.2"c 99.3° 99.9‘I .03

Exposure Test 97.8" 98.8c 98.9‘ 99.6‘| .13

Total 97.0" 98.0c 98.2c 99.5‘ . 13

‘ Standard error of the mean.

5"“ Means within a row with unlike superscripts
differ (P < .05).

122
Table 15. Calculated Savings By The Use of An Inoculant

Corn Price Slbu. (as is)

 

1&8 2.25. 2.3.0 1.3.6
Savings with an inoculant, Slton .25 .33 .42 .50

123
ensilement and fermentation period appears to have little effect on those parameters that

have economic significance.

CONCLUSION

Evaluation of successful storage practices for high moisture corn (HMC) should be
based on return per unit of investment. From the standpoint of microbial inoculants;
nutrient density, nutrient preservation and stability are the critical parameters for
consideration. Average daily gain (ADG), dry matter intake (DMI) and feed efficiency
(FE) are common measures of feed quality or nutrient density. Improvements in these
performance parameters could justify utilization of microbial inoculants for ensilement
of HMC.

Nutrient recovery could potentially justify the use of products such as lactic acid
producing bacteria (LAB) used to aid in the preservation of ensiled feeds. In this study,
dry matter conserved was utilized as the parameter to evaluate preservation.
Improvements in dry matter recovery (DMR) could increase profits simply by reducing
the amount of feed lost during storage. Other measurements such as gross or digestible
energy recovery could also be used, but are more difficult to measure.

Stability after aerobic exposure is a critical parameter to be measured. Silage
additives which improve aerobic stability would allow more flexibility in feeding systems
through extended bunk-life of HMC.

Fermentation characteristics such as chemieal and microbial measurements are useful

in explaining the observed results from feeding and storage studies; however, chemical

124

125

and microbial measurements do not dictate profitability. In this author’s Opinion,
elevated lactic acid content alone does not always prove to be a desirable characteristic.
The final decision of whether or not to use a microbial inoculant should be based on
economic returns and the conditions which these returns are manifest.

In trials one and two from the first experiment, nutritive value was similar between
control and inoculated corn as measured by ADG, DMI and FE. Dry matter recovery
was similar for both treatments in trial one; however, DMR was lower in corn treated
with inoculant as compared to control corn in trial two. Stability after aerobic exposure
was measured in trial two and greater temperature rise and microbial growth were
detected in corn treated with LAB than the control.

Dry matter recovery was evaluated in trials one and two of experiment two though
feeding quality and aerobic stability were not measured. In trial one, lactic acid content
was increased and pH decreased in both ground corn treatments. Corn stored in the
whole form underwent less fermentation and thereby, incurred less DM loss. Inoculation
of corn in both forms increased lactic acid concentration and reduced pH. Unfortunately,
inoculation also reduced DMR. Dry matter recovery was unchanged by the addition of
water, inoculants, non-protein nitrogen or acids.

Experiment three demonstrated benefits of using LAB during anaerobic storage.
Microbial inoculation increased lactic acid content and decreased pH in the dry corn
treatments (78 and 74% DM). Microbial inoculation also improved DMR (P < .001) by
.5 % over control corn after 67 d of fermentation. Corn stored at 78% DM had a greater

DMR than corn stored at 74%, which was greater than corn stored at 66 or 63% DM.

126
Storage temperature did not affect DMR, but corn stored at 20 °C appeared to be more

stable than corn stored at 4 or 12 °C as measured by temperature rise above ambient after
48 h of air exposure. Inoculation of corn stored at 4 or 12 “C during fermentation was
less stable after air exposure than corn stored at higher temperatures. Calculation of DM
recovery from harvest to consumption by the animal indicated a .3% increase in DMR
with inoculation. Corn stored at 78% DM had a greater combined DMR than 74, 66 or
63% DM com. After calculating the economic value of .3% improved DMR with LAB
addition and the cost of applying a commercial microbial inoculant, use of microbial
cultures is marginal.

The recommended moisture content for ensiled corn is 25-30%; however, DMR was
demonstrated to be greater at the lowest (22 %) moisture level studied. In addition, the
dryer corn appeared to be more stable after aerobic exposure.

Small laboratory silos were utilized in experiments two and three. The validity of
using laboratory silos to study fermentation as it occurs in large farm silos has been
questioned. However, because of the lack of replication in large farm silos, small
laboratory silos have been utilized to identify treatments which may enhance fermentation
in larger silos. Experiments two and three could be used to suggest potential treatments
for large silos. Even though the trials did not prove LAB inoculation to be economically
beneficial, further evaluation in large farm seale silos may be warranted. Farm silos are
not as air tight and DMR should be measured from the total silo. Note that DMR, in the
first experiment, was measured from buried dacron bags. In experiment 2 several

additives were evaluated on corn ensiled at 20% moisture with no difference in DMR.

127

Again, different results may occur in larger silos.

Experiment three indicates that com stored with greater dry matter content had
greater DMR. Previous research has indicated DMR ean be greater with drier corn;
however, the risk of heating and earmelizing the corn within the silo is also greater.

Corn with greater dry matter content was more stable after aerobic exposure, while
LAB inoculation adversely affecting stability. Corn stored at 20 °C throughout
fermentation was more stable than corn stored at 4 or 12.5 °C. Storing small laboratory
silos at a constant, controlled temperature can be accomplished easily; however, this
application on large silos is impractical. Future research is needed to evaluate the role
which external or ambient temperature plays on fermentation and subsequent aerobic
stability in farm-scale silos.

In summary, these studies suggest that corn stored with greater dry matter content
allows for large quantities of recoverable HMC which is more stable during oxygen
exposure. Corn inoculated with LAB is less stable than control corn and did not show
economical benefit.

Future research should focus on improving the factors that increase output and
thereby profitability. Previous research has shown that HMC fed to growing-finishing
steers, as opposed to dry corn, has exhibited equal or larger daily gains, improved
digestibility and fwd conversion efficiency while decreasing dry matter consumption.
Corn stored at elevated moisture content has increased acid and soluble nitrogen content,
which can decrease DMI.

Improvements in feed conversion and ADG may be feasible. Preserving corn with

128

greater energy density or less proteolysis may be possible. Soluble and ammonia
nitrogen have been shown to increase in HMC as moisture content increases. Elevated
soluble nitrogen content has been shown to decrease intake of high moisture feeds.
Therefore, strategies reduce nitrogen solubilization and proteolysis may enhance nutrient
preservation and dry matter intake.

Experiment three and other trials have demonstrated that DMR ean be maximized
with lower moisture content by limiting the fermentation. As the fermentation process
continues, more carbon is potentially lost as earbon dioxide. Consequently, the results
of these trials would indicate that DMR could be maximized by limiting fermentation.
However, many of the trials have been conducted using air tight laboratory silos and
results from large scale silos have shown problems with preservation at moisture contents
below 25 % moisture. The key is to control fermentation to ensure preservation without
excessive DM loss. Research in monitoring and controlling fermentation in this manner
would be useful.

Aerobic stability is an area which needs more research. High moisture corn can
deteriorate rapidly after oxygen exposure. Differences in deterioration rate has been
shown between corn samples collected for evaluation. The explanation for these
differences is not fully known. Links have been made between stability and the chernieal
constituents of the corn. High moisture corn which has undergone heating in the silo and
contains elevated levels of butyric acid and appear ammonia have appeared to be quite
stable. Corn samples which contain high acetic acid content have been associated with

improved stability. Microbial characteristics post-ensiling were not identified in these

129

experiments and requires more testing to elucidate the characteristics associated with
deterioration. The key is to produce high moisture corn with extended stability without

sacrificing nutritive value or conservation efficiency.

APPENDIX

130
Model development to predict chemical, microbiological and dry matter recovery of

ensiled and deteriorating high moisture corn.

The data from Experiment three was utilized to form the following regression tables.
Corn was harvested at four dry matter contents (63.4, 66.4, 74.0 or 78.0% DM). At
each moisture content, corn was rolled, inoculated with either water or a lactic acid
producing microbial inoculant and 3 kg placed into polyvinyl-chloride silos (10.2 cm dia.
x 45.7 cm). Each moisture by inoculation treatment combination was stored at 4, 12.5
or 20 °C. For each treatment combination, triplicate silos were evacuated 3, 7 or 67 d
post-ensiling. Subsamples were collected for chemieal and microbial analysis.

Tables one and two show regression coefficients, standard error of the estimate and
probability for the fermentation characteristics. The tables were generated using the
General Linear Model Procedure for Regression (SAS Institute, 1987). The variables
used in the regression were dry matter (DM), day of evacuation (D), storage temperature
(ST), inoculation (I; 0 represented control, 1 represented inoculation), DM, D’, ST’,
DM*D, DM*ST, DM*I, D*ST, D*I and ST'I. Chemical and microbial characteristics
were expressed as the percent of dry matter and the log of colony forming units per gram
of wet corn, respectively.

Tables three and four represent the regression coefficients, standard error of the
estimate and probability of the change in chemical and microbial characteristics after
aerobic exposure. After sub-sampling, corn evacuated from silos 67 d post-ensiling,

were placed in plastic-lined styrofoam containers to evaluate stability during oxygen

131

exposure. Samples collected from silos 67 d post-ensiling also represent time zero for
aerobic stability. Corn was removed from the containers after 48 h of aerobic exposure
and subsamples collected for chemical and microbial analysis.

Tables three and four were generated using the same procedure as tables one and
two; however, the variable of evacuation day did not apply in the aerobic stability model.
The variables used in the regression model were DM, ST, 1, DM“, ST’, DM*ST, DM‘I,
and ST*I. The input values for statistieal analysis for the aerobic stability regression
tables were ealculated by subtracting the 48 h values from time zero values. Chemical
and microbial characteristics were expressed as a percent of dry matter and the log of
colony forming units per gram of wet corn, respectively. Temperature values were

calculated by subtracting 48 h temperature from the average ambient temperature.

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