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thesis entitled

Prevention of Mold on High Moisture Hay
With EmphaSis on the Fatty Acids as Fung—
icidal Agents

presented by

Bernard D. Lewis

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PREVENTION OF MOLD ON HIGH MOISTURE HAY
WITH EMPHASIS ON THE FATTY LCIDS AS FUNGICIDflL AGENTS

by
Bernard.David Lewis

A.THESIS

Submitted to the School of Graduate Studies of Michigan State
College of Agriculture and Applied Science in partial
fulfillment of the requirements for the degree of

iASTER OF SCIENCE

Department of Bacteriology and Public Health

1951

69/:i7‘//5/
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TABLE OF CONTENTS

Acknowledgements
List of Tables

List of Graphs

I. Introduction . . . . . . . . . . . . . .
II. Historical Review

A, Methods of Preservation . . . .
B, Fungi Involved in Bay . . . . . .

III, Experimental Procedure

A. Isolation and Identification
of Fungi . . . . . . . . . . . .
B, A Study of Mold Inhibiting.Agents

IV. Conclusion . . . . . . . . . . . . . . .
V. Literature Cited . . . . , . . , . . , .

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LIsr OF TABLES

Table I
The ten commonest fungi isolated from
hay in Lansing, Michigan area , , , , , . , , . . , .

Table II
Fungi isolated from hay as compared with
genera reported present in soil . . . . . . . . . . .

Table III
Physical properties of organic acids . . . . . . . .

Table IV
Inhibition of monobasic and dibasic
aCidS on mgi O O O O O O O O O O O O O O O O O O 0

Table V
Variation in hydrOgen ion concentration
of tryptose glucose agar after addition
of organic acids . . . . . . . . . . . . . . . . . .

Table VI
Commercial fungicides tested on high
mOiSture hay O O O O O O O O O O I O O O O O O O O C

Table VII
Concentrations of acids and days requiring
mold development on hay . . . . . . . . . . . . . .

IO

17

21

26

31

33

LIST OF GRAPHS

Graph I

Concentrations of prOpionic acid mycostatic

to several fungi . . . . . . . . . . . . . . . . . 24
Graph II

Relationship of days of incubation to percent
concentration of acid . . . . . . . . . . . . . . 35

A KNO‘JLED GEE-Elm S

The author wishes to express his sincere thanks to
Dr. Walter L, Mallmann, under whose constant supervision, per-
sonal guidance, and unfailing interest this investigation was

undertaken.

Grateful acknowledgement is also due to Mr. Elbert
S, Churchill for his helpful suggestions and assistance in one

way or another.

The investigator also extends his sincere thanks to
Mr. Robert Kleis and other members of the Agricultural Engineer-
ing Department at Michigan State College for their assistance and

the supply of materials used in this study.

The writer appreciates the financial support of the J.
I. Case Company for the fellowship which they provided for this

study.

INTRODUCTION

The methods used today in the curing of hay result in
considerable loss of nutritive value while the hay is being reduced
in moisture content to a point which will prevent heating and molding
during storage,

Several qualifications must be met in the production of
quality hay. These qualifications are: (a) purity of the hay, (b)
retention of a high percentage of leaves, (c) pliability of stems,
(d) color, (e) absence of moisture and moldiness.

In the late eighteen hundreds, it was found that 25 to
50 percent of the dry matter in hay would be lost by exposing the hay
to rain. This represents a considerable loss of the most digestible
portions of hay; When.weather conditions are unfavorable during the
haying season, the longer the hay is exposed the greater the loss
encountered. Besides loss due to weather conditions, the present
methods of moisture reduction cause the fragile leaves to become dry
and they are easily lost. This results in a.heavy loss of protein.
For these reasons, a way for improving the curing process has long
been sought.

The specific purpose of this study was to explore the
possibilities of using chemical agents to inhibit the deveIOpment of
microorganisms responsible for the spoilage of most baled hay, and

the possible application of the agents in the preservation of baled

.2-

hay with moisture contents in excess of 20 percent hy'weight. Par-

ticular attention is paid to possible use of the short chain fatty

acids. This is an entirely new approach to the prevention of mold

deveIOpment in hay.

HISTORICAL REVIEW

A. Methods of Presegyation

Most of the literature dealing with the prevention of
mold development in stored hay attacks the problem.from.a physical
point of view. Most workers have used methods such as drying and
heating, while little experimentation seems to have been carried out
with chemical treatment.

It has long been recognized that high relative humidity
and moisture content are the primary factors in the control of mold
growth in stored products. McHargue (17) found that molds were able
to develop on corn.having 15 percent moisture, if no ventilation was
provided. Thom.and LaFevre (29) gave a critical value of 13 percent
moisture for mold growth on corn. Wright (36) stated that "the rela—
tive humidity, rather than the moisture content of the material, is
the primary factor affecting mold growth". He found that samples of
artificially dried grass, stored at 13 percent moisture content,
could be held for periods greater than one year without mold‘develop-
ment.

Based on moisture contents, methods have been develOped
to prevent molding by reducing the moisture. For centuries, hay has
been field dried before being placed in storage. Methods of speeding

the drying process, such as the use of forced air, have been employed

in the hay mow.1

Heat, as a drying agent, has been employed to produce
quality hay. Hendrix (8) pointed to the fact that the heat due to
respiration and bacterial action in hey can drive off considerable
amounts of moisture. Terry (26) suggested that low temperature
(cold storage) could be tried to prevent spoilage in hay. He also
indicated that a hay pasteurizer, having a temperature above 125° F.
might be used. The latter idea actually has been applied by Cheveley
(3) in the use of hot air dryers.

The use of chemical methods to prevent molding in hay
has not been widely explored. Henry (9) stated, "salt and lime
scattered over damp hay (in the mow) tends to prevent fermentation
and checks the growth of molds". In storage problems, similar to the
one afforded by hay, chemical means of preservation.have been used.
Rigler and Greathouse (22) used the fatty acids in the prevention of
root rots. Humphrey and Fleming (12) listed several oils and salts
which have been used to preserve wood against fungi. ‘Mallmann and
Michael (18) showed the value of chlorophenols, and related compounds,
in preventing mold development in paper. Horsefall (11) described

the action and uses of most of the modern fungicides.

 

l. The workers here are too numerous to mention. Some references
are Journal Agricultural Engineering 1947, 28: 141-144, 257-258,
289-290, 294.

Shepherd et al. (25) used sulfur dioxide and other
gases for the preservation of forage products. Similar work was
carried out previously by Klotz (14) using nitrogen trichloride,

and other gases, as fungicides in the storage of fruit.

B. Fungi Involved in Hay

Examination of the literature on hay and other grasses
revealed no complete studies of fungi which cause moldiness on hay.

Thom and Raper (30) discuss members of the aspergilli
that would appear in this problem. Almost all the aspergilli are well
adapted for growth on a medium.such as moist hay. Their frequency in
stored hay led Cohn (A) to believe that they were the cause of "heating"
in damp hay.

Thom (28) reports the presence of penicillia in almost
every type of substratum. Many Studies (17) of molds as a cause of
deterioration in corn have shown members of the fienigilligm group as
the destructive agents. Ruschman (23) found that the destruction of
the flax fibers was due to penicillia. Thom.identified Penicillium
rggugiozti and its related strains as the characteristic molds of
silage.

Hay contains an abundance of carbohydrate material. A
great deal of work has been done on the decomposition of carbohydrates
by fungi, including celluloses, hemicellulose and pectins. ‘Ward (32)
studied the pathogenicity of penicillia as wood-destroying fungi.'Dox

and Neidig (6) showed that several of the aspergilli and penicillia

were able to destroy the pentosans found in large quantities in
soil and manure.

Many species of soil inhabiting molds are capable
of transferring cellulose and lignin portions of plant material.
A comprehensive idea of these activities, and of their importance

in the economy of nature, is summarized by Thaysen and Bunker (27).

EXPERIMENTAL PROCEDURE

A. Isolation and Identification of Fungi on Hay
1. Laboratory Procedure (Hay Fungi)

Isolation and identification of the molds on hay were
carried out so that an over-all picture could be formed as to the
types of fungi involved in this problem.

The bales of hay used for the isolation were obtained
from farms in the vicinity of Lansing, Michigan. The bales, from
which isolations were made, all showed visible moldiness. This hay
was the first cutting of the spring.

The isolations of the molds were made by susmnding
small samples of the bale in sterile water. Various dilutions of
these samples were plated in potato dextrose agar, pH 4.5, or Saba-
rouds maltose agar, pH 4. 5. The plates were incubated for four days
to two weeks at room temperature. In this way adequate time was
allowed for the development of slow growing species. .

- The penicillia isolated in this study were identified
by the aid of Thom's "The Penic_il_lia" (28). The aspergiZLli were
identified from descriptions in Thom and Raper's "Manual of the Asper-
gflli" (30). The rest of the cultures were identified only as to

genera, by use of the key in Gilman's "Soy, Fungi" (7).

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2. Results and Discussion

In Table I are presented the ten commonest molds iso-
lated from.30 bales of hay. These molds represent over 300 isola—
tions. The most frequently isolated fungi were found to be members
of three groups: Muggg, ggpgrgillgg and Penicillium. An attempt was
made to differentiate between the fungus flora of high moisture hay
and hay having a low moisture content. The number of samples was not
sufficient to show any definite trend. However, the results obtained
indicate that the same molds were predominant in.both types of samples.

Thom.and Raper (30) point out that the Agpgggillgglglgg-
ggg group is most conspicuous upon concentrated material, such as drya
ing plant products. The Agpgrgillgg nigg; group, which is probably the
most common of the aspergilli, is a.predominant cause of mildew. Agpgg-
gillug fumigatgs is an extremely cosmopolitan mold and occurs with
particular frequency on vegetable material, undergoing slow decomposition,
and upon imperfectly stored grains.

The presence of the penicillia is to be expected. Cereal
grains and their products are a.favorable host material for this group
of organisms. Their presence is directly connected with the presence of
moisture. It has been found that penicillia are unable to grow at the
low percentage of moisture at which members of the A. glaggus group are
still active.

Mucors are found in greater abundance in the soil than
any other group of soil fungi. Compared with other fungi, isolated in

this study, mucors were found to grow most rapidly on the media used.

-10..

TABLE II

FUNGI ISOLATED FROM HAY AS COE-fiPARED WITH
GEE-ERA REPORTED PRESEI’JT IN SOIL*

 

 

 

Hay Fungi Soil Fungi
l. Mucor 1. Absidia
2. Rhizopus 2. Mucor
3. Aspergillus 3. Rhizopus
4. Rhodatorula and 4. Zygorhynchus
other yeasts 5. Saccharomyces
5. Penicillium. 6. Chaetomium
6. Scopulariopsis 7. Monilia
7. Cephalosporium. 8. Oidium
8. Trichoderma 9. Sporotrichum.
9. Alternaria lO. Botrytis
10. Cladosporium. ll. Aspergillus
11. Sporotrichum_ 12. Penicillium.
l2. Verticillium. 13. Scopulariopsis
13. Fusarium. l4. Verticillium
l4. Helminthosporium 15. Cephalosporium
l5. Chaetomium. 16. Trichoderma
16. Oidium. l7. Acrostalagmus
l7. Sterile white 18. Zygodesmus
mycelium l9. Dicoccum
18. Pullularia 20. Cephalothecium
21. Basisporium
22. D ematium
23. Acremoniella
24. Cladosporium.
25. Alternaria
26. Fusarium
27. Melanconium
28. Coniothyrium
29

. Sclerotium
. Sterile red and white
mycelium

3O

 

* The soil genera are from a list compiled by Waksman, S. A.,
Soil Fungi and Their Activities. Soil Science. 2:139.
1916.

.11..

Their extreme proliferation proved to be a handicap, in that they
masked slower growing organisms.

Workers in the field of soil fungi have found results
similar to those in this paper. Waksman (33) lists the most widely
spread groups of soil fungi as the M3992, Aspergillgs, Penigillium,
Trichoderma and Rhizopgs. Table II shows the main groups of fungi
found in hay as compared to the main groups found in soil by Waksman
(33). All genera isolated from.hay are seemingly represented in soil.
In both cases, the predominant groups are the mucors, the aspergilli
and the penicillia.

Werkenthin (34) concluded that there is a rather constant
and characteristic fungus flora in the soil. Aspergilli seemed to be
the predominant fungi in southern soils, while penicillia and mucors
are found more extensively in northern soils, and occur only occasion-
ally in southern soils. Jensen (13) showed four typical groups present
_ in the soil of Denmark. These are Egggg, Eggigilligm, Aspergillgg, and
Tgighgdgzma. In addition, he found Fusarium.and Alternaria present
frequently in field soils. Other fungi found occasionally in field soils
are Hormodendrgm, gephalgspgrigm.and Botrytis.

The reports, of the above mentioned workers, show that a
parallel may be drawn between the groups found predominantly in soil
and those present in.hay, Table II. It is reasonable to assume from
these data that the types of fungi causing mold on hay are the predomp

inating fungi in the soil upon which the hay is growing.

3. Laboratory Procedure (Soil Fungi)

Isolation of soil fungi was attemped to establish
whether there actually is a correlation between the fungus flora
found in hay and that found in the soil. For these tests, soil
samples were collected from the fields in which the hay, previously
used in isolation, had been grown. The soil samples were collected
in sterile flasks, under aseptic conditions, and were brought immedi-
ately to the laboratory. The soil samples were then placed in sterile
water blanks and plated by the dilution plate method. Sabarouds dexp
trose agar, pH 4.5, was used for plating. All plates were incubated
at room temperature and held for three weeks observation. The result—
ing colonies were then picked and purified by the dilution plate
method. 7

Thirty soil samples were collected and tested. It was
not anticipated that the results would include all genera or Species
that were present in the soil from which the samples had been taken.
However, it was hoped that these random.samples would indicate the

most common fungi to be found in these particular soils.
4. Results and Discussion

The results obtained showed three main groups to be
present in an overwhelming majority. As in the case of hay, these
three groups proved to be the mucors, penicillia and aspergilli. 0f
the thirty samples investigated, thirty contained aSpergilli (thirt-

een of which were ispergillus niger), and ten contained penicillia.

-13-

The result of this study indicates, within certain
limitations, that three predominant types of fungi grow in soil
and in hay. The predominance of these three main groups does not
rule out the implication of other groups as causative agents in
the molding of bay. The area of sampling in this experiment was
too limited to make a blanket statement to the effect that the
elimination of any specific groups would solve the entire mold
problem in hay. However, the results of the data compiled show a
more or less constant flora is present in hay and in the soil upon
which members of this flora are growing. The large variety of
fungi found points to the need of controlling all mold growth on

hay, if the problem is to be solved.

B. A Study of Mold—inhibiting Agents

Inherent characteristics in mold-inhibiting materials
made most compounds unsuitable for use in this problem. In selecting
the material, the main facts to be taken into consideration were: (I)
toxicity; (2) solubility; (3) methods of application; (4) cost of
material to the farmer.

The inhibiting agent had to be toxic to fungi, yet be
totally non-toxic to animals. This fact ruled out a great many of
the fungicides known today, especially such as the heavy metals, cOp-
per, mercury, arsenic, etc. Because of this fact, attention was
turned to the use of the fatty acids which are naturally occurring in
many substances and have been used as mycostatic preservatives for a
long time.

Other reasons for experimentation with the fatty acids
can also be enumerated. They are relatively soluble and, therefore,
could be easily applied in a solution. Their cost is comparatively
low. This is an essential factor for so extensive a problem as the
one encountered in the protection of the hay. The fatty acids are also

easily produced on a commercial basis and thus readily obtained.
1. A Discussion of the Fatty Acids

The disinfectant value of strongly dissociated mineral
acids, such as sulfuric and hydrochloric, depends upon the number of

free hydrogen ions present. The weak organic acids, however, exert a

-15-

more toxic effect than.would be indicated by the degree of dissocia—
tion.

In determining the mode of action of the fatty acids as
disinfecting agents, various theories have been presented. Many work-
ers have attributed the action to a particular entity on the basis of
their experimental work. However, the effect of an acid seems to be a
collective one in which several attributing factors must be taken into
consideration.

The factors which must be considered.when discussing the
effect of organic acids upon microorganisms can be enumerated under the
following headings:

1. The hydrogen ion concentration.

2. The characteristic anion.

3. The undissociated molecule.

4. The surface tension.

5. The molecular weight and size of the molecule.

6. Specificity of certain acids for certain micro-

organisms.

Acetic acid in vinegar is one of the most ancient preserva.
tives. Wolf and Shunk (35) found that it was more toxic to the six spe-
cies of bacteria investigated than.was hydrochloric, sulfuric, citric,
tartaric, malic, or formic acids. In the experiments of Bitting (l),
acetic acid was as toxic to fungi as benzoic, boric, or salicylic acids.

Itkilledfianisillmamsaaddiamaziasalmi, anindiaalasiis
under conditions in which citric, lactic, malic or tartaric acids only

-16-

slightly retarded their growth.

The toxicity of acetic acid to fungi has been discussed
by many workers. Doran.(5) indicates that the toxicity to fungi by
acetic acid is due largely to the undissociated molecule and partially
to the hydrogen concentration. Kahlenberg and True (15) found that
undissociated acetic acid is toxic to molds. According to True (31),
the 02H302 ion is not toxic. However, Wolf and Shunk (35) found that
the limit for the growth of Bagterium tabaggm was pH 5.9, when the
medium was adjusted with acetic acid, and pH 4.6 when the medium was
adjusted with malic acid. They concluded that the hydrogen ion alone
is not responsible for the toxicity of acetic acid.

Kahlenberg and True (15) found that formic acid was the
most toxic to fungi of the fatty acids, being strongly dissociated.
Prepionic, butyric and valeric acid exhibited a similar inhibitory act-
ivity and were slightly more active than acetic, although the latter is
more strongly dissociated.

The relationship of molecular structure to mycostatic
power of the fatty acids was demonstrated by Hoffman, Schweitzer and
Gaston (10). By using substituted acids, they showed that, by altering
the position of the substituted group, the fungicidal power would be
altered. Monochloroacetic, alpha-chloropropionic and di-chloropropionic
acids showed less disinfecting power than normal propionic acid. The
introduction of chlorine on the alpha carbon decreased the mycostatic
power of the acid with the same effect on mono-chloroacetic acid. A
chlorine atom.on the carbon next to the carboxyl group affects the myco-

static properties of the acid, whereas the same substituent on the beta

-17-

 

 

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-18-

carbon atom has no effect.

Langmuir (16) pointed out that halogen substitution
had an appreciable effect on the dissociation constant of a fatty
acid. The theory is rather widely held that the biological activity
of an acid is dependent on the dissociation constant; that is, the
less ionized the acid, the more powerful its biological effect. The
result of Langmuir's work showed that the relative mycostatic power
of acetic, monochloroacetic, propionic, alpha-chloropropionic acids,
when compared with their respective dissociation constants, are in

agreement with this theory.

Bruenn (2), by adding varying amounts of a salt contain—
ing a common anion to acetic and lactic acid, determined that a de-
crease in the number of free hydrogen ions resulted in a decrease of
disinfectant power. He concluded that the disinfectant power of the
organic acids depends upon the hydrogen ion concentration only. Norton
and Hsu (19) agree that the disinfectant action of acids is due to
their hydrogen ion. Decreasing the number of free hydrogen ions in
formic acid, and increasing the undissociated molecule, by the addit-
ion of a common ion, decreases the toxic action of the acid for bac-
teria. These workers increased the number of free anions without
changing the hydrogen ion concentration or the number of undissociated
molecules. They determined that the acid anions act upon the hydrogen
ions as positive catalyzers, increasing their germicidal powers.

Paul, Birstein and Reusz (20) reported that the organic
acids, acetic and butyric, were more toxic for bacteria than hydro-

chloric, when equal isohydric solutions were considered. This they

-19-

explained on the basis of the additive toxic effect which the anion
and undissociated molecule might exert.

Reid (21) indicated that there is a relationship be-
tween the surface tension of the acids and their disinfecting powers.
He demonstrated that the lowering of surface tension is inversely
proportional to the length of the carbon chain. Therefore, as the
antiseptic properties of an acid increases, the surface tension of
the acid decreases.

The dibasic acids, oxalic, malonic and succinic, decrease
in toxicity to fUngi as the series is ascended. Pans, mentioned by
Reid (21), noted that, with the exception of oxalic and malonic, all
dibasic acids were less active against Escherichia 99;; and Salmonella
typhoga than were the normal fatty acids. He concluded that there was
little relationship between the hydrogen ion concentration and growth,
but that the kind of acid, as well as the acidity, was responsible for
their germicidal action.

All the literature discussed in the preceeding paragraphs
emphasizes the fact that the exact mode of action of the fatty acids is
undecided. Too many of these workers have disregarded the fact that
the mode of action of these acids is a combination of the various fact-

ors presented, rather than the result of a single factor.

2. Laboratory Experiments on the Effect

of Some Fatty Acids on Fungi

The fungi selected for these tests were obtained from

'isolations previously mentioned. They were chosen as examples of the

-20-

predominant groups which.were found by the author, in hay from the
Lansing, Michigan area.

An agar plate method was used for testing the effect
of the various acids on the organisms. In all experiments carried
out, tryptose glucose extract agar (T.G.E.) was used. The pH of the
media, before the addition of the acids, was 6.0-6.2. The acids
were diluted in sterile water, using the medium as the last diluent.
The final volume of all plates was ten ml.

Suspensions of conidia of the test fungi were made by
washing the cultures with sterile water. One—tenth ml. of the suspen-
sions was then transferred, by pipette, to the plate containing acid
concentration ranging from 0.1-1.0 percent. The plates were incubated
at room temperature, and results were recorded every day for three
weeks (Table IV). Plates on which well developed colonies were ob-
served were recorded as positiveged Where only a slight growth or dwarf
colonies could be observed with the naked eye, pluseminus (X) was
indicated. Plates showing no growth at the end of three weeks were

considered to be negative (-).
3. Results and Discussion

The data in Table IV show that as the carbon chain of the
monobasic acids was lengthened, there was an increase in the mold in—
hibiting effect. 'There is one noticeable exception in the results for
‘valeric acid. The inability of this acid to show increased inhibition
to fungi at higher concentrations can be explained by its insolubility

in water. Table IV also illustrates the specificities that the acids

-2l-

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-22-

have for certain fungi. The data in this table show that an acid
concentration greater than 0.5 percent would be required to assure
inhibition of the most common molds.

In discussing the results recorded in.Table IV, it is
interesting to note that at the end of the three week observation
period, the (5) colonies were no larger than they'were when growth
was first observed at four to six days. Upon transplanting portions
of these colonies into nutrient broth or on sabarouds agar, there was
observed a natural abundant growth in three days. All controls in the
above experiments were positive in 72 to 96 hours.

When valeric, butyric, propionic and acetic acids were
buffered (using a KHgPQQand NaOH system) to a pH of 3.5, the inhibit-
ing concentrations of the acids were almost the same as in those of a
nonébuffered solution (Table IV). This indicates that in the monobasic
acids, the undissociated molecule is more important than the hydrogen
ion as a cause of toxicity towards the fungi tested here. These data
show a relationship between inhibiting power and surface tension. Table
III indicates that the surface tension of the acids decreases as the
carbon chain becomes longer. If two acids are compared and one is more
active as a surface tension depressant than the other, the one which in-
duces the lower surface tension will be present in the greater concentra—
tion at the interface between the fungus and the medium and therefore will
have the greater effect.

Graph I shows the necessary pH and concentrations of prop-

ionic acid which.were mycostatic to Aspergillqg giggg, Penicillium. re-

.23..

guetans and Rhizopus niggigang. These results were obtained by buffer-
ing the acid to various pH levels and determining the molar concentra-
tion required to inhibit the fungi at that pH. The points on the graph
represent the lowest concentration of acid which inhibited at the part-
icular pH. These results are similar to those of other workers.1

When an acid was added to the medium.for testing its fung-
icidal properties, a change in the pH of the medium occurred as recorded
in Table V. All pH readings were measured by’a Beckman line pH meter.
An examination of the data presented in.Tables IV and V clearly indicated
that inhibition of the fungi, by fatty acids, varies inversely with the
dissociation constants of the acids. In contrast, it should be noted
that the dibasic show the opposite results. This could be explained by
the extremely low pH of these acids, making their action more like that
of the mineral acids, or by the theory that branched carbon chains will
not have the inhibiting properties of straight carbon chains.

The sodium.and calcium.salts of propionic acid have had
use in industry as mycostatic agents.2 Using the plate method pre—
viously'mentioned, results were obtained which showed that the pH of a
medium.must be well below 4.0 befOre any inhibition could be detected.
The organisms used in this test were: Aspergillus niggr, Penicillium

freguetans and Ehizgpgs nigzigans. It would appear from the results

 

l. Hoffman, C., Schweitzer, T. R., Gaston, D. Fungistatic
properties of the fatty acids and possible biochemical

significance. Food Res. 6:539 1939

2. Macy. H., J. C. Olson. Prel nary observations on the treatment
of parchment paper with sodium or calcium propionate. Jour. Dairy
Sci. 22:527. 1939.

Molar Concentration

.10
.09
.08
.07
.06
.05
.01;
.03
.02

.01

 

 

2 3 h S 6 7

Hydrogen ion Concentration

Concentrations of propionic acid mycostatic to Aspergillus niger,
Penicillium frequetans and Rhizopus nigricans.

 

 

 

.25-

obtained, that the propionic ion was the inhibiting factor, and the
presence of the calcium.and sodium ions was insignificant. It can
be seen that nothing would be gained by using a salt, at a low pH, to
obtain results that would be more efficiently produced by using the
corresponding acid.

After observing the effect of the fatty acids on fungi,
it was decided to apply these acids to the problem of moldiness of
hay. From.the laboratory results, there was definite indications that
the acids might inhibit mold formation during the storage period in
the curing of hay.

For ease of application, an aqueous solution of the acids
was desirable. This somewhat limited the acids which would be used.
As was previously pointed out, increasing the length of the carbon
chains increased the inhibiting powers of these acids. However, the
problem.af solubility limited the length of the carbon chains which
could be used. Acids having carbon chains longer than.valeric are too
insoluble in water to give concentrations which would act as inhibiting
agents. For this reason, only the acids from.acetic through valeric

were used for treating hay.

-26-

 

 

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4. Laboratory Experiments Using Fatty

Acids as Mold-inhibitors on Hay

The hay used for these experiments was obtained from
various fields in the vicinty'of Lansing, Michigan. The hay was mowed
and field dried in windrows, until a.moisture content, ranging from.30
to 40 percent, was reached. When the proper moisture content was
reached, the hay was divided into 300 gram portions. Both chopped hay
pend regular hay were used.

The test solution was applied to the hay by spraying with
a paint type, spray gun, using air pressure of 30 pounds. The spray
gun was held approximately two feet from.the hay. Known volumes and
concentrations of the test materials were used. The hay was then placed
in two quart, wide mouth jars. It was found that 300 grams of hay (35
percent moisture) would solidly fill one of these Jars, hence simulat-
ing as closely as possible the condition of a.bale. Some jars were
covered with paper, allowing an air passage, while others were covered
with glass lids. The jars were incubated, both at room.temperature and
at 37° C. Daily checks were made for the appearance of visible mold or
deterioration of the hay.

a. Control test 1. Hay samples, ranging from.35

to 40 percent moisture, were placed in jars and in-

cubated at 37° 0., while other samples were placed

in jars and allowed to stand at room temperature.

None of this hay was treated in any way. The results

—28—

obtained showed that hay with 35 percent moisture
possessed visible mold at the end of 48 hours in-
cubation. The jars covered with paper showed the
same amount of mold as did the jars covered with

a glass cap. Therefore, it can be assumed that

the passage of air had little effect on the devel-
Opment of mold in this test. Within one week, all
jars were completely filled with mycelia and beads
of moisture, due to respiratory activity of both
mold and hay. A putrid odor was also observed.

b. Control test 2. Tests were made in which the
moisture content of the hay'was reduced below 30
percent. It was found that hay containing less than
20 percent moisture would occasionally produce visible
mold spoilage. Below 15 percent, all samples showed

negative mold development.

The results of these control tests are similar to work re-

ported by Terry (26), who found that hay in which the moisture content

was reduced below 20 percent, in not more than three—and-oneéhalf days,

was free of mold. Terry also stated, "When the temperature is eighty to

ninety degrees Fahrenheit, the drying process must be completed in less

than two days, if mold is to be avoided".

Some known commercial fungicides were tested by the method

described above. These compounds have been used in.mold problems simp

.29..

ilar to that encountered in.hay. The compounds used were: D.H.A.S.l,
Thiourea, and a commercial compound, V.H.C.92, specifically sold for
the prevention of mold on high moisture hay. Aqueous solutions of
D.H.A.S. and Thiourea, ranging from.one-tenth to one percent, were
sprayed on 300 grams of hay. V.H.C.9 was applied in powder form to
another 300 grams of hay, according to the manufacturer's instruction.
After treating the hay with these chemicals, all samples were placed
in jars and incubated in a manner similar to that of the previously
mentioned controls.

The results of the above experiment are presented in
Table VI. These data show that the concentrations of the test mater-
ials were too low to be effective in the prevention of mold development
on hay; Since Thiourea and.D.H.A.S. produce a toxic effect in animals,
when used in higher concentrations, and since these concentrations
would be very costly to use in large quantities, it seemed inadvisable
to attempt any further work with these compounds.

Table VI shows that V.H.C.9 was ineffective in the con-
centrations used. In these tests, as much as 50 grams of compound was
applied to 300 grams of hay. These quantities were far in excess of
those deemed necessary, by the manufacturer, to prevent mold develop-

ment.

 

1. Dow Chemical Company, Midland, Michigan.
2. VHC9 H. D. Campbell 00., Rochelle, Illinois - Advertising
pamphlet - Hudson Chemical Co. New Crop Preserving VHC9.

.30-

In succeeding experiments, using the spray method on 300
grams samples of hay, the effect of the fatty acids on hay fungi was
tested. Hay with 35 to 40 percent moisture by weight, was used in all
experiments. The data in Table VII show that a definite inhibiting
force is present in these acids which indicates that they could be used
in this particular prOblem. Butyric acid would be the best acid because
of its inhibiting properties, according to these results, since necess-
ary concentration of this acid, in aqueous solution, would be lower than
any of the other acids. However, the odor of both butyric and valeric
acid would be a.detriment which would eliminate any wide use by farmers
in practical application. Propionic acid, in concentration over two
percent, showed results equal to those of the other acids. The odor of
this acid would not minimze its use; in fact, four days after spraying
there was a lack of any residual odor on the hay.

In comparing the data of Table IV with those of Table VII,
there is a direct correlation between the inhibiting qualities of the
acids on test organisms in plate media, and on the organisms in their
normal environmental conditions. The only deviation was in the effect
of valeric acid upon the fungi in the bay. The inability of valeric
acid to inhibit fungi at higher concentrations can be explained by its
lower solubility in aqueous solutions; this prevented an even distribu-
tion of the acid on the hay with the spray method of application.

In making up the solution to be sprayed, it was found
that 150 to 200 ml. of each solution was needed to give satisfactory

results on 300 to 400 grams of hay. The amount did not vary with the

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concentrations, indicating that there must be a total coverage of the
hay surface to prevent molding.

Graph 2 shows the relationship of percent of concentra-
tion of the acids to the length of time in which visible moldiness
would be observed. As the length of the carbon chain of the fatty acids
increased, the concentration required for the inhibition of mycelial
development decreased. The one exception, as was previously pointed
out, was valeric acid; only the lowest concentration of this acid acted
in accordance with the carbon chain theory. This, again, due to the
insolubility of the acid.

The addition of aqueous solutions raised the moisture
content of the hay up to 70 to 80 percent by weight. The addition of
great amounts of moisture would rule out the practicability of this
operation in the field. However, if hay with a moisture content of 70
to 80 percent can be prevented from.molding, then.by some other method
of application, such as vaporization, the acid could be applied without
adding to the moisture content of the hay.

At the end of three months, the moisture content of the
hay in the glass jars was still approximately 35 percent. At this time,
the hay began to develop visible mycelial patches. 'When this was ob—
served, the total contents would become moldy within three to four days.
This indicated that the acids were not fungicidal, but rather, myco-
static in nature. Under natural conditions, it is probable that a bale
of hay would dry much faster. The static period would be long enough so

that, if the acid were applied, and the hay allowed‘to dry to a moisture

.33..

TABLE VII

CONCENTRATIONS 0F ACIDS AND DAXS RE3UIRING
MOLD DEVELOPMENT ON HAY

 

 

0.5% 1% 2% 3% 4% 5% Control

 

3 days
Acetic x x x x x g x
Propionic § § - - - - x
Butyric - - - — — - x
Valerie - - - - - - x
6 days
Acetic x x x x x x x
Propionic x x E - - - x
Butyric 5 - _ - - - x
Valeric E - — - - - x
{f
9 days
Acetic x x x x x x x
Propionic x x x - - - x
Butyric x — — — - - x
Valerie x x x f - - x
15 days
Acetic x x x x x x x
Propionic x x x - - - x
Butyric E - - - _ x
Valerie x x x E - -

 

x . . . Heavy mycelial development
g . . . Scattered mycelial development

- . . . No visible mycelia

-34.

content below 20 percent in three to four weeks, there would be no

visible moldiness.

5. Field Tests Using Fatty Acids

as Mold Inhibitors

The procedure, used in the laboratory tests, was followed
on a larger scale in the field. However, tests which.were carried out
under natural conditions introduced problems that could not be entirely
foreseen during laboratory work.

In the first test, the application of the acid was made
with the spray gun used in the laboratory procedure. The acid was ap-
plied to windrows of hay which had a.40 percent moisture content. This
hay was then.baled immediately. The coverage and penetration of the
acid in this method was far too inadequate, as shown by the fact that
all bales molded considerably within one week.

To overcome the problem of coverage, a new means of ap-
plication was devised. Attachments were made on the mowing apparatus,
and the acid was applied as the hay was being cut. The new procedure
in the field was as follows: A five percent solution of pr0pionic
acid was sprayed from.a series of jets on a pipe, which extended par-
allel to the cutter bar, three feet above the ground and eight feet
ahead of the bar. A fine fog of the acid was sprayed ahead of the
cutter bar, giving good coverage of the hay. The hay was windrowed
and cured, then baled at 35 percent moisture content.

The results of this experiment showed no inhibition to

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-36—

mold formation in the test bales. Within a few minutes after spraying,
there appeared to be no residual acid on the hay. The acid seemed to
volatilize rapidly during the earliest stages of curing, thus affording
no protection against mold development in the bale. Since the hay was
high in moisture content, there was considerable heating in these exe
perimental bales.
It was impossible to carry out further field studies

after this attempt, due to the fact that the end of the haying season
had arrived. The weather conditions became unfavorable, and there was

a lack of standing hay for any'more tests.

CONCLUSION

The problem of mold development in hay, as pointed out
in this paper, is a complex one, both from the standpoint of contrib-
uting factors and methods of control. In high moisture content hay,
the host material is extremely compact, the amount of fungi very high
and the moisture and temperature conditions exceedingly beneficial to
the growth of these fungi.

The mold population of hay has been shown to be wide and
varying, containing, in greatest numbers, the typical cosmopolitan
fungi found in the soil upon.which the hay is growing. Any chemical
method of treatment, to prevent mold development, must eliminate all
the various types of molds present on the hay; chemicals, which show
a specificity for only certain groups of these fungi, will be of little
value in solving this problem.

In the chemical treatment of hay, the action of the chemp
ical need only be mycostatic in nature. If the mold present at the
time of baling could be held in a state of inanimation, until a critical
moisture content of the hay could be reached, the problem of mold in hay
would be eliminated.

The laboratory experiments in this thesis indicated that
the short chain fatty acids have sufficient mycostatic powers to be use-
ful in preventing the development of mold in high moisture hay. By use

of the fatty acids, high moisture content hay could be baled and placed

-38-

in the barn, without fear of mold deterioration in the hay.

Propionic acid proved to be the most practical of the
fatty acids for actual use. This acid is low in cost, free from
unpleasant odor, easily obtained, readily soluble and proved effect-
ive in laboratory tests for the preservation of hay. It is necessary,
however, to develOp a.practical method of application of the acid to
"the hay before baling, without materially changing the moisture con-
tent of the hay. The method develOped must insure that volatilization

of the acid should not take place before the bale is formed.

10.

11.

12.

13.

-39-

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