STUDIES ON EFFECT or CIT-RIC ACID 0N m m
CELLULOSE DIGESTION BY’ RUMEN MlCR-DORGANISMS
FROM NORMAL AND
TRAUMATIC GASTRITIS ANIMALS

Thai: for the Degree cf M. S.
MICHIGAN STATE UNIVERSITY
Brij Mohan r’Mitru-ka
19621

1HE9I’S

STUDIES ON EFFECT OF CITRIC ACID ON IN VITRO CELLULOSE
DIGESTION BY RUMEN MICROORGANISMS FROM NORMAL

AND TRAUMATIC GASTRITIS ANIMALS

BY

Brij Mbhan Mitruka

A THESIS

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

MASTER OF SCIENCE
Department Of Microbiology and Public Health

1962

ABSTRACT

STUDIES ON EFFECT OF CITRIC ACID ON IN VITRO CELLULOSE
DIGESTION BY RTMEN MICROORGANISMS FROM NORMAL
AND TRAUN TIC GASTRITIS ANIMALS

by Brij Mohan Mitruka

A study was made of in 21552 cellulose digestion in order to deter-
mine the effects of citric acid when inocula were obtained from normal
fistulated animals. Alfalfa leaf meal, SolkawFloc and timothy hay were
used as sources of cellulose. It was demonstrated that there was no
statistically significant difference between the percentages of cellulose
digested with 0.2 percent, 0.1 percent, 0.05 percent and 0.01 percent
concentrations of citric acid added in the test fermentations as compared
to equivalent amounts of dextrose added in the control fermentations.

A study of in_vi££g_fermentation of cellulose by rumen micro—
organisms drawn from traumatic gastritis animals was made to determine
the effect of 0.1 percent citric acid on cellulose digestion and
volatile fatty acid production. The fermentation mixture consisted of
alfalfa leaf meal or Solka-Floc as the source of cellulose, rumen liquor
drawn from traumatic gastritis animals, and 0.1 percent citric acid or
0.1 percent dextrose. No significant difference was found in percentages
of cellulose digestion and volatile fatty acid production by adding
0.1 percent citric acid in the test fermentation. A wider variability
in the results was encountered with the percentage of cellulose
digestion and volatile fatty acid production by rumen microorganisms
drawn from traumatic gastritis animals than by inocula from normal

animals.

Bri j Mohan Mitruka
Determination of citric acid disappearance from the fermenters
showed that most of the citric acid was metabolized by rumen micro-

organisms after 16 hours of fermentation.

ACKNOWLEDGEMENTS

The author wishes to extend his sincere appreciation to Dr. C. K.
Smith of the Department of Microbiology and Public Health for his con-
stant stimulation, patience and guidance during this investigation and
the preparation of this manuscript.

The many helpful suggestions of Dr. M. P. Rines and Dr. G. R.
Moore of the Department of Surgery and Medicine are acknowledged with

much gratitude.

ii

TABLE OF CONTENTS

Page
ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . ii
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . v
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . vi

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . .

In vitro fermentation methods . . . . . . . . . . . . . . . . . 3

Determination of cellulose . 9

Volatile fatty acid determination and

citric acid determination . . . . . . . . . 11

Rumen microflora and their activity in in vitro fermentation . . 17
Rumen microflora . . . . . . . . . . . . . . . . . . . . 17
Isolation and culture media . . . . . . . . . . . . . . . . . l8
Microbial species in the rumen . . . . . . . . . . . . . . . 19
Physiology of rumen microflora . . . . . . . . . . . . . . . 21
In vitro activity . . . . . . . . . . . . . . . . . . . . . . 21

Mechanism of cellulose breakdown . . . . . . . . . . . . . . 25

Factors affecting in vitro cellulose digestion . . . . . . . . . 29
Citric acid utilization . . . . . . . . . . . . . . . . . . . . 36

EXPERIMENTAL AND RESULTS . . . . . . . . . . . . . . . . . . . . . 40
Section I
Experimental . . . . . . . . 40
Effect of citric acid on in vitro cellulose digestion
by rumen inocula from normal animals . . . . . . . . . . . 40
Animals used . . . . . . . . . . . . . . . . . . . . . 40
Collection of samples . . . . . . . . . . . . . . . . . 40
Substrates used . . . . . . . . . . . . . . . . . . . . 40
Fermentation procedure . . . . . . . . . . . . . . . . 41
Determination of cellulose . . . . . . . . . . . . . . 42
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Section II
Experimental . . . . . . . . . 51
Effect of citric acid on cellulose digestion by
cattle with clinical traumatic gastritis . . . . . . . . . 51
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Section III
Experimental . . . . . . . . . . . . . . . . . . . . . 57
Volatile fatty acid determinations . . . . . . . . . . . . 57
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

iii

Table of Contents, Continued

Section IV
Experimental . . . . . . . . . . . . . . . . . . . .
Colorimetric determination of citric acid . . . .

Materials and methods . . . . . . .
Preparation of test samples .
Preparation_of standard samples .
Procedure . . . . . . . . . . . .

Results . . . . . . . . . . . . . . . .

DISCUSSION

Percent cellulose digestion by inocula from normal and
traumatic gastritis animals with dextrose and citric

acid added . . . . . . . . . . . . . . . . . . . . . . . . .

VFA production as criteria of in vitro cellulose digestion .

Citric acid disappearance . . . . . . . . . . . . . .
SUMMARY..

LITERATURE CITED

iv

Page

67
67
67
68
68
68
69

75

Table

LIST OF TABLES

Percent of cellulose in substrates

Effect of various concentrations of dextrose and
citric acid on the in_vitro digestion of cellulose

Percent of cellulose digested in_vitro in inocula
from normal animals in 8 hour and 24 hour
fermentation periods . . . . . . . . . . .

Percent cellulose digested by inocula taken from
9 clinical cases of traumatic gastritis in 8
hour and 24 hour fermentation periods .

Percent in_vitro cellulose digestion by normal
and traumatic gastritis animals with 0.1 percent
dextrose or 0.1 percent citric acid . .

Total VFA produced during in vitro cellulose
vfermentation by inocula taken from cow K-l39

Total VFA produced during in_vitro cellulose
fermentation by inocula from traumatic
gastritis animals .

Percent of citric acid disappearance in vitro
with inocula taken from normal animals

Percent of citric acid disappearance in_vitro

with inocula taken from traumatic gastritis animals .

Page

41

44

50

54

56

66

67

Figure

10.

LIST OF FIGURES

Effect of 0.2 percent citric acid on the percent of
the cellulose digested by inocula from cow K-139
with alfalfa leaf meal, SolkamFloc and timothy
hay as substrates . . . . . . . . . . . .

Effect of 0.1 percent citric acid on the percent of
the cellulose digested by inocula from normal
animals with alfalfa leaf meal, Solka-Floc and
timothy hay as suhstrates . . . . . . . .

Effect 0.05 percent citric acid on the percent of
the cellulose digested by inocula from cow T-26
with alfalfa leaf meal, SolkawFloc and timothy
hay as substrates . . . . . . . . . . . . . . . .

Effect of 0.01 percent citric acid on the percent
of the cellulose digested by inocula from cow T-26
with alfalfa leaf meal, SalkaoFloc and timothy
.hay as substrates . . . . . . . . . . .

Percent of cellulose digested with 0.1 percent
dextrose or 0.1 percent citric acid by inocula
taken from traumatic gastritis animals . .

Volatile fatty acid production during cellulose
fermentation of alfalfa leaf meal substrate
when the inocula was used from cow K-139

Volatile fatty acid production during cellulose
fermentation of SolkauFloc substrate when the
inocula was used from cow K-139 . .

Volatile fatty acid production during cellulose
fermentation of alfalfa leaf meal substrate when
the inocula was used from traumatic gastritis
animals . . . . . . . . . . . . . . . . . . .

Volatile fatty acids production during cellulose
fermentation of SolkauEloc substrate when the
inocula was used from traumatic gastritis animals . .

Citric acid disappearance during cellulose fermentation
.of alfalfa leaf meal substrate by inocula taken
from normal and traumatic gastritis animals . . . . .

vi

Page

45

46

48

49

53

60

62

63

65

70

~List of Figures, Cantinugg
Figure Page
11. Citric acid disappearance during cellulose
fermentation of Selka~Floc substrate by

inocula taken from normal and traumatic
gastritis animals . . . . . . . . . . . . . . . . . . . . ?1

vii

INTRODUCTION

The utilization of cellulose has long been recognized as a pri-
mary factor in the nutrition of ruminants. Since ruminants are an
'expensive eXperimental subject for use in preliminary investigations
several types of artificial rumens have been devised for ig_¥i££g
studies of fiber breakdown and other problems relating to ruminant
digestion. It is believed that the rate of cellulose digestion in_
zitgg by the microorganisms of the rumen may be used as a measure of
cellulose digestion in_!£!g.

Since unusual types of bacteria digest the food in the rumen,
it is necessary to ascertain that the same bacteria are functioning
in digestion in the artificial rumen. The most pepular criterion of
activity of microorganism in the artificial rumen is the percentage of
breakdown of pure cellulose (Burroughs, Headley, Bethke and Gerlaugh,
1950; Stranks, 1956; Davey, Cheeseman and Briggs, 1960). The end
products of fermentation, volatile fatty acids (VFA), have also been
used as criteria of activity (Gray, Pilgrim and Weller, 1951; Warner
1956; Barnett and Reid, 1957). Studies have also been made using
similar systems on the effects of various substances on 12_vi££2_ruminant
fermentation. Burroughs E£.§l: (1950) studied the effects of cereal
grain and high protein feeds and Burroughs, Latona, DePaul, Gerlaugh
and Bethke (1951) studied mineral influences. Wasserman, Duncan,
Churchill and Huffman (1952) investigated the effects of antibiotics.

The purpose of this study was to test the effects of citric

2
acid on the in_xi££g activity of the rumen microorganisms from normal
animals and from animals suffering from traumatic gastritis. Citric
acid was selected as a test material because of its well known role as
an energy source and easy availability at low cost.

The objective was attained by determining the percentage of
in yi££2_cellulose digestion and VFA production. The disappearance of

citric acid was observed to insure the validity of the approach.

REVIEW OF LITERATURE

In vitro fermentation methods

Tappiener (1888) was the first to investigate the in_yi££2_fer-
mentation of cellulose by rumen microorganisms. He found that paper
pulp and cotton wool, when incubated under anaerobic conditions with
the fluid contents of the rumen dissolved slowly with the evolution of
methane and carbon dioxide and the formation of fatty acids. Analyzing
the distillate of the medium from prolonged $2.XE££2 fermentation of
cellulose by rumen contents he stated that acetic acid and butyric
acids were the main products of the fermentation. Markoff (1911, 1913)
using the same technique as described by Tappiener (1888) stated that
the average molecular weight of the fatty acid products of the fer-
mentations of cellulose was the same as that of butyric acid. Further
work on in xitrg study of rumen fermentation was mainly on YEA pro-
duction by cellulose digestion. Barcroft, McAnally and Phillipson
(1944) used fistulated animals from which samples were drawn for
VFA determination. Similarly, Daniell, Hitchcock, Marshall and
Phillipson (1945), and Gray (1947) analyzed the rumen contents drawn
frequently from fistulated animal.

The early workers did not compare the conditions lfl.!§££2.t°
those which occur 12_vixg_until Marston (1948) deve10ped the technique
to simulate more closely the natural environment in the rumen. He
considered the low pressure of oxygen, the high carbon dioxide tension,

the supply of phOSphate and inorganic nitrogen from the saliva, the

U

buffering capacity of the saliva, the temperature of approximately

40 C and the residual contents of the rumen which provide a massive
inoculum of organisms already selected by the environment. Cellulose
prepared from beech-wood or filter paper was suspended in buffer solu-
tion and inoculated with a suspension of rumen microorganisms which
had been separated from 3 to 5 liters of rumen fluid. Anaerobic con-
ditions were maintained by passing pure nitrogen over the fermentation
mixture.

Marston (1948) did not remove the nongaseous fermentation end-
products and as they accumulated they might have been expected to slow
and eventually inhibit the rate of digestion. To test the influence of
such an accumulation of break-down products, Louw, Williams and Maynard
(1949) compared cellulose digestion after incubation with fresh-rumen
fluid (700 ml) in a closed vessel and in a semi-permeable bag sus-
pended in a large volume of aqueous growth medium. They showed that
their semi-permeable system was capable of maintaining good rumen
papulation during 24 hours of fermentation.

Burroughs g£__l, (1950) devised an artificial rumen which con-
sisted of 500 ml glass fermentation flasks incubated in a waterbath at
40 C. A three hole stOpper was inserted in the flasks for carbon
dioxide inlet and outlet and for regulating the pH of the contents.

The fermentation was carried out continuously in a 36 hour period and
subsequent inocula consisted of oneahalf the residue of the preceding
fermentation.

Gall and Glaws (1951) compared an all glass impermeable apparatus
of Burroughs gthgl, (1950) and a permeable type as described by Louw

_e_§.§_1_. (1949). Rumen fluid was added to cellulose or grass in a salt

5
solution in these systems and neutralized freqaently. The fermentation
was carried out for 24~28 hours under anaerobic conditions. They-found
that in the impermeable system, accumulation of cellulase may account
for complete cellulose digestion rather than viable bacteria. Poor
culturability, alteredmcram reactions and lack of anaerobic organisms
were found in seven trials with the impermeable system. They con-
cluded that the process in the permeable system was more nearly like
rumen digestion.

Huhtanen and Call (1952) madified the artificial rumen of Louw
st 31. (1949), using a small cellophane sac suSpended in a 4 ounce screw
.cap jar which contained a solution similar in mineral composition to
sheep saliva. The sac containing the substrate and rumen fluid was held
.in place by screwing the cap onto the sac. The whole artificial rumen
was sufficiently small to be placed in an incubator. The sac consisted
of dialysis tubing, about 6 inches in length and tied at one end with a
firm knot to form a sac with a volume of at least 10 m1. They used
10 m1 of rumen fluid which had been filtered through two layers of cheese
cloth as inoculum in the sac. The miniature artificial rumen used by
Huhtanen.§£_al, (1952) is particularly useful in routine work where
large numbers of determinations of fiber digestion are desired.

McBee (1953) used the manometric procedure for evaluating the
total activity of rumen microorganisms. The basis of his method is
the assumption that the rate of fermentation of any substrate by the
microorganisms in the rumen fluid is pr0portional to the total activity
»of all organisms in the rumen capable of attacking that substrate and
the compounds derived from it. The total numbers and morphological

types of organisms were ignored in his study. The results obtained

by-MeBee were influenced by the method used for obtaining the rumen
content, the fraction of rumen fluid used, the buffer system in the
warburg vessel and the time of collection of the sample relative to
the time of feeding. .Johns (1951) carried out fermentations, using
‘washed suspension (resting~cells) of rumen organisms in a flask '
attached to a mercury filled manometer connected to an adjustable.res-
ervior which was adjusted to keep the gas at atmospheric pressure. It
was so arranged that after inoculation under sterile conditions the
apparatus-could be filled with sterile nitrogen. The volume of gas
produced by fermentation included that indicated by the mercury-mano-
meter and the dissolved gas in the liquid (mainly C02) which was deter-
mined in the VanSlyke manometric gas analysis apparatus. .The gas in
.the manometer was analyzed by means of a Haldane gas analysis apparatus.
Doetsch and Robinson (1953) used the same technique as described
.berbBee (1953) except that the inoculum used was a suspension of
washed cells. A number of substrates were used and the gas production
was measured manometrically using the warburg apparatus. Volatile
fatty acid were also determined. Henderson, Horvat and Block (1954)
used a simplified method for the study of cellulose digesting activity
-of rumen microorganism EEIXEEEE: They measured the cellulose activity
'by the loss of weight of parchment paper strips incubated with the
frozen culture of the rumen microorganisms blended with a warmed
nutrient solution containing a number of salts in standard bacterio-
logical test tubes. The loss in dry weight of the parchment strips
'was considered to represent cellulose digestion. Bentley, Johnson,
Hershberger, Cline and beon (1955) used the sediment of the strained

centrifuged rumen juice, for the inoculum in their study on cellulolytic

factor activity of short chain fatty acids by rumen microorganisms
$2,!i552, The sediment from one liter of juice was suSpended in 200 m1
of phosphate buffer (pH 7) which contained 0.01 percent cystine hydro-
chloride. Eight ml of this suspension were used in fermentation
.flasks containing cellulose, urea, glucose and mineral solutions. The
pH was adjusted to 6.7 to 6.9 by saturated sodium carbonate solution.
Carbon dioxide bubbled through the flask contents, served to establish
anaerobiosis and to agitate the suspensions. At the end of 30 hours

of fermentation the volume was adjusted to 100 ml and the disappearance
of cellulose was determined on 10 m1 of the reaction mixture by the
method of Crampton and Maynard (1938) modified by Hersberger, Engle,
Bentley and Moxon (1955).

Dehority, Bentley, Johnson and Moxon (1957) used an in 33552
rumen fermentation technique essentially the same as described by
Bentley 25.51, (1955) except that the urea concentration was changed
from 0.168 g to 0.126 g per 100 ml. Also an improvement in the pro-
cedure for the collection of the rumen microorganism was made by drawing
a line one inch from the bottle of the celluloid liner placed inside
the Sharples centrifuge. Only the sediment above this line was used
for inoculation of the 33,21532 fermentation flasks, by these workers.
warner (1956) has classified in_!i££2 rumen procedures as follows; (a)
Undiluted or slightly diluted rumen liquor incubated with the substrate
in an all glass impermeable system, this technique was applied by Pearson
and Smith (1943), Quin (1943), and Gray 25 _1. (1951); (b) Whole rumen
liquor diluted to half strength with mineral solution incubated with
substrate in an impermeable system, this system was used by Burroughs

SE a}, (1950), and MeBee (1953); (c) Various fractions of rumen liquor

8
used in an impermeable system, e.g. centrifuged cells and washed cell
suspension, this was used by McNaught (1951), Cheng, Hall and Burroughs
(1955), and Bentley, Johnson, Vanecko and Hunt (1954); (d) Rumen liquor
usually whole or undiluted incubated with a substrate in a semipermeable
container and dialysing against a mineral solution; this system was used
by Louw gt a}, (1949) Huhtanen and Gall (1952), and Wasserman gt al,(l952).

Johnson (1958) improved the method of inoculum preparation of
Bentley g£_§l, (1954). The basal medium was the same as described by
the latter investigators, except that the cellulose level was raised to
2 g per 100 m1 of medium. Quicke, Bentley, Scott and beon (1959)
applied the in_vi££g technique of Bentley g£_§13 (1955) modified by
Dehority g£_§l, (1957) and Johnson g£_§g, (1958) in their evaluation
of cellulose digestion with forages, with some further modifications.

Davey g£_§l, (1960) designed an improved artificial rumen for
continuous control during prolonged operation. The important feature
of their apparatus is the high degree of control which can be exer-
cised over the progress of in_yi££2_ruminal fermentation, permitting
eXperiments-of considerable duration. The criteria used for the
validity of their fermentation apparatus include quantitative and
qualitative bacteriological data, volatile fatty acid production, pH
levels, and cellulose digestion rates. These were examined by them
with a variety of diets using cows on similar diets as control.

Stewart, Daryl, Werner and Seeley (1961) have recently reported
preliminary tests of reliability of continuous culture for studying the
activities of mixed microbial pOpulation of the rumen in 23532, They

designed an apparatus for continuous culture (5.5 liter) maintained in

an 8 liter glass vessel at a constant temperature of 39 C. The culture

9
was stirred with Lucite (methyl methacrylate) paddles, and the sampling
device consisted of a suction flask connected to half-inch glass tubing
‘which extended down into half the depth of the culture. They tried to
>simulate the conditions of the normal rumen by their continuous culture
apparatus and suggested that the normal rumen fermentation can be repro-

duced at a biochemical and cellular level.

Determination 2£_cellulose

There are two common ways by which determination of cellulose
can be accomplished. In the first of these, the cellulose is hydro—
lyzed and the increase in reducing power due to release of glucose
units is determined. This method is not applicable in the present
study because of the heterogenous nature of rumen fluid and the prob-
ability of the development of interfering substances during hydrolysis.
In the second method, non-cellulosic materials are removed and the
residual cellulose can be determined by volumetric or gravimetric
methods. None of the methods of cellulose determinations are entirely
satisfactory (Salsbury 1955). .However, there are some methods in the
‘second group which give reproducible results and which are applicable
to the type of study presented here.

Kurschner and Hanak (1930) used a method for the quantitative
estimation of cellulose, in which the sample was freed of non-cellulose
organic constituents by digestion with an acetic acid-nitric acid
reagent. The treatment involved boiling the samples with the reagent
for twenty minutes.

Knrschner and Hoffer (1931) substituted alcohol-nitric acid as

the digestion reagent and the sample was boiled with the reagent for

10
two and sometimes-three successive hour periods.

Crampton and Maynard (1938) modified the method of Kurschner
and Hanak (1930) by using alcohol instead of water for the first washing
to.free the cellulose from the digesting reagent. .They used 15 ml of
80 percent acetic acid and 1.5 m1 of concentrated nitric acid and boiled
this gently for 20 minutes. The sample and liquid were transferred to
a 50 ml centrifuge tube with about 20 ml alcohol. This was centrifuged
for 10 minutes and then the residual_fiber was transferred to a crucible
and, by using suction this was washed with hot benesene, hot alcohol,
and ether.

Viles (1948) developed the anthrone procedure used in the deter-
mination of the undigested fiber and for colorimetric analysis of starch
and cellulose at a wavelength of 625 mp. In this method sulfuric acid
(60 percent by-volume) is used to digest the material prior to analysis.
It is then treated with water and anthrone reagent. .He found that 60
percent sulfuric acid give optimum.resu1ts for rapid disolution of cellu-
lose. Cotton is the only-form of cellulose referred to in his work.

Huhtanen, Saunders and Gall (1954) used the method described by
Viles (1948) for cellulose determination, with modifications. They
used a preliminary extraction of the water soluble carbohydrates present
in the sample followed by a conversion of the remaining carbohydrate
material into a form that will react with the anthrone to give a colored
product. This colored product could be read in a Beckman spectrOphoto-
meter at a wavelength of 625 mp.

Henderson g£_gl, (1954) used a simplified method of cellulose
determination. They incubated weighed strips of parchment paper be-

fore fermentation. At the end of fermentation the paper strips

11
were removed, washed in running water, air dried and reweighed. They
found as much as 40 percent of the weight of the strips was lost by
digestion and that the strips had retained sufficient strength to allow
washing, drying and weighing. Hershberger _£H_l. (1955) described a
rapid volumetric method for the determination of cellulose with rumen
microorganisms i2_vi££g. In this method cellulose was suSpended in water,
centrifuged and the supernatant fluid decanted. The cellulose in the tube
was suspended in 6.5 ml of glacial acetic acid and 1.5 m1 of concentrated
nitric acid and placed in a boiling water bath for 20 minutes. After
cooling the tubes were centrifuged for 10 minutes, the top of cellulose
leveled and after that recentrifuged for 5 minutes. The volume of
cellulose in the tube was determined and compared to the control tubes.

The authors claimed the advantage of rapidity of their volumetric

method over the gravimetric method of Crampton and Maynard (1938).
Volatile fatty acid determination and citric acid determination

Volatile fatty 2233 determination. An early investigation on
quantitative analytical methods for the determination of individual
volatile fatty acid (VFA) in a mixture has been based upon such dif-
ferences as rate of distillation or partition between immiscible
solvents. Ramsey and Patterson (1945) were the first to devise a
method for chromatographic separation of saturated VFA (Cl to C4).
They used silica gel previously saturated with water containing a
suitable indicator in its alkaline form in a column as in the ordinary
chromatography. The chloroform solution of the volatile acid was
added to the column which was then develoPed by washing with chloro-

form containing suitable amounts of n-butanol. The position of each

12

acid in the column was indicated by a color change in the indicator.
The different bands containing one acid (or a mixture of isomeric acids
in case of butyric) were then eluted from the column and collected
separately. Elsden (1946a) introduced a technique based on partition
chromatography for the separation and estimation of lower members of the
saturated series of fatty acids. He used wet silica gel impregnated
with bromocresol green. A small sample of the mixture of acids dis-
solved in chloroform was introduced at the top of the column and after
its penetration into the gel, a suitable mixture of butanol and chloro-
form was allowed to percolate through the columns. Elsden was able
to estimate prOpionic and butyric acids in samples subjected to previous
distillation to quantitatively remove the acetic acid. Acetic acid
could not be estimated by the column. This method has been used by
:Elsden (1946b) and modified by Elsden, Hitchcock, Marshall and
Phillipson (1946) with excellent results in the analysis of rumen
contents.

Paper chromatography has also been applied to the separation
and quantitative determination of relatively non-volatile organic acids.
The volatility of lower fatty acids however limits the direct applica-
tion of paper chromatography techniques for their separation and identi-
fication. Fink and Fink (1949) overcame this difficulty by chromato-
graphing the acids as their'hydroxamate derivatives and Spraying the
deve10ped chromatograms with ferric chloride. The derivatives were
athen visible as purple-spots on a yellow background but accurate quanti-
atative determination was not possible. Brown and Hall (1950) and Reid
.and Lederer (1951) devised a similar procedure by which lower fatty

acids from 02-C7 could be separated, identified and estimated. They

l3
employed the ascending deve10pment for 20-24 hour as described by
'William and Kirby (1948). The solvent used was butanol saturated with
aqueous 1.5 N ammonia. .The acids migrated on the-chromatogram in the
form of ammonium salts. Bromocresol purple in ethanol containing
formaldehyde was used as a spraying agent. Using this technique lactic
acid and formic acids could not be separated from acetic acid when
lactic acid was present in significant amounts and it had to be removed
by a preliminary distillation.

Keeney (1955) used a direct method for the chromatic determination
of Cl-C6 fatty acids in rumen fluid, as described by Harper (1953) for
cheese samples. Keeney (1955) used silicic acid which had been mixed
with an ethylene glycol solvent and then slurried with butanol-hexane
to prepare the columns. Two m1 of rumen fluid acidified with one drop
_of 50 percent sulfuric acid were mixed with 2 g of dry silicic acid and
this mixture was transferred to the column. The solvents used in Keeney's
method were a series of nmbatanol concentrations in n—Hexane.

Emery, Smith, and Huffman (1956) used the method as described by
Harper (1953), for volatile fatty acid determination in rumen fluid,
with some modification. They used 13 g of dry silicic acid and 7.5 m1
of 1.6 M potassium phosphate (pH 6.3) buffer for the buffered section.
-The sulfuric acid section of Harper (1953) was omitted by these workers
and they found no effect on the results of their exPeriment. Further-
more, they prepared the sample by weighing 6 to 7 g of rumen liquid
directly on to 10 g of dry silicic acid on a watch glass and thoroughly
mixed this in a mortar. Fifty m1 of chloroform were used to form a

rslurry and this was used as a vehicle to transfer to the column.

14
The use of celite in preference to silica gel columns has been
-suggested by Bueding and Yale (1951) and by Gray (1947), because of un-
predictable chromatographic prOperties encountered with various prepara-
tions of the silica gel. Gray (1947) reported losses when passing the
first sample of acids through celite columns. These results were re-
jected by Wiseman and Irvin (1957). They found that this loss could
be prevented by an addition to the stationary phase of either 3 g

of ammonium sulfate or 0.5 ml of 0.1 N H,SO Bueding (1951) used

2 4'
this phosphate buffer with celite column and quantitative separations
of VFA were accomplished by the pH variables of the buffer present in
the non-mobile aqueous phase and the butanol concentration of the eluent.
Wiseman and Irwin (1957) designed a method for determination of organic
acids in silage. They used celite columns with an internal indicator
for the quantitative separation of silage acids including butyric and
succinic acid. They found that the aqueous internal phase employed
minimal amounts of sulfuric acid to prevent retention of organic acids,
.and-sugar was added to increase elution resistance. The eluting
solvents used were mixtures of acetone with Skellysolve B. The method
was designed to eliminate steam distillation or ether extraction of
acids and mechanical equipment for the collection of fractions. They
found single zone-collections made possible a reduced number of titra-
tions with increased accuracy. Hungate, Mah, and Simensen-Mogens (1961)
applied the method of Wiseman and Irwin (1957) in the determination of
butyric, prOpionic and acetic acid in the rumen of lactating cows. Two
ml of acidified liquid were mixed with the cap material on the t0p of

a Wiseman-Irwin column, and the acids eluted with solvents were titrated

with alcoholic sodium hydroxide. They found that butyric acid was

15
eluted with 1 percent acetone in mixed hexanes,.pr0pionic-acid with 5
percent acetone-hexane mixture and acetic acid with 10 percent and then

-20 percent-acetone in hexanes.

Citric acid determination. Several methods for estimation.of

 

citric acid utilize the Furth and Herrman (1935) reaction in which color
is formed in the presence of pyridine and acetic anhydride. Saffaran
and Denstedt (1948) applied this reaction to the colorimetric determina—
tion of citric acid in animal fluids and tissues. Babad and Shtrikman
(1951) modified the procedure to estimate citric acid in milk-serum.
In both procedures the sample-was held for 10 minutes at 60 C.in the
-presence of an excess of acetic anhydride, pyridine was added and the
.mixture'held for further 40 minutes periOd at 60 C and then cooled in an
ice bath. The two heating periods were accurately timed to minimize
variability. Reducing the amount of-water added with the sample was
»feund to lower the intensity of the yellow color but enhanced the re-
»producibility (Babad 25.51:, 1951).

Saffaran and Denstedt (1948) found that temperatures higher than
60 C decreased color intensity and stability while lower temperatures
gave greater sensitivity but required longer holding periods for full
color developments. Addition of pyridine to the hot solution caused
a vigorous reaction that made it difficult to keep the tubes stoppered
during the subsequent holding period in the procedure of Babad _£'_l.
(1951). Reinart and Nisbett (1957) modified the method to reduce the
-violence of the reaction at 60 C whereas Murthy.and Whitney (1957)
recommended.the use of glass stoppers instead of rubber stappers to

-prevent possible contamination of the sample.

16

Marrier and Boult (1958) improved the method of color develop-
ment-using lower reaction temperatures and this led to-a procedure at a
.reaction.temperature of 32-C for direct determination of citric acid
in milk. They found that the reaction was-strongly exothermic, and
that a constant reaction temperature was essential to obtain reproducible
~results. They also found that rapid color formation at temperatures be-
low 60 C could be induced by introducing more water with the sample;
however, the water and acetic anhydride concentrations were difficult
to control if they were allowed to interact before the addition of
pyridine. On the basis of these observations pyridine was added to
~the sample prior to acetic anhydride and as color development was not
affected the need for timing the pyridine step.was obviated. All
-reagents were added at room temperature and the tubes were placed in
.water bath at 32 C immediately after adding the anhydride. In this
manner they controlled the vigorous nature of the reaction and even
~at the reaction.temperature of 60 C it was no longer necessary to
-stopper the-tubes.

Chromatographic separation of citric acid in plant and animal
tissues was attempted by a number of workers. The reader is referred
to papers by Bulen, Varner and Burrel (1952), Phares (1952), Varner (1957),
Jones, Dowling and Skroba (1953), Bush, Hurlbert and Potter (1952),
Owens, Smith and Wright (1943), and Bryant and Overall (1953). None of
the chromatographic techniques for the estimation of citric acid were
applicable in the present study due to the lactic and succinic acid in
the rumen samples. Citric acid could also be determined by the method
of Natelson, Pincus and Lugovoy (1948) modified by Elliott (1957) in

which colorimetric determination of citric acid is based on the oxidation

17
of citric acid to pentabromoacetone and conversion of the later to a
.colored complex. The reader is also referred to the procedure of

wail-Mslherbe and Bone (1949) modified by Taylor (1953).
-Rumen Microflora and their activity in_in vitro fermentation

Egggn_microflora. The ruminant differs from other mammals in
that its food is subjected to microbial fermentation in the rumen
~before it passes into the true stomach and intestinal tract where most
mammalian digestion occurs. The environment of the rumen is well
adapted for the maintenance of a large and diverse microbial population.
.The temperature is held relatively constant at about 39 C. The pH
.of the ingesta is usually slightly acid and is held relatively con-
-stant by the influx of food, water and heavily buffered saliva (Masson
~and Phillipson, 1951). -The Eh of the ingesta is held at a low level
due to intense-microbial activity and the low oxygen tension of the

~gaseous phase (Huhtanen gt 1., 1952 and Bryant, 1959). Many different

groups of microorganisms have been isolated from the rumen, however it
is now generally believed that the organisms of functional significance
in the rumen are protozoa and bacteria, capable of growth under the
anaerobic conditions prevailing in the rumen. Although a large number
of bacteria of diverse morphological characteristics can be demonstrated
»readily in the rumen contents by microscopic examinations only a small
number of these bacteria have been grown in pure culture and described
as to their morphological-and physiological characteristics. Hastings
(1944) recognized that many of the bacteria in the rumen.can not be

cultivated by the more common bacteriological techniques, because of

their fastidious growth requirements.

18

Isolation and_culture media, As early as 1928 Bechdel isolated
an organism from.the- rumen which was identified as Flavobacterium L12
EEEEE: This bacterium was grown on nutrient agar under anaerobic con-
editions. Since then pure cultures of other species have been isolated
from.the rumen many of these having been cellulose decomposing bacteria.
Hungate (1947) described an anaerobic technique and an agar medium con-
taining minerals, carbonic acid bicarbonate buffer, and sterile rumen
fluid for the isolation of cellulolytic bacteria. He showed that large
numbers of non-cellulolytic bacteria also could be grown on this medium.
Gall, Stark and Loosli (1947) isolated rumen microorganisms from-rumen
.contents diluted as much as 100 billion times.

Sijpesteijn (1949) published anaerobic techniques for isolating
cellulose decomposing bacteria from the rumen of cattle. The cultural
procedure described by Gall gt_al, (1947) is satisfactory for the
isolation of many rumen organisms but this technique had several weak-
nesses which made the isolation of several more delicate rumen organisms
‘rather difficult. Gall, Hendrickson, Thomas and Loosli (1949) used a
rich organic broth medium and emphasized the necessity of continued
maintenance of anaerobic conditions and speed in preparing cultures of
bacteria from high dilutions of rumen contents. Sijpesteijn (1951)
used a similar medium as described by Hungate (1947) that contained
yeast autolysate in place of.rumen fluid to cultivate cellulolytic
ecocci.

Huhtanen gt. 1. (1952) devised an improved technique for the

isolation of rumen bacteria from high dilutions of rumen.f1uid. They
designed a procedure to create a.suitable pH and Eh in both the dilu-

tion blanks and broth and to control the production of acidby the more

19
rapidly growing organisms. They found the ability to culture rumen
bacteria increased and growth of several delicate anaerobics typical

of the adult roughage fed ruminant is encouraged by this method.

Microbial species ig_the rumen. Huhtanen and Call (1953) iso-

 

lated and purified nine types of curved rods, and related rod-types from
the rumen. They described the rods and divided them into three main
groups based on morphology; large curved rods, small curved rods and
straight rods. All the organisms were nonSpore-forming obligate
anaerobes, which attacked fiber and produced short chain fatty acids,
carbon dioxide and methane.

Bryant and Burkey (1953) using culturing techniques similar to
Hungate (1950) found that on rumen.f1uid glucose cellobiose Agar (RGCA)
medium there were higher colony counts and larger colonies were obtained,
than from the agar medium described by Gall gt a}, (1947) or a tomato
juice yeast extract trypticase agar with the carbondioxide—bicarbonate
buffer system. Counts of 1,100 million, 180 million and 380 million
per m1 of rumen fluid were obtained from a single sample of rumen
contents cultured on three media respectively. They also found that
higher colony counts were obtained from the solid portions of rumen
contents obtained by squeezing the liquid off through cheese cloth
than from the liquid fraction. Furthermore, they found that out of 20.2
billion bacteria per ml seen under the microsc0pe, only 1.7 million
bacteria per ml were viable. They suggested that many of the bacteria
either were not viable when cultured or were not capable of growing
under the cultural conditions used. It was of interest that none of

the common groups of bacteria such as members of the family

20
Enterobacteriaceae, enterococci or Spore forming anaerobes were found in
the rumen under the conditions applied by Bryant and Burkey (1953).
Hungate (1944, 1950, 1952) isolated Clostridium.cellobiOParus,

Bacteriodes succinogenes and cocci. Sijpesteijn (1949, 1951) isolated

 

Ruminococcus flavifaciens. Johns (1951) isolated Veilonella gazogenes

 

an anaerobic, pr0pionic acid producing coccus from the rumen of sheep.
Gall-§£_§l, (1951) have described pure cultures of lactic acid bacteria
but refrained from assigning names.

Hungate (1952) isolated 25 strains of cellulolytic bacteria and
divided these into 4 groups: Bacteriodes; butyric rods, cocci and Spore
formers. He suggested that the cellulolytic flora are not a constant
association of organisms but that any strain might predominate depending
upon the particular animal and the particular time.

Mackay and Oxford (1959) and Nana, Oxford and Masson (1954) and
Mann and Oxford (1955) have characterized several different kinds of
bacteria isolated from a young calf and a goat. The organisms identified
included, species of coliforms, lactobacilli, streptococci and Gram-
negative, facultative anaerobic, urease positive rods. Hungate (1957)

isolated Ruminococcus albus from the rumen of a cow.

 

An extensive study on the bacterial species of the rumen by Bryant
(1959) produced evidence that the rumen contains a great variety of
bacterial Species, many of which are present in large numbers in one
animal held under one set of conditions. Bryant concluded that the
groups of bacteria correSponding to definite Species are sometimes easily

identified, e.g. the genera Butyrivibrio and Ruminococcus and the succinic

 

 

 

,acid producing Bacteriodes, but the great variability in characteristics

among strains in these groups suggests that Species Specific patterns

21
will be very difficult to find. Furthermore, he stated that based on
their function in making the energy of the ration available to the
ruminant, the cellulolytic bacteria are often the most important
ruminal microorganisms. Based on numbers present and the rate of

cellulose digestion the genus Ruminococcus is one of the most important

 

cellulolytic groups.

Physiology g£_rumen microflora. A symbiotic relation exists
between the ruminant and bacteria found in the rumen in that the host
supplies the bacteria with materials to be utilized and the bacteria
in turn breaks down these substrates to products which may be absorbed
into the blood stream (Thorton, Howie, Baker, Phillipson, Synge and
Elsden, 1952). Notable among the functions of the bacterial flora are
the digestion of cellulose, production of VFA and synthesis of protein
and vitamins of the B-complex. A number of reviews on the functions of
rumen bacteria are available and the reader is referred to the papers
by McElory and Cass (1940), Wagner, Booth, Bohstedt and Hart (1941),
Pearson and Smith (1943), Owen §£_al, (1943) Baker and Harris (1947),
Conrad, Hibbs, Pounden, and Sutton (1950), Quin and Clark (1951)
MbNaught (1951), Arias, Burroughs, Gerlaugh, and Huffman (1951),
Nasserman,§£“gl. (1952), Dehority, E1~Shazly and Johnson (1960),
Huffman (1953), Salsbury, Heifer and Luecke (1961), Hinders and Ward
(1961) and Bryant and Robinson (1961), for more detailed information on

the subject.

-In;vitro activity. Biological and chemical criteria have been
used in attempts to establish the validity of the results obtained

with the artificial rumen systems. Biologically, attention has been

22
paid to motility of the protozoa and predominant morphological or cul-
tural types of bacteria. Chemically most attention has been paid to
the preportion of cellulose digested; though other relative values have
been used, little attention has been given to the actual rates of
digestion of the total substrate. It is essential to ensure that the
microbial multiplication which takes place should proceed in a fashion
.similar to that found in the rumen of the living animal. When the
environment or substrate is not that found in the rumen of the animal
from which the rumen liquor inoculum was taken, then the results of
this metabolism may bear little or no relation to events in the rumen
22mg.

Pearson and Smith (1943) used undiluted or slightly diluted rumen
fluid incubated with substrate in an all glass impermeable system as
used by Quin (1943) and Gray g£._l, (1951). On the basis of their finda
ings they claimed that their system remained normal for 2-4 hours of
fermentation and that, numbers and types of mainly the iodOphilic bacteria
.showed no-significant changes during that time. ‘With the same technique
MtNaught and Owen (1949) showed that the concentration of O-phenanthroline
which suppressed urea utilization $2.XEEE2 corresponded to a concentra-
-tion of ferrous ion of the same order as that found in 2322: With a
similar system with rumen liquor free of protozoa McNaught (1951)
found that the C02/CH4 ratio in the evolved gases had an average value
similar to that found in_yiy2_though the range of values was larger.

Quin (1943) and McAnally (1944) showed that the rates of gas
output following addition of glucose was similar in yiyg_and i2_yi££g.

but the incubation periods observed were very short.

1. (1950) claimed that there were no marked changes

Burroughs 25

23
in numbers, size or predominant types of bacteria throughout, but, that
—some of the types of protozoa failed to survive. However the main
criterion of normal rumen function relied on by those workers was the
‘high degree of cellulose digestion obtained. They used 3 g per liter
of cellulose per artificial rumen contents in early experiments. Gray
35 El: (1951) successfully applied a number of criteria of normal rumen
function to their lg yigggpsystem. They microsc0pically observed the
activity of the protozoa, the rate of methane produced to fodder supplied,
and the digestibility of cellulose and pentosans. These total digestions
-were-found to be similar in yiyg and i3 yiggg but the authors noted that
_the rates of digestion of these substrates and of methane production
were only-one-half of those found in_yi££2, They used as buffer
ammonium-carbonate equivalent to 103 mg N/ 100 ml, a concentration.of
NH3 very rarely found in.the rumen of healthy animals even when fed a
diet rich in a readily attacked protein.

,Bentley e£_gl, (1955) found that rumen microorganism required
the presence of certain VFA, particularly valeric acid, for digestion
.of cellulose $2.22E32: However, Bryant and Doetsch (1955) working with
cultures of Bacterioides succinggenes, a cellulose digesting organism
isolated from.the-rumen, reported that this organism required a com-
-bination of a.branched chain fatty acids, isobutyric, iso-valeric or
DL-OC - methyl-n-butyric acid, and a straight chain fatty acid,
valeric or caproic acid for growth on a purified medium. Allison,
Bryant and Doetsch (1958) investigated the volatile fatty acid require-
.ment.of-several-strains of-Ruminococcus flavifaciens and Ruminococcus
glhgg, both anaerobic cellulolytic cocci isolated from the-rumen.

_They found that a mixture of acetate, isobutyrate and valeric acid

24
e.isomers and also a mixture of acetate and isovalerate greatly stimulated
the growth of these organisms.

warner (1956) using an artificial rumen system-of-Louw £5 31, (1949)
.showed.that in the presence, though not in absence,.of-substrate the
~numbers~of bacteria and protozoa were maintained approximately constant
in the-artificial rumen for 7 hours. -For the microbial population to
remain normal in numbers and activity, it was shown to be necessary to
vuse-as test substrate in_yigggn.only substances similar to diet fed to
animal from which the rumen liquor inoculum was taken.

Dehority, 35,31, (1960) studied the-cellulolytic fractions of
bacteria by differential centrifugation. They found that the fraction
_of bacteria still in suspension at 15.x G but sedimented at 3000 x
G contained the majority of the cellulolytic rumen bacteria. Fermenta-
tion studies with the 3000 x G sediment showed that after incubation
with cellulose as the source of carbohydrate a predominance of Gram-
.negative micrococci were present along with a lesser number of very
{small Gram-negative rods. These observations indicated that the Gram-
.negative cocci are the principal cellulose digesting organisms which
proliferate ippyiggg~under these conditions. These same kind of
. organisms were also found in large numbers in the inoculum obtained from
the rumen. They also-reported that the microorganisms showed a.strict
~requirement for both valeric-acid and biotin.

vStewart gg'gl, (1961) using a continuous culture method for
pinpyisygpfermentation found that the values for pH, rates of total VFA
production, proportions of VFA, and numbers of coliform and amylolytic
organisms were similar to values which have been.reported by Belch

.and Rowland (1957) and Reid, Hogan and Briggs (1957). The rates of

25
-growth\of oligotrich protozoa in the in yiggg experiments using hay-
concentrate substrate were equal to-the fastest rates of growth reported
rig.y1532.by other workers (Hungate, 1942, 1943 and Coleman, 1958).

They found the apparent growth of oligotrichs in their experiments with
an all hay substrate consistent with that reported by uxford and Sugden
(1953). Both holotrichs and oligotrichs showed good motility in their

experiments.

Mechanism 2E cellulose bgegydown

 

Ruminants do not possess a salivary enzyme for degradation of
cellulose nor is there any Such enzyme secreted into the rumen. Thus
the degradation of cellulose is clearly a function performed by the
flora found in the rumen.

Henneburg (1919) first reported detailed microscopic studies of
disintegration of plant tissues in the rumen. An important finding
was that the areas of erosion on the surfaces of plant materials were
»usually occupied by cocci. This method of approach'nas extended by
the Studies of Baker (1939} who, using polarized light, examined
microsc0pically cellulosic material undergoing digestion by rumen
bacteria. The most characteristic result of the attask on plant tissues
was found to be the excision of clear cut cavaties or lacunae in which
iodiphilic organisms were enclosed. An important finding by Baker
(1939) was that the morphological characteristics of the rumen micro»
organisms were correlated with their functional activities.

Norman and Fuller (1940)_stated

"Since~cellulose is completely insoluble in water and obviously
.therefore to accomplish decomposition any organism must-possess

an extra cellular enzyme-system capable of producing some
-soluble product that can pass into the cell. It seems to be

26
some indication that two types of hydrolytic system may be
concerned, one accomplishing the primary attack, perhaps
breaking cross linkages or accomplishing.substantial chain
-shortening and the other bringing about subsequent hydrolysis
of these fragments."

They concluded that it seems likely that exoenzyme-production is
only stimulated by direct contact with the fiber in the case of a wholly
insoluble substrate. Baker and Harris (1947) found that the rumen micro-
organisms reach the susceptible plant structure by invading the middle
lamella of the adjoining cells penetrating into the lumen of fibers and
invading the fractures in the plant material caused by prehension and
rumination. Disintegration of plant material then takes place adjacent
to the microorganism with the result that cavaties are formed. At
first these cavaties generally follow the form of microorganisms but
as digestion proceeds the cavaties enlarge and form corrosion figures
which lack the birifrigence shown by the neighboring plant material.

Hungate (1944) has suggested that the apparent requirement for
close proximity of the cellulose digesting cell to its substrate could
be explained by the secretion of an exocellular cellulase. The close
association of cell and substrate would be required for the organisms
to benefit from the breakdown products of the-cellulose.

Considering the available observations Sin (1951) gave a ten-
tative scheme for the enzymic breakdown of cellulose. He postulated
a separate enzyme Cx' The postulation of Cxenzyme was based on the
observation made by Reese (1950) that the carboxy-methyl cellulose was
attacked not only by filtrates from cellulolytic microorganisms but
also by those from non-cellulolytic fungi. According to Sin (1951)

the linear polysaccharide is degraded by Cx enzyme directly into

glucose without formation of cellobiose as an intermediate. Sin (1951)

27
extended his scheme for breakdown.of cellulose step wise. -He stated
that the first step was the rupture of Vanderwall's forces in the
'amorphous region of the cellulose-and of the hydrogen bonds in the
-crystalline region. -Second step would be the-rupture-of the~cross
linkage which are assumed to join the linear chains-of the anhydrous
glucose units. For the third step, the breakdown by the Cx enzyme of
linear polysaccharide into units which can diffuse into the cell. He
reserved the term 'Cellulase' for the enzyme bringing about the initial
change in the native cellulose molecule.

‘Levinson, Mandles and Reese (1951) used glucose oxidase for the
determination of glucose and beta-glucosidase followed by the deter-
mination of glucose formed with glucose dehydrogenase for the deter-
mination of cellobiase-and found that cellobiose was the principal
produce of cellulose degradation. The cellulolytic organisms studied
aproduced large amounts of Cx enzyme which diffused into the medium.
‘Only small amounts of B-glucosidase were found outside the cell. They
found that the cellobiose resulting from Cx activity can enter the cells
as rapidly es-can-glucose. >They hypothesized that the principal final
product of 9x activity is cellobiose and the presence of cellobiose in
the medium is not a prerequisite to utilization of cellobiose by the
.organisms They stated that this hypothesis is a correction of the
hypothesis previously made in the same laboratory by Reese (1950) that
glucose appeared to be the final product of Cx activity.

‘Reese and Gilligan (1953) separated the 9x enzyme into three
components by differential adsorption onto cellulose and Kaolin. The
number of components varied depending upon.the species of micro-

.organisms used and the conditions of growth. They could not reach

28
the conclusion whether these-components represented individual enzymes
or were-due.to interaction of a single-enzyme with other elements-of
the=system.

-Kitts-and Underkofler (1954) using rumen organisms in an-anaerobic
fermentation found that unlike aerobic fungi these-rumen organisms do
not liberate cellulase into the medium. Using undiluted rumen fluid to
which an inhibitor was added the yields of glucose-obtained were of a
magnitude-of 2—3 mg/ml in 24 hour-when a substrate concentration of
16 mg/ml of Solka-Floc was used.

Whitaker (1953, 1954) has concluded.that Myrothecium verrucaria
cellulase is a single enzyme capable of hydrolyzing native cellulose
to glucose. Kooiman, Roelofsen and Sweeris (1953) agree with Whitaker
except that they involve a separate enzyme (Cellobiase) in the
rhydrolysis of cellobiose to glucose. Jermyn (1952) demonstrated the
presence of several p-glucosidases some of which he believes act on
-comparatively long chains as found in.carboxy—methyl.cellulose (CMC).

.The recent deve10pments on mechanism-of cellulose hydrolysis

were summarized diagramatically by Reese (1956) as follows:
‘ C C
Native-cellulose l 3 Linear.cellulose :x
A,B,C :eté

rchains

Cellobiose

p-gluc. glucoge p-glucosidase A,B,C etc (3 transferases).

He-explained that the components of cellulolytic systems may differ
largely in their preference for chains of a particular length. If
this is true then there may be components bridging the gap between
ethe-cellulases and p-glucosidases. Such components would have maximum
activity on the short chains (6 to 10 units long) and lesser activity

as the chains become longer or shorter. Not enough is known about

29
C1 to characterize it. He prOposed the existence-of C1 enzyme when
Cx was considered to be a single enzyme. He deduced the presence

of C enzyme from the fact that some organisms unable to attack native

1
-cellulose did produce enzyme capable of hydrolyzing degraded cellulose
(e.g. CMC). Reese (1956) stated that it is uncertain whether C1 is
that form of Cx acting primarily on the longest chains or whether it
may indeed act on some minor linkage or minor component, in native
cellulose.

Reese (1956) concluded that glucose is certainly the end product
of the complete hydrolysis of cellulose by the enzymes whammy-glucosidase
is present in the cellulolytic complex. Much of the glucose results

fromqfi-glucosidase action on cellobiose, but it is still uncertain

whether glucose may be produced in the absence of p-glucosidase.

Factors affecting in,zi£gg_cellulose digestion

Several factors could be of significant importance in in 33552.
cellulose fermentation. These factors include: Lignification and
.stage of maturity of substrate, amount of cellulose, particle size,
rumen liquor source, amount of rumen liquor, pH adjustments, addition
of trace minerals, length of fermentation, various techniques used and
the nitrogen source.

Kammtra, beon and Bentley (1958) reported the effect of lignifi-
cation and stage of maturity of plant in in vi££2_cellulose digestion.
They observed that the amount of cellulose in the flasks affected the
amount of cellulose digested by the microflora in the fermentation
flasks because of the amount of cellulose available to them. Further-

more, the maturity of the forage affects the in_vitro digestion of

‘30
cellulose in plant material. With first stage alfalfa, an average-of
83.6 percent of cellulose was digested in 30 hours whereas only 46.7
percent of the cellulose was digested in alfalfa in the third stage
of growth. Lignin pg£_§£_does not appear to influence the activity
of rumen organisms in_!i££25 however, the lignin inhibits cellulose
digestion by an encrusting action based upon the deposition of lignin
around the cellulose during the growth of the plant. These observa-
tions were similar to those reported by Norman (1935), Crampton and
Forshaw (1939), Dyer, Weaver and Comfort (1954), Ely, Kane, Jacobson
and Moore (1953) and Trimberger, Kennedy, Turk, Loosli, Reid and
Slack (1955) on the maturity of forages and their feeding values and
digestibility. The effect of lignin on digestion of fiberous plant
material in_!i££g_has also been.reported to be similar by Baker and
Harris (1947) and Howie and Baker (1952).

Perlin, Michaelis, and Mcfarlane (1947) and Norkans (1950)
using cellulolytic microorganisms showed that the amount of cellulose
breakdown was inversely proportional to the degree of crystallinity of
the cellulose fraction. Furthermore, they showed that by changing the
crystalline cellulose to amorphous cellulose they increased the rate of
microbial decomposition of the cellulose. Ranby and Immurgot (1955)
have demonstrated that the physical constants are not identical for
cellulose from different sources.

Stewart and Schultz (1958), Salsbury, Vanderkolk, Baltzer and
Luecke (1958) and Quicke, Bentley, Scott and Moxon (1959) found little
difference in cellulose digestion by various rumen liquor sources.
However, Huhtanen _£_ 1. (1952) and Asplund, Berg, McElory and Pigden

(1958) have observed qualitative and quantitative differences in

31
.inpzitrg.digestion.for.rumen liquors from steers fed different diets.

.McLeOd and Brumwell (1954) using purified substrate and washed
cell suspensions as described by Cheng.g£_al, (1955) reported that the
amount.of-rumen liquor may have an appreciable influence on the extent
of in zitgg digestion of cellulose or dry matter. This information was
confirmed by Church and Peterson (1960) who applied the same procedures
as described by Barnett and Reid (1957).

In many of the ig_xi££2’eXperiments reported, pH adjustments have
been made in the range of 6.4 to 6.9. Wegner, Booth, Bohstedt and Hart
(1940) suggested adjustment between 6.0 to 7.5 whereas Meites, Burrel,
and Sutton (1951) have reported an Optimum pH between 4.53 and 7.35
for cellulose digestion. Kitts and Underkofler (1954) and Stanley and
Kesler (1959) working with isolated rumen cellulose preparations
found Optimum cellulase activities at pH 5.5 to 6.0. Reis and Reid
(1959) observed that Optimum pH for ammonia accumulation varied between
6.0 to 6.7 on different diets and appeared to be correlated with the
range of pH normally encountered in the rumen on each diet. Virtanen
((1946) found that mesOphilic bacteria fermented finely ground wood
dust more-completely than coarsely ground dust, possibly due to less
interference by lignin.

Church and Peterson (1960) confirming the results of Reis and
Reid (1959) and Baker, Quicke, Bentley, Johnson and Moxon (1959) in-
dicated little difference in the digestion of purified wood cellulose
when-separated into different particle sizes. However they reported
that the fermentation of some substrates (e.g. alfalfa hay) may be
'altered appreciable by fine grinding. -They suggested that fine

grinding may have altered solubilities of the other carbohydrate

32
.constituents with the resultant change in the cellulose digesting flora.
Cheng;g£_gl, (1955) and Dehority g£_al. (1957) reported that increasing
levels of dried grass in the fermentation flasks, depressed the per-
centage of digestibility of dry matter and cellulose. Church and
Peterson (1960) also found the-same effect Of dried grass added to
their cellulose fermentation. Huhtanen and Elliott (1956) found that
cellulose digestion was not influenced by the quantity of alfalfa added
within the ranges studied and with the technique used.

Baumgardt, Cason, and Markley (1958) found that the relation
between in zitrg and in zizg_cellulose digestion was not significant
when all forages were considered, but the relationship was highly
significant for the grass hays.

>The mineral supply to rumen microorganisms is vital to efficient
cellulose digestion in_xi££g, Phosphorous and iron have been named
as-two minerals most likely to affect the cellulose splitting organisms.
Little, Cheng and Burroughs (1958) found that a balanced mineral
nutrient solution is needed by the rumen microOrganisms for Optimum
cellulose digestion and the increase reached a maximum at a concen-
tration.of 12.5 ppm. The addition of 7.5 ppm Of zinc or manganese,
or 15 ppm of magnesium also increased cellulose digestion but to a
lesser extent than could be Obtained with the addition of iron.
Cobalt and cOpper failed tO stimulate cellulose digestion at any concen-
tration tested and they were found to be inhibitory to cellulose
digestion at levels of 2.5 ppm and above. Selenium was found to decrease
cellulose digestion when added to the basal medium by these workers.
Church and Peterson (1960) demonstrated that changes in mineral medium

-concentration within the ranges used have only negligible effect on

33
lfly!!££2.d1393t1°n of dry matter and cellulose, or VFA production.

Cardon (1953) reported that a high intake of salt adversely
affects cellulose digestion.

Using alfalfa leaf meal as a substrate, Huhtanen and Elliott
(1956) showed the following rate of cellulose digestion: at 6 hours
17 percent, at 12 hours 34 percent; at 18 hours 40 percent, and at 24
hours 49 percent. The 24 hour period was recommended as suitable for
the in_!i££2_fermentation by those workers. Lefevere and Kamastra (1958)
compared cellulose digestion in_yi££2_and in yizg_with 22 different
rations and found the 48 hour in_!i££2_fermentation period as most
nearly simulating in_yizg_cellulose digestion.

(LeFevere and Kamastra (1960) using the technique of Bentley,
Lehumkuhl, Johnson and Hershberger (1954) in the in_zi££g_digestion of
_cellulose found that 24 hour fermentation was below that obtained in 2139,
Cellulose digestion coeficients Obtained in_!i££2 with sheep and cattle
rumen fluid as inoculum were lower for rations containing 25% roughage
than for those containing 75 percent roughage.

The factors affecting in gi££2_cellulose digestion may be vari-
able due to the different techniques employed by the several workers.
Hungate (1947), Sijpesteijn (1951) and Bryant and Berkey (1953) used
pure-cultures for some studies on cellulose digestion. Cheng gE_§l,
(1955) modified this technique by centrifuging and washing the rumen
microorganisms. .Bentley §£_gl, (1954) employed centrifuged unwashed
bacterial cells from rumen fluids while Wasserman g£_al, (1952) and
Huhtanen-£5 a}, (1954) worked with whole unaltered rumen fluid. Some
authors have pooled the rumen liquor samples from several animals

(Barnett, 1957) or washed suSpensions Of microorganisms (Doetsch and

34

Robinson, 1953) as a.means of reducing variability between inoculums.
Arias g£_al, (1951) found that the degree to which energy require-

meats-of:the-rumen microorganisms were fulfilled depended upon.their
ability to utilize urea or other ammonia supplying compounds. .They
,ysuggested that.the probability exists that there is specific need for
small amounts-Of readily available energy, such as that from dextrose,
.sucrose or starch when the principal energy source is cellulose, to
bring about the most efficient utilization of urea in the fermentation.
Huhtanen and Elliott (1956) found that the addition of urea or glucose
alone markedly inhibited the in,gi££2_breakdown of cellulose in
timothy hay substrate but, when they were added together, normal
activitijas restored. The effect was less marked or absent with
alfalfa leaf meal. Bloomfield, Muhrer and Pfander (1958) studied the
.relation Of the source Of-energy to urea utilization by rumen micro-
.organisms and found that lactic acid, pyruvic acid and alanine stimulated
ourea utilization indicating a possible necessity for a functional group
on the alpha carbon. Starch was found to be~more-effective than the
'simp1e~sugars,,fructose was found to be superior to other hexoses, and
equal amounts of glucose and fructose were-superior to similar amounts
~of-sucrose as sources of energy.

.Salsbury, Hoffer and luecke (1961) found that a ration of
cellulose, cornstarch and urea was the simplest combination capable of
maintaining the cellulose digesting ability of the rumen microflora
for three days. Cellulose plus soyabean protein was found more effec-
tive than cellulose plus urea. .Furthermore, when the ration consisted
of only cellulose,.starch hnd urea,a.source-Of amino acids-was required

to maintain cellulose digestion for more than 3 days. Determination

35
of molar concentration of VFA indicated that feeding ground cellulose
in addition to hay and-supplement lowered the ratio of acetic to
-pr0pionic acid in the rumen.

Barnett (1957) reported that by the use Of chemically untreated
feed-stuffs and whole filtered rumen liquor the Operative microflora
may be kept sufficiently active in_zi££g_for a suitable length of time.
He concluded that the microbial population in an artificial rumen
bears no relationship at the end of run to the original pOpulation.

He also stated that different animals on the same diet have approxi-
mately the same microbial pOpulation but different animals on different
diets show considerable variation. LeFevere and Kamastra (1958) found
that there was no significant difference in cellulose digestion
inpyi££2_and in_!izg_when the animals used were on the same ration.
_However-when.the animals used as sources of animal fluid were on
different rations, the cellulose digestion values in_!i££3_with the

. same basal medium were of different magnitudes. Greater cellulose
digestion was indicated for rations high in roughage both in_gi££2_

and in_zizg,

Donefer, Lloyd and Crampton (1961) reported that the digestion
of Solka-Floc cellulose was much more affected by source and type of
inoculum than were any of the forage substrates. Timothy hay digestion
Mwas stimulated by casein hydrolysate supplementation only when re-
-suspended cell inoculum was used.

Burroughs g£_al, (1950), Ruf, Hale, and Burroughs (1953) and
Bentley gt a}, (1955) demonstrated cellulolytic factor activity in
,hot water extracts of alfalfa, timothy hay, ladino clover, cow manure,

autolysed yeast, dried brewers yeast and distilled dried fermentation

.36
asoluble molasses. Hall, Cheng, Hale and Burroughs (1954) found that
the partial hydrolysate (acid, alkali and enzymatic) of casein.or soya-
bean protein, chicken feathers, hair and blood-meal also stimulated
cellulose digestion. Huhtanen, Saunders and Call (1954) noted that the
addition of 50 mg Difco yeast extract or enzyme hydrolysed casein to
theirinyzitrg system increased the digestion of Solka-Floc by as much
as 100 percent over the control.

Wasserman at _l, (1952) showed that antibiotics interfered with
1§_yi££g_cellulose digestion. The effect of the antibiotic chloro-
tetracycline hydrochloride on cellulose digestion was further investi-
gated by Huhtanen and Elliott (1956) who reported that this form Of
antibiotic when fed had no correlative inhibitory action on cellulose
digestion over the test period. -Gas production by rumen.microorganisms
increased greatly after a few days on the antibiotic regime. Lodge,
Miles, Jacobson and Quinn (1956) showed that the tetracyclines tend to
depress cellulose digestion over a period of time and that resistant
strains-of organisms appear in the.rumen.

Contradictory evidence has also been.forthcoming on the.inf1uence
of added fat and steroids on cellulose digestion. Brooks, Garner,
Muhrer and Pfander (1954a) reported that steroids are generally shown to
~stimulate cellulose digestion while Brooks, Garner, Muhrer and Pfander

(1954b) reported that the position of fat is still in diSpute.

 

-Citric acid utilization
There are two major stages in the process by which energy becomes
available to ruminants; a.preliminary conversion of the protein, or

metabolism of carbohydrates and fats of the diet via acetyl CO A or an

37
intermediate of the tricarboxylic acid cycle and the subsequent oxida-
tion of these relatively simple compounds.

The direct utilization of citric acid by rumen microorganism is
not known, however citric acid utilization by other microorganisms has
been reported by several workers. Early recognition of citrate-utili-
zation in milk and in glucose-citrate-broth by Hammer (1920), Hucker
and Pederson (1930) and Van Beynum and Pette (1939) led to the apprecia-
tion of the role of Streptococcus lactis, Streptococcus citrovorous

and Streptococcus paracitrovorous in better flavor deveIOpment by the

 

production of acetoin and diacetyl. In most cases, however, a ferment—
able sugar also-was required to obtain citrate fermentation. Brewer
and workman (1938) found pyruvate in citrate-glucose fermentation of
§, paracitrovorous, but were unable to find citrate as its precursor.

Citrate fermentation in Aerobacter aerogenes was formulated by Deffner

 

and Franke (1938, 1939) as follows:

~4 citrate ----- 9. 7 Acetate +-5 C02 + Formate-+ Succinate.
Carbon dioxide and acetic acid were the major products determined.
Brewer and Werkman (1939, 1940) obtained evidence that oxaloacetate
and pyruvate are intermediate in citrate fermentation by S, paracitro-
lvorous. ’Campbell and Gunsalus (1944) showed that energy can be derived
from-citrate in the absence of carbohydrate fermentation and lactate
production by homofermentative lactic acid bacteria. They found that
at pH 7.0 lactic acid is not produced, but acetate, carbon dioxide
and formic acid were the main products. At an acid pH, acetylmethyl-
-carbinol-and lactate become major products. The citrate cleaving enzyme

has been shown to be present in-Streptococcus faecalis, A, aerogenes and

.Escherichia coli by Dagly and Dawes (1953) and Grunberg—Monago and

38

Gunsalus (1953). The cleavage of citrate in.microbia1 fermentation is
:catalyzed by a.distinct-enzyme which.does not involve-coenzyme A as
'shown by Wheat, WOng and Ajil (1954), Wheat and Ajil (1954, 1955) and
Smith, Stamer and Gunsalus (1956).

lnyziygpstudies on feeding trials with sodium citrate in the
(ration.of lambs was reported by Packett, Butcher and Fordham (1960).
They stated that addition of 1.63 percent equivalent citric acid to the
diet,.produced 47 percent increase in gain. The gain-enhancement was
‘greatest during the initial period but gains were continuously greater
throughout the experiment. -Increase in feed consumption, feeding
efficiency, salt intake and water consumption were greater according
~to these workers.

Heany, Pritchard, and Sylvestre (1961) used sodium citrate buffer
vwith feeding roughage rations (pelleted) in steer feeding trials.
They expected sodium citrate would provide a readily fermentable anion
-and a-residual cation. They used.citric acid also to provide only the
-readin fermentable anions. They found that sodium citrate increased
the intake of pellets in all periods but citric acid had no effect.
Furthermore, they reported that there was a tendency for the consumption
-of pellets to increase during the period of fermentation. When com-
‘pared with consumption of chopped hey the consumption of pellets was
greater in all groups, the sodium citrate groups showing the greatest
increase.

Gray and Anthony (1961) reported the effect of citric acid and
citric acid plus yeast extract fed with Bermuda Hay in zigg digestion
.trials among eight steers. They found that there was no statistically

significant difference among the treatments in either phase. -However,

39
steers receiving-citric acid supplement did have an increased average

daily gain.that approached-significance.

EXPERIMENTAL AND RESUEIS
Section I
Experimental

Effect 2: citric acid on in'vitro cellulose
digestion by_rumen inocula from normal animals

 

 

 

The experiments were carried out using normal cows in an attempt
to determine the in_vitro cellulose digestion using different substrates
and to estimate a.suitable concentration of citric acid to be used

in the following studies.

Animals used, .Inocula were obtained from three nine-yeareold
Holstein cows (A-l30, T-26, and K-l39) maintained on a ration of seven
pounds of alfalfa hay,and four pounds of grain mixture. All three
-animals had been fistulated and fitted with-screw-capped lucite fistula
plugs similar in construction to one described by Hentschl, Berry and

Huffman (1954).

Collection 2f samples. vInocula were obtained 2-3 hr after the

 

animals had been fed. .The upper portion of the rumen contents was
.removed through the fistula to approach the area of the ventral sac

_of the rumen. The rumen fluid was then squeezed into a polyethylene
bottle through two thickness of cheese cloth. The bottle with the

rumen sample was kept under tepid water and was brought to-the laboratory
as soon as possible. ‘The sample was then transferred to a glass battle,

and aerated with.C0 while immersed in a 39 C waterbath.

2

Substrates used. Substrates used in these studies were air

 

dried alfalfa leaf meal, Solka~Floc and timothy hay. (See Table 1.)

41

.TABLE 1. ~Percent of cellulose in.substrates

W
G used to obtain

 

.Substrate 'Cellulose percentage 0.2-g Of cellulose
Alfalfa leaf meal 22.5 0.80
Solka-Floc 95.2 0.20
Timothy hay .31.6 0.60

 

Alfalfa leaf meal and Solka-Floc were used throughout these
'studies, while timothy hay was used only in the preliminary experiments.
The concentrations of citric acid tested were 0.2 percent, 0.1
percent, 0.05 percent and 0.01 percent (w/v) of citric acid in distilled
water. These concentrations were made from stock solutions-stored at
4 C and were brought to a temperature 39 Cxin a water bath. Dextrose
'was used as the standard at.the same concentrations as citric acid in
.each run. urea was used as the source of nitrogen. The concentration
~of urea.used throughout the studies was 0.1 percent as recommended by

,Salsbury (1955).

Fermentation prgcedure. The fermentation procedure-used in these
,studies was similar to that described by Huhtanen~_£__l. (1954). As
inocula 20 m1.of rumen fluid were placed in Visking cellOphane tubing,1
6-1/2 inches in length and 1 inch in diameter; these sacs were placed
in.4 oz screw-capped bottles. -Twelve fermentation vessels of each
:substrate were set with the test concentrations of citric acid and 12

with the same concentrations of dextrose as a standard. Each experi-

ment was performed in duplicate. Composition of the mineral mixture

1Obtainable from the Visking Corporation, Chicago, Ill.

42
used was one-half strength artifical saliva as described by McDougall
(1948). Eighty ml of the mineral mixture plus citric acid or dextrose
-were used outside the Visking tubing in each individual fermentation
.vessel. .The fermentation jar containing the substrate, rumen fluid and
citric acid or glucose in the Cellophane bag was sealed and placed in a
water bath at 39 C. .Fermentations were stopped at 0, 4, 12, 16, 20

and 24 hours by freezing the fermentation vessel at -10 C.

Determination 2f cellulose. Cellulose was determined by the
modified method of Crampton and Maynard (1938). In these experiments
the mixture in the cellOphane bag was transferred to a 150 ml Berzelieus
beaker, the bag was rinsed with distilled water and the solution evap-
.orated to dryness at 105 C. The dried.sample was treated with 15 m1 of
80 percent acetic acid and 1.5 m1 Of concentrated nitric acid. A glass,
round bottom flask, filled with cold water was placed on the beaker and
the contents refluxed on a hot plate for 20 minutes. The beaker was
removed from the hot plate, the sides of the beaker and bottom of the
flask were rinsed with 95 percent ethyl alcohol, and the beaker was set
.to cool. The contents of the beaker were then filtered through a Selas
40XF sintered porcelain crucible and the crucible was dried 18 hours at
105 C. It was then cooled in a desiccator over calcium sulfate and
weighed to determine the weight of the crucible with cellulose. The
:cellulose in the weighed crucible was then muffled for 2 hr at 700 C,
cooled again in~a desiccator and weighed to determine the weight Of
the crucible and ash. The percent cellulose digestion was calculated

from.comparison with the residual cellulose in the zero hour samples.

43
Results
‘The percentages of cellulose digested in 8 hr and 24 hr fermen-
.tation-periods are shown in Table 2. When alfalfa leaf meal, Solka~
Floc and timothy hay were-used as substrates, the concentrations of
dextrose-and citric acid used were 0.2 percent, 0.1 percent, 0.05 percent
and 0.01 percent. There was a decrease in the percent of cellulose
digested when 0.2 percent citric acid or 0.2 percent dextrose was used
in all the.substrates. Citric acid gave higher percentages of cellulose
digestion in 24 hours of fermentation when.compared to equivalent
samounts of dextrose, although there was only slight change observed
with added citric acid.

The-change in the percent of cellulose digestion.with these con-
—centrations of citric acid with alfalfa leaf meal, SolkaaFloc and
timothy hay was not statistically significant at the 5 percent level as
tested by the t-test (Stearman, 1955).

Figure 1 shows the average percent of cellulose digestion of
‘Solka-Floc, alfalfa.leaf meal, and timothy hay using 0.2 percent added
citric acid or dextrose when the inocula were taken from cow K-139. The
percentage of digestion obtained in 24 hours of fermentation was lower
using Solka-Floc as substrate. An increase in the percent of cellulose
digestion with all three substrates was observed when.citric acid was
added, but this increase was not significantly different from that of
.the standard.

Figure 2 shows the percent cellulose digestion with the same
~substrates, after adding 0.1 percent citric acid or dextrose. The
percent-of cellulose digestion.in 24 hours was in agreement-with that

of previous workers (Huhtanen gt. 1., 1954, Huhtanen and Elliott, 1956)

44

TABLE 2. Effect of various concentrations of dextrose and citric acid
on the ig.vitro digestion of cellulose

 

Cellulose Digestion*

 

 

 

 

 

 

Dextrose Citric acid
Time of [concentration** concentration**
fermen-
,Substrate -tation 0.2 0.1 0.05 0.01 0.2 0.1 0.05 0.01
Alfalfa
leaf meal 8 hr 10.5 20.1 25.4 25.4 16.0 21.4 18.8 23.8
24 hr 34.7 46.5 49.9 50.9 45.9 47.5 46.9 52.1
Solka-Floc 8 hr 0 8.6 10.8 6.1 3.0 0.3 7.8 5.5
24 hr 50.5 67.2 55.3 55.8 49.7 67.8 57.9 60.0
Timothy
‘hay 8 hr 1.8 5.96 8.86 10.5 5.8 5.75 7.6 7.6
24 hr 34.8 25.9 44.2 35.3 33.3 29.5 35.7 33.3
Average
of all
substrates 8 hr 4.1 11.6 15.0 14.0 8.2 12.5 11.4 12.3

24 hr 40.0 46.5 49.8 47.3 42.9 48.3 46.8 48.5

*Average values of-experiments expressed in percent.

**Concentration of citric acid and dextrose expressed in percent.

6O 1"

55 ..

50 0

4o 4.

35 {L

 

PERCENT CELLU LOSE DIGESTED

15.1-

10.—

 

Fig. 1.

3O ..

20 l

454

LEGEND:
0—0 0. 2% dextrose + substrates

x---x 0. 2% citric acid + substrates

 

 

 

I _- -—

L

7
12 l

O".
N‘
vb

2

O\-1r-

"F”
4 8

FERMENTATION TIME IN HOURS

Effect of 0. 2 percent citric acid on the percent of the cellulose
digested by inocula from cow K-139 with alfalfa leaf meal, Solka-
Floc and timothy hay as substrates.

Each point represents an average value of three duplicate experi-
ments. The perpendicular lines represent the range of variation
with the substrates with dextrose and citric acid.

PERCENT CELLULOSE DIGESTED

701

65"

60~

55-1

50‘

45..

35‘

304

25*

20 "

15 J-

10 ‘-

 

Fig. 2. Effect of 0. 1 percent citric acid on the percent of the cellulose di-
gested by inocula from normal animals with alfalfa leaf meal,

46

LEGEND:
0—0 0. 1% dextrose + substrates

x----x 0. 1% citric acid + substrates

 

 

-A— i I
l 1 .1

4 8 1'2 16 2

o‘-

FERMENTATION TIME IN HOURS

Solka-Floc and timothy hay as substrates.

 

Each point represents an average value of six duplicate experiments.

The perpendicular lines represent the range of variation with the

substrates used with dextrose and citric acid.

ll,

4;
in this field. When Sclkileoa‘waS used as the safstrete the increase

di

{D

m

in cellulose digestion with d citric acid'was observed up to 8
hours of fermentation and with timothy hay as a substrate this increase
was noted after 8 hours. The increase in persent cellulose digestion
with 0.1 percent citric acid is not statistically sigzificent at the

5 percent level with all the substrates used.

Figure 3 shows the average perceit cellulose digestion with
alfalfa leaf meal, SolkaaFlaz and timothy hay using 0.05 percent citric
acid or dextrose. Solkaofloz and timothy hay gave a slight increase in
percent cellulose digestion with added citric acid. gaing SolkauFioc
as a substrate the percent of cellulose digested was lower in the 24

hours fermentation period when citric axid was adied as compared to

I

added dextrose.

Figure 4 shows the average percent of the cellulose digested
with the same substrates using 0.01 percent added citric acid or dextrose.
Citric acid increased the percent of cellulose digestion with alfalfa
leaf meal as compared to adied dextrose. The percent of cellulose
digested with added citric acid was lower in 94 hours of-fermentation
when Solka-Floc was used as a substrate.

Table 3 shows the percent of the cellulose digested in Solka-
Floc and alfalfa leaf meal in 8 hour and 24 hour fermentation periods
using 0.1 percent citric acid or dextrose with inocula from different
animals. The difference in percent of the cellulose digested with
~citric acid or dextrose adied was not statistically significant after

8 hours of fermentation. A'wide variation was observed.

U'l
g1

45“
4e-
3e»

sol

25"

I

201b

PERCENT CELLULOSE DIGESTED

15“”

10"

51-

 

48

LEGEND: ' __

0—0 0. 05% dextrose + substrates

x ------ x 0. 05% citric acid + substrates

 

 

 

 

—:L—

:1)
4 8 12 16 20 24

qu-

FERMENTATION TIME IN HOURS

Fig. 3. Effect of 0. 05 percent citric acid on the percent of the

cellulose digested by inocula from cow T-26 with
alfalfa leaf meal, Solka-Floc and timothy hay as
substrates.

Each point represents the average value of three duplicate
experiments. The perpendicular lines represent the range
of variation with the substrates used with dextrose and
citric acid.

7O

65

60

55

50

45

4O

35

3O

25

20

15

PERCENT CELLULOSE DIGESTED

lO

 

,4?

 

 

 

 

 

 

LEGEND:
o—————O O. 01% dextrose + substrates .2:
x ------ x 0. 01% citric acid + substrates
/
I -..

I I - _ ‘1- l l l L

I I I T l r
4 8 12 16 20 24

FERMENTATION TIME IN HOURS

Effect of 0. 01 percent citric acid on the percent of the cellu-
lose digested by inocula from cow T-26 with alfalfa leaf meal,
Solka-Floc and timothy hay as substrates.

Each point represents the average value of three duplicate
experiments. The perpendicular lines represent the range of
variation with the substrates used with dextrose and citric aCid.

50

 

 

 

 

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-- -- -- -- o.am a.sm s.mm o.~m caucuses
Hooum
m.He a.sa e.HH m.e. ~.ee m.ee m.Na ~.oa AmmH-av
announce
zoo
s.ee m.me o o e.me o.ee m.mm a.e~ Ammn-av
caucuses
.soo
m.me a.sm e.HH a.a e.ee e.ee m.ma H.0H Ae~-ev
sausages.
soc
-- -- -- -- s.om H.ee e.wa a.sfi Ae~-ev
flfimumHO—w.
sou
m.ee o.ae c.w o.m e.me «.me o.m~ a.NH AomH-<v
swoumaom.
sou
uaoouom unmouom unmouom unmouom unsouom- unmouoe unmouoe unmouon Hmafln<
H.o scum H.o H.o snow H.o H.o scum H.o H.o seas, H.o
cauuwu ononuxon. owuuwu ooouuxon. ownuuut omouuxon owuuau. amouuxon
on am A we a He,e~ an.mt
ooaa-maaom Hams mama seamea<

 

 

wooeuon.nowumuaosuom.Hn.¢N cam H: w :w
mfimsasm Hashes Scum ofisoosm.hn ouuu> NM vouoomww ouoasaaoo mo unsouom .m mam<H

 

51

Section 11

Experimental

cattle with clinical traumatic gastritis

 

Traumatic gastritis, a widely occurring disease in ruminants,
is caused by metallic objects swallowed by the animal incidently or
with the feed. The "hardware" is usually lodged in the ventral part of
the reticulum, where it penetrates or causes continuous irritation.to
the animal. This may slow the rumen contractions and may cause death
,of the animal if there is penetrationthrough the diaphragm and
vpuncturing.of the heart. The animal displays anorexia,.rumen.contractions
are slower and weakened, there is rise in body temperature and tenderness
in the region of the xiphoid cartilage. The treatment is removal of the
foreign body by rumenotomy.

Rumen samples from nine cases of traumatic gastritis from the
Michigan State University cattle clinic, were obtained during rumenotomy.
They were treated with 0.1 percent citric acid or dextrose as were the
—regular rumen samples. The percent of cellulose digestion, VFA production
and citric acid disappearance were studied. The animals used were eight
Holstein cows from 3 yr-12 yr of age and a yearling steer-of the Holstein
breed. The history of all the animals in these studies was similar.

‘The animals were anorexic.with a body temperature above 102.5 F and they
had slow rumen movements. Traumatic gastritis was diagnosed in all
.cases. Rumenotomy was performed in the clinic and the foreign bodies
werexremoved.

Cellulose fermentations were carried out using the methods
described above. Substrates tested in these studies were alfalfa leaf

.meal and Solka-Floc. .The experiments were designed so that both

52
-substrates could be used at the same time and treated with inocula from
-the same animal. The concentration of citric acid or glucose tested
was 0.1 percent in all the experiments. The urea concentration of 0.1
percent was kept constant.

The fermentations were stepped at 0, 4, 8, 16, 20 and 24 hours,
the material within the sac and the artificial saliva were mixed and
centrifuged at 800 X C after the fermentation period. A twenty m1
sample of supernatant fluid was stored at -10 C for later chromatographic
analysis of volatile fatty acids and colorimetric determination of
citric acid. The sediment of the fermenter was treated for residual

cellulose analysis as described previously.

Results

Figure 5 shows the average values of percent of the cellulose
digested with 0.1 percent citric acid, using alfalfa leaf meal and
Solka-Floc as substrates at different periods of fermentation. The dif-
ference observed in both substrates due to 0.1 percent added citric
acid was not significant; however, with alfalfa leaf meal a slight
increase in the percent cellulose digested was noted until 16 hours
of fermentation.

Table 4 shows the percent cellulose digested in 8 and 24 hour
fermentation periods using alfalfa leaf meal and Solka-Floc as sub-
-strates when the inocula were taken from nine clinical cases of
traumatic gastritis. The average difference in percent of the cellulose
digested in 8 hour fermentation with 0.1 percent citric acid or dextrose
added was tested using the t-test and was found not to be significant

at the 5 percent level. A wide variation in percent cellulose digestion

53

 

 

 

70 '7
LEGEND:
65 + f
x-——--x alfalfa leaf meal + 0. 1% dextrose /
60 .- o ------ o alfalfa leaf meal + 0. 1% citric acid /
*——* Solka-Floc + O. 1% dextrose /
o 55 a _ . . /
a A ----- A Solka-Floo + O. 1% Citric ac1d /
:3 so - x
O / ’1‘
5 45 t / z W” ’ I
m 6'
8 4O Jr ../'/
.4 6’
/
3 35 ..
U 30 “ 47 /
E I / ///’/ /
m 25 '- fl/"'// /
U / /
a3. /
33 20 " / /
// /
15 l’ / /
l/ /
10 db I ’- "1
l’/
5 J, ///
O ‘r 4v 5 i ‘L i
. 4 8 12 16 20 24

FERMENTATION TIME IN HOURS

Fig. 5. Percent of cellulose digested with O. 1 percent dextrose or 0. 1
percent citric acid by inocula taken from traumatic gastritis
animals.

Each point represents the average value of nine duplicate
experiments.

54

 

 

 

 

 

 

c.¢o w.mo m.m o.HH w.c¢ o.oq m.m~ o.HN owmno><
¢.wn H.mn q.m m.oH o.~m m.¢q 5.0N N.mN m
H.mm m.~o m.mH N.HN ¢.¢m w.o¢ c.0N w.mm w
o.mn w.mm N.©H m.m w.wq m.o¢ m.wm mm.mm m
m.ho w.om m.m m.w m.mc c.0q «.mH m.mw c
¢.mo w.mo o.n H.mH ¢.Hn n.Hm H.mm o.mm m
m.mm m.oo m.HH H.mH w.m¢ m.om m.mm o.mw d
o.mo ¢.Hm m.¢ H.o a.sq H.mq m.HN m.m~ m
a: an an n. w.oq N.¢¢ N.m mm.N N
N.hq w.~o m.m N.m w.o¢ m.c¢ m.mw o.mH H
ammonia uaoouom ucoouom unocuom unmouoa uuoohoe unmouoa unmouom .02 some
H.o meow H.o H.o wfiom H.o H.o cwom H.o H.o cwom H.o
owuuwo omouuxon ownuwu omouumom ofiuuwo omouuxon owuuwo omonuxon
a: «N HA w H£.¢N an m
oonumxHom Home mmoH mmamma< oumuumnsm
meowuoe,:owucuuoahom woo: cm can soon w ca owuwnummw .
owumeomuu mo mommo Hmowowfio m Eoum aoxmu Manson“ he woumomwc omoHoHHoo unmouom .¢ mummy

55
in both alfalfa leaf meal.and Solka~Floc at 8 hour fermentation was
observed. 'This variation may possibly be due to the difference in
activity of rumen microorganisms obtained from different cases of
traumaticzgastritis.

In Table 5 the average percent cellulose digestion is compared
in normal and traumatic gastritis animals with alfalfa leaf meal as
substrate. There was not a significant difference at 8 hr of fermenta-
stion. At 16 hr of fermentation, the percent cellulose digestion in
traumatic gastritis animal was lower than that in normal animals. In
24 hr there was no difference in percent cellulose digestion between
normal and traumatic-gastritis animals.

‘When Solka-Floc was used as a substrate there was a considerable
difference in percent cellulose digestion between normal and traumatic
gastritis animals. In normal animals it was lower than_in traumatic
gastritis animals at 4 hr and 8 hr of fermentation; while at 16 and 20
hr of fermentation, there was no difference in cellulose digestion
between the two groups. In both normal-and traumatic gastritis animals
there was no statistically significant difference in the cellulose diges-

tion with both alfalfa leaf meal and Solka-Floc.

56

 

 

 

 

0.e0 0.50 H.00 ~.e0 n.0e o.0e 0.50 0.00 use; em
0.0a 0.ae 0.00 o.me 0.0a 0.0e 0.00 a.se use; ow
0.0m ~.0m 0.He 0.~e m.em 0.0m H.me H.0e use: 0H

-- -- a.oa 0.NH -- -- «.mm a.sm “so: NH
m.a o.HH m.oH 0.0 s.m~ 0.HN 0.HN H.o~ use: 0
0.0 n.0 m.~ H.m 0.0 0.0 H.ma 0.5 uses e
fivfi UN: UQOHUNQQ UM UN QQOHUNUQ UHUN . QmOHUMHUQ VH UN QMOHUNUQ GOflUGUd—QH—Mom
unease unease unease, ensues

nHmaqumimwuwuummw, manages «Hmawsm ouuwuumow mHmsaso

owumaomua Hmauoz owumaboua Hmahoz
uo~s-mxaom Hmma.0mma neamua<

 

 

woven meow owuuwo accused H.o Ho_omouuxon unmouoe ~.o suds
macaque mfiueuumow owumsomnu was Hashes he sowumowwc oeoHoHHoo ouuw>gmw uaouuom .n.mAm<H

 

57

Section III
Experimental

Volatile _M A_c_i_cl SE4). Determinations

Volatile fatty acids were determined by column chromatography,
based.on the method described by Bueding (1951),-with modifications.

Samples prepared from the fermentation mixtures of 0, 4, 16
and 24 hr with 0.1 percent citric acid or dextrose added, were applied
to a column.of celite and sulfuric acid. Acetic, pr0pionic and
butyric acids were separated by development with hexane-butanol solvent.
A standard column was run with known amounts of acetic, pr0pionic and
butyric acids and a recovery of 98.4 percent was-obtained. VFA were
determined in each sample with Solka-Floc and alfalfa leaf meal as
.substrates and fermented by inocula from normal and traumatic gastritis
animals.

Celitel-was washed twice by decantation with two volumes of
purified diethyl ether. .It was air dried overnight and then dried at

»110 C for 4 hr. Six grams of this celite were used to prepare a column.

2SO4 and 10 N H2304 solutions

were made from Analytical Reagent grade H2804 of 35.5 N strength.

-Sulfuric acid solutions. 0.2 N H

.Ethanolic potassium-hydroxide. 1 N KOH.was made by dissolving
(w/v) potassium hydroxide pellets (Reagent grade) in 95 percent ethanol.
0.01 N KOH was made by diluting 1 N KOH with 95 percent ethanol and
~standardized against 0.01 N potassium~biphthalate. 0.01 N standardized

“ethanolic KOH was prepared fresh for each experiment.

 

IJohns-Manville No. 535.

58
Indicator. 0.1 percent bromothymol blue in ethanol was used as

an indicator.

’Solvent. 1, 3, 5 and 25 percent butanol concentrations were
prepared (v/v) in n-Hexane (Skelly solve B). Each solution was

-equillibriated with 0.2 N H SO three times for 6 hours and the H280

2 4

-was separated by means of a separatory funnel.

4

Columns. Chromatographic tubes, 20 mm (inside diameter) x 400 mm,
“sealed‘with a.sintered glass disc at the bottom were used in these
analyses. The columns were prepared-using 6 grams of celite. The
~celite was weighed in-a beaker and 2.6 m1 of 0.2 N H2804 were added
-and mixed until completely homogenous. Sufficient hexane (Skelly solve
B) was added to wet the celite. Hexane was added to the column until
.the column was approximately oneahalf full. The celite was then added
.and the columm was packed lightly with a solid glass rod. rFifty ml of
hexane were passed through the column using_3 pounds of nitrogen pressure.
One inch of hexane was left on the column above the celite. ‘Ten m1 of
the sample of the supernatent fluid preparation of the fermentation
described previously were pipetted into a 100 m1 beaker and was adjusted
.to a pH of 10-12 by 1N KOH (Asplund §__§l,, 1950) and dried on a steam
bath. The dried sample was dissolved in 2 m1 of 12 percent H2804 and

1 m1 of this solution, acidified with 3 drOps of 10 N H2804, was used

for the chromatographic analysis.

Chromatographicprocedure

One ml of the sample was mixed with 2 grams of dried celite
-until completely homogenous. This was then transferred quantitatively

to the column and packed evenly. Traces of celite were transferred by

59
wiping the beaker, Spatula and inside walls of the colcmn'xith the glass
wool plug. The flow rate mas controlled to approximately 5 ml per minate
and five ml fractions of effluent were collected in individual 20 mm x
150 mm test tubes. The effluent was titrated with 0.01 N KJH using two
drops of 0.1 percent bromothymol blue as an indicator. Bgzyric,
prOpionic and acetic acids were elated with 1 percent, 3 percent and
25 percent butanol in hexane reapectively. The butyric acid was eluted
using 100 ml of 1 percent tetanol in hexane, prOpionic acid was eluted
with 125 m1 of 3 percent b;:anol in hexane and acetic acid was eluted
using approximately 100 m1 of 25 percent butanol in hexane. After an
acid was completely eluted the next solvent was ad ed. The blank value
was subtracted from the titrations for each tube to obtain the amount

of VFA eluted.

Results

The titration values for the individual ¥FA were added and
calculated as mM percent of the acid per fermenter. Eash experiment
was performed in duplicate. The blank determinations were made with
each lot of reagents and apprOpriate correctiors made.

Figure 6 shows predictio: of mM :f‘?EA per fermeLter with
alfalfa leaf meal as substrate in O, 4, 16 and 24 hour of fermentation
when inocula were used from Cow K~139° It was feand that batyric acid
was increased threeafold in 4 heurs of fermentation over the initial
concentration in the fermentation containing dextrose, while with
citric acid the increase was twosfold as great as the initial concen-
tration. rDuring the 4-16 hour period of fermentation there was little
difference in the butyric acid production between the fermenters with

and without added citric acid. The prOpionic acid prodaced in 4 hours

 

 

6O

 

 

    

 

 

3. 6 .-
//Q\
3. 2,, x’ x
I, \\\
[I \\
/ \
/ \
/ \
2. 8"F /’ \\\
I’ o
/
3" fl
2 Acetic acid
[-4
E:
U 2.
a:
£11
[14
g 1. .1
1 x——x Fermentation with O. 1% dextrose added
0 ------ o Fermentation with O. 1% citric acid added
1. 2“
O. 8‘-
A
-----———---o— ------------ -o
Propionic acid
0- 4‘ T'TT/K
““““ ~o
Butyric acid
0 J' ' ‘r t : fi-
4 8 12 16 20 24
TIME IN HOURS
Fig. 6. Volatile fatty acid production during cellulose fermentation of

alfalfa leaf meal substrate when the inocula were used from
cow K- 139.

61
-of fermentation was 50 percent of the total prepionic acid produced in
the 24 hour period in both dextrose and citric acid fermentation. There
'was no significant difference in the propionic acid production between
the dextrose and the citric acid fermentations.

Acetic acid production with alfalfa leaf meal as substrate was
markedly higher in the fermentation with 0.1 percent citric acid than
in the 0.1 percent dextrose. The difference in acid production between
the two fermentations was greatest at 16 hours of fermentation. The
fermentation with 0.1 percent citric acid contained 3.43 or 3.4 mM percent
while the control with 0.1 percent dextrose contained 2.59 or 2.6 mM
percent. Figure 7 shows VFA production in O, 4, l6 and 24 hours by
inocula from Cow K—l39 using Sclka-Floc as substrate, with 0.1 percent
dextrose or 0.1 citric acid. Butyric acid production was similar until
16 hours of fermentation in both cases. During the 16—24 hour period of
fermentation the production of butyric acid was higher in the fermenta-
tion with 0.1 percent dextrose.

PrOpionic acid produced was not significantly different in the
two fermenters in the 0-16 hour period of fermentation. During the 16-24
hour period there was increased production of prOpionic acid in the
0.1 percent dextrose fermentation.

There was an increase in acetic acid production during the 0—16
hour period with citric acid fermentation as compared to the 0.1 percent
dextrose fermentation but they were similar at 24 hours.

Figure 8 shows VFA production by inocula from traumatic gastritis
animals with alfalfa leaf meal as substrate. Here butyric acid increased
.until 16 hours of fermentation in both 0.1 percent dextrose and 0.1 percent
citric acid samples. There was an apparent utilization of butyric acid

in both fermentations during the 16-24 hour period. There were no

62

3. 2T

LEGEND:

x—x Fermentation with O. 1% dextrose added
2. 8" . . . . .

o ------ o Fermentation With 0. 1% Cltrlc ac1d added
2. 4"

2.0"

1.6-

mM PERCENT

1.21

0.4»

 

———————— o
' Butyric acid
1 I

4 8 12 16 20 3‘4

 

 

 

 

TIME IN HOURS

Fig. 7. Volatile fatty acid production during cellulose fermen-
tation of Solks-Floc substrate when the inocula were used
from cow K-l39.

mM PERCENT

63

  
  
 
 

Acetic acid

Fermentation with O. 1% dextrose added

Fermentation with O. 1% citric acid added

Propionic acid

-
_
-
“

 

_ ' n n 1
0 ' ' ‘ t i

4 8 12 16 20 24
TIME IN HOURS

 

Fig. 8. Volatile fatty acid production during cellulose fermen-
tation of alfalfa leaf meal substrate when the inocula were
used from traumatic gastritis animals.

64
significant differences observed between the two fermentations at 0, 4,
16 and 24 hours.

The prOpionic acid produced was maximum in.4 hours of fermentation
in both dextrose and citric acid fermentations and then demonstrated
apparent utilization. While the acetic acid produced was higher at the
4 hour period in dextrose fermentation than in citric acid fermentation,
during the 4-24 hour period of fermentation acetic acid production did
not change in either fermentation. Mast of the acetic acid production
in these fermentations was completed during the 0-4 hour period.

Figure 9 shows VFA production in traumatic gastritis animals with
Solka-Floc as substrate at O, 4, l6 and 24 hours of fermentation. There
was no difference in butyric acid production with 0.1 percent citric
acid as compared to 0.1 percent dextrose in the 0-16 hour period of
fermentation; however, lower amounts of butyric acid were noted at 24
hours with-the citric acid-sample.

Frapionic acid production was increased slightly in both cases
during the 0-16 hour fermentation period; however, during the 16-24 hour
period a marked increase in prOpionic acid production in the dextrose
fermentation over that in the citric acid fermentation was observed.
Acetic acid production was found higher in citric fermentation in the
0-16 hour period; however, there was no measurable amount of acetic acid
produced in the 0-4 hour fermentations in either the dextrose or citric
acid samples.

‘The total amount of VFA produced in O, 4, l6 and 24 hours of
fermentation.with alfalfa leaf meal and SolkaaFloc is presented in
‘Table-6. It shows that the 0.1 percent citric acid fermentation in-

»creased the total VFA production during the 4-16 hour period as

65

3' ‘27 LEGEND:
x——x Fermentation with O. 1% dextrose added 0
I
2' 8 'L o ------ o Fermentation with O. 1% citric acid added !

m M PERCENT

 

 

 

 

J
r

4 8 12 16 20 24

TIME IN HOURS

Fig. 9. Volatile fatty acid production during cellulose fermentation
of Solka-Floc substrate when the inocula were used from
traumatic gastritis animals.

66
compared to 0.1 percent dextrose when alfalfa leaf meal was used as
.substrate. There was no significant difference in the amounts of VFA
at 24 hours of fermentation.

TABLE 6. Total VFA produced during in_vitro cellulose fermentation
by inocula taken from cow K-139

 

 

 

 

Alfalfa leaf meal Solka-Floc
Fermentation
time in hours Dextrose Citric acid Dextrose Citric acid
0 1.885* 2.010 0.946 0.915
4 2.720 2.730 1.090 1.125
16 3.635 4.335 1.395 1.650
24 3.660 3.605 3.655 3.045

 

*Acids expressed in mM percent.

When Solka-Floc was used as a.substrate the total VFA production
during 16 hours of fermentation was similar in both groups; however, at
the end of the 24 hour period the total VFA production was lower with
-added citric acid than with dextrose.

Table 7 shows the total amount of VFA production at 0, 4, l6
and 24 hours of fermentation with alfalfa leaf meal and Solka-Floc as
substrates when the inocula used were taken from traumatic gastritis
animals.

VFA production was lower during the 0-4 hour period due to the
citric acid addition when alfalfa leaf meal was used as a substrate.
During the 4-16 hour fermentation period VFA production of the citric
-acid fermentation was increased over that of the dextrose fermenta-

tion. After 24 hours of fermentation there was no significant

67
difference in the amount of VFA in the two-samples. The difference in
-VFA production between dextrose and citric acid fermentations was-not
'measurable until after 4 hours of fermentation when Solka-Floc was used
as a substrate. During the 4-16 hour period the citric acid fermentation
produced more VFA and from 16-24 hours the VFA production in both dextrose
and citric acid fermenters was marked but essentially the same.

TABLE 7. Total VFA produced during ig_vitro cellulose fermentation
by inocula from traumatic gastritis animals

 

 

 

 

 

 

Alfalfa leaf meal Solka-Floc
Fermentation 0.1 percent 0.1 percent 0.1 percent 0.1 percent
time in hours Dextrose Citric acid Dextrose Citric acid
0 1.490* 1.460 1.343 1.233
4 3.809 3.495 1.305 1.275
16 3.465 3.750 1.635 1.745
24 3.685 3.575 4.360 4.260

 

*Acids expressed in mM percent.

Section IV

Experimental

Colorimetric determination 2£_citric acid

 

Citric acid in the fermentation mixtures at 0, 4, 16 and 24 hours
was determined colorimetrically based on the Furth and Herrman reaction

(1935) modified by Hartford.1

Eguipment

la) A calorimeter equipped to measure color intensity at 420 mu

 

1Personal communication, 1961.

68
b) A water-bath set at a temperature of 32 i_0.25 C.
c) Self-filling 10 m1 microburette from both acetic anhydride

and pyridine.

Reagents

a) Reagent grade acetic anhydride and Baker analysed reagent-
pyridine solution.

b) Citric acid stock standard solution of 69.9069 grams of trisodium
citrate dehydrate (Reagent grade) made up to one liter with
distilled water. This solution is equivalent to 50,000 pg
citrate per m1. Dilute standards were made from this stock

solution and stored at 4 C.

 

Preparation g£_test samples

The supernatant fluids of the centrifuged fermentation mixtures
at O, 4, 16 and 24 hours, stored at ~10 C were brought to room temperature
and recentrifuged at 800 x G for 10 minutes. One ml of the supernatent

fluid was used for the colormetric determination of citric acid.

Preparation 2£_standard samples

 

 

Dilutions were made from the stock solutions containing 50,000
pg citric acid per m1. Dilutions containing 5, 10, 25, 50, 75, 100,
125, 150, 200, 250 and 350‘pg citrate per ml were used for standard
curve, plotting Optical density versus pg equivalent of citric acid

monohydrate of the above concentrations.

Procedure
One ml of test sample, standard or blank was added to a colori-

meter tube. Then 1.30 ml of pyridine were added and the tube was swirled

69
briskly. After similar additions had been made_to all tubes inta
.series, 5.70 ml of acetic anhydride were added and the tube swirled
again and immediately placed in the constant temperature (32-C) water
bath for 30 minutes. Color intensity at 420 my was read with the
blank set at 0.000 Optical density. Blank determinations were run with
each experiment. The-standard curve was made by taking the average
rof five repeated trials at different times with each concentration of
citrate as described above. The citric acid content of the fermenta-

tion mixture was estimated with reference to the standard curve.

Results

One thousand,pg;citric acid per ml was added to the 0 hr fermen-
ter and the colorimetric reading gave the initial concentration of
-citric acid for the test fermentations. »The,pg of citric acid recovered
at 4, 16 and 24 hours of fermentation~was subtracted from the 0 hr
.reading to obtain the citric acid disappearance during these periods of
fermentation. It is shown (Fig. 10 and Fig. 11) that the 0 and 24 hour
fermentation-samples, without citric acid and dextrose added, had
essentially the same colorimetric reading. The calculated amounts of
-citric acid with reference to the standard curve are shown in Table 8.
This also shows the percent of citric acid disappearance in 0, 4, 16
and 24 hours of fermentation with Solka-Floc and alfalfa leaf meal when
inocula were used from normal animals. Seventy-four percent of the
[total citric acid disappeared in 4 hours of fermentation with alfalfa
leaf meal and Solka-Floc with inocula from normal animals. At 16 and
24 hours of fermentation the remaining citric acid disappeared. ‘The
Arate-of percent disappearance was slightly lower with Solka-Floc than

with alfalfa leaf meal.

p g . CITRIC ACID

70

1200 L
LEGEND:

\ x————x Normal animals

\ >:< ------ :zc Traumatic gastritis animals

\ \ o———o Blank

800 "

700 '"

I

600 ‘

500 'J'

400 "'

300

200 ‘

 

100 *-

 

: “FTC—”"5““?

A
I l

0 4 8 12 16 20 24
FERMENTATION TIME IN HOURS
Fig. 10. Citric acid disappearance during cellulose fermen-

tation of alfalfa leaf meal substrate by inocula taken
from normal and traumatic gastritis animals.

pg CITRIC ACID

1200

1100
1000
900
800
700
600
500
400
300
200

100

Fig.

 

71

 

\ LEGEND:
( \\\
\‘w x x Normal animals
\\
t \x * """ * Traumatic gastritis animals

Blank

\ O-————O

 

11.

4 8 12 16 20

FERMENTATION TIME IN HOURS
Citric acid disappearance during cellulose fermentation

of Solka-Floc substrate by inocula from normal and
traumatic gastritis animals.

72

 

 

 

n.~m o.mmm o.mNH . nm.om m.mom o.m- «N

n.Hm o.mHm o.mmH mm.mm m.mmm o.wHH 0H

mm.wo m.mwo m.mom mN.¢m m.~¢m o.mmm a

o o o.omoH o o m.nnoa o
concomamuwp ooumoadmmflo He won ooumoaammwo oouooaaomflo HE and mason a“ mafia
udoouom owed owuuwo owes uwuuwu udoouom owes ofluuwo ofium ofiuuwu. cowumucoahom

mo ml mom; mo wafl mow;
oonamxHom Home mooH mmHme<

 

 

 

 

1

 

mHmeam Hmauom.aoumycoxmurmfisoocfl sows

onuw>.mm coccuooammmwo uwom,uwnuflo mo uncouom .w mqm<H

Table 9 shows the percent of disappearance of citric acid in
.inocula from traumatic gastritis animals. .In both alfalfa leaf meal

.and Solka-Floc substrates only 16.2 percent and 13.2 percent-respectively
of citric acid had disappeared at 4 hours of fermentation, while at 16
hours 93.9 percent of the citric acid had disappeared with alfalfa

leaf meal as substrate as compared to 68.2 percent with Solka-Floc.

After 24 hours all of the citric acid had disappeared from the fermen-
tation samples with alfalfa leaf meal as substrate while 93.2 percent

of the citric acid had disappeared with SolkaaFloc as substrate.

Figure 10 shows a comparison of the citric acid disappearance
with alfalfa leaf meal as substrate in normal and traumatic gastritis
animals. In normal animals 75 percent of the total citric acid dis-
appeared in 4 hours of fermentation as compared to traumatic gastritis
animals where only 16 percent of citric acid disappeared from the
fermentation mixture.

In Figure 11 citric acid disappearance is shown with Solka-Floc
-substrate in normal and traumatic gastritis animals. Two-thirds of
the total.citric acid disappeared during 4 hours of fermentation in
normal animals while in traumatic gastritis animals the rate of dis-
appearance was slow and most of the citric acid did not disappear until
the 4-16 hour period of fermentation. After 24 hours of fermentation
93.2 percent and 92.5 percent of the citric acid disappeared in traumatic

gastritis and normal animals respectively.

74

 

 

 

 

 

m~.mm m.Nmm o.oo~ m.ooH mood o.nNH om

mm.mo m.mwo 0.0mm mm.mm m.mmm m.NmH 0H

mN.mH m.NmH o.oooH N.oH o.NoH o.omm q

o o m.NmHH c o o.NmHH o
nonmoQQMmfio woumoddmwwo He won poumomdmmwo ooumoadmmwo He won mason a“ mafia.
acouuom owns owuuwo owom oauuwo unmouom omen owuuwo ofiom owuuwu dowumucoauom

mo m1 mo wax mo wax moiwl
oofimumxaom Home mama oMHmMH<
mfimawcm mwuwuuomw-uwumabmuu Eoum
:oxmo mfiouocfi sows onuw>.mw ouomumoaaoufio poop owuuwo mo ucooHom .m mamma

VDISCUSSION

Percent cellulose digestion by inocula from normal
land traumatic gastritis animals with dextrose
and.citric acids added

 

 

 

The data of percent cellulose digestion by both normal and
traumatic gastritis animals show that no significant difference is
effected by the addition of 0.1 percent citric acid. .(Tables 2,

3 and 4). It is interesting to note the decrease in the percent of
cellulose digested in all substrateswhen 0.2 percent citric-acid was
uused.in norma1.cows. The percent of cellulose digestion in all three
=substrates was considerably-lower than that.reported by previous
”workers for igygigggpcellulose»digestion studies (Huhtanen and Elliott
1956, Salsbury 1955). One-tenth percent dextrose maintained the
normal percent of cellulose digestion in 24 hours. This is in agree-
ment with the findings of Salsbury (1955), Burrough gt 1. (1950),

fand Huhtanen £2. 1. (1954).

Among the three substrates used, (alfalfa leaf meal, Solka-Floc
and timothy hay) the cellulose of alfalfa leaf meal was most rapidly
attacked, timothy hay was intermediate and Solka-Floc was most re-
~sistent. _This is in excellent agreement with the results-obtained
by Huhtanen and Elliott (1956).

The difference in percent cellulose digestion by the addition
of 0.1 percent citric acid was not significant as compared to that with
0.1 percent dextrose. Dextrose or citric acid in 0.05 percent con-

centration lowered the percent of cellulose digestion in 24 hours of

fermentation when Solka-Floc was used as a substrate. The 0.01 percent

76
concentration of citric acid tended to increase the percent of cellulose
digested as-compsred to the 0.01 percent dextrose; however these.values
'were-belOWrthose of the 0.1 percent level. Salsbury (1955) reported
that the percent cellulose digested increased gradually as the glucose
econcentration was increased from 0.005 to 0.15 percent, but the dif-
ferences obtained in his experiments were small. He found agreement
among replicates best when no glucose was added and when 0.2 percent
glucose was added.

Hoflund, Quin and Clark (1948) have shown that small amounts of
glucose stimulate cellulose digestion, but the methods used were too
emperical to permit the estimation of the percent cellulose digested.
Arias SE 51, (1951) reported that the addition of readily available
carbohydrate at low concentrations stimulates cellulose digestion.
These workers used serial transfer of the fermentations according to
the method of Burroughs g£_§1, (1950) and averaged the results. They
.suggested the possibility that the stimulating effect was actually the
overcoming of depressing forces such as subOptimal conditions or
nutritional deficiences.

The small amount of citric acid added in the fermentation mix-
tures shows greater though not significant increases in cellulose
digestion, than that with dextrose. This indicates that citric acid
can be degraded by the rumen microorganisms as a readily metabolizable
energy source. As can be seen in Table 2 the effect of citric acid
noted was only evident at 8 hours-of fermentation, but, after 24 hours
the change was slight or none. This is possibly because citric acid
was utilized in the 8 hour period of fermentation.

There were wide variations in the percent of cellulose digestion

77
as can be seen in Table 3 when the inocula were taken from different
'animals. The variation was marked when inocula were-used.from cow
A-130 with alfalfa leaf meal as substrate and from cow K-139 with
Solka-Floc-as-substrate. It seems likely that the microbial pOpula-
tion of the rumen might vary according to the type of forage used as
substrate. Variations were less in the fermentation trials with the
other experimental animals and different forages with 0.1 percent citric
acid, while the variations were rather larger when 0.05 percent and
0.01 percent citric acid concentrations were used.

When the inocula were used from traumatic gastritis cases and
the activity of rumen microorganisms was tested with alfalfa leaf meal
and Solka-Floc, variations in the percent of cellulose digested in
the Solka-Floc substrate were greater at 8 hour of fermentation as
compared to that with alfalfa leaf meal. This could be explained on
the basis of the finding by Donefer 25 El: (1961) that digestion of
.Solka-Floc was much more affected by the source and type of inoculum
.than were any of the other forages tested.

The average percent cellulose digestion in the 8 hour fermen-
tation period with alfalfa leaf meal as a substrate was higher than
that with Solka-Floc;,however, in the 24 hour period the average percent
cellulose digestion with Solka-Floc as substrate was greater. These
results are in agreement with those reported by Salsbury (1955) in that
the rate of digestion of Solka-Floc was slower in the first part of the
incubation and faster in the latter part, while the reverse was true
with alfalfa leaf meal. This may be due partially to the fact that the
alfalfa leaf meal provide nutrients not furnished by Solka-Floc and

the lack of crystallinity of the alfalfa leaf meal cullulose. The

78
addition of 0.1 percent citric acid as compared to the Solka-Floc‘fermen-
tation increases in the percent.of cellulose digestion-and it more
closely approximates that observed at 8 hour of fermentation.with alfalfa
leaf meal. It-is possible that the rumen microorganisms were not
adapted to the Solka-Floc substrate and the lower activity and retarded
rumen movements due to traumatic gastritis. Solka—Floc is not the
usual feed of cattle and warner (1956) has reported that wide varia-
tion of cellulose digestion can be seen with feeds other than the
normal feed of the animal whichprovide the inocula “till-11932, studies.
.Furthermore he suggested the use of a substrate-similar to the feed
of the animal that provides the inocula for ip;giggg,fermentation to
achieve proper activity of the rumen microorganisms. With alfalfa
leaf meal as substrate the addition of 0.1 percent citric acid increased
the percent cellulose digestion over that of dextrose in the 8 hour
fermentation period. Since-the Crampton and Maynard (1938) method of
the determination of cellulose digestion also includes some hemicellulose,
the faster initial rate with alfalfa leaf meal may reflect a more rapid
digestion of hemicellulose.

In the 24 hour fermentation period the average percent cellulose
digestion by 0.1 percent citric acid was essentially the same in both.
This may be explained by the fact that citric acid had disappeared by
.24 hour of fermentation. In any event the change due to 0.1 percent
citric acid was not significant as calculated by the t-test as can be
seen.from Fig. 5.

Table 5 shows that, after.4 hours of fermentation there was a
-considerable increase in cellulose digestion due to 0.1 percent citric
acid with alfalfa.leaf meal with inocula from normal animals while

there was little change_in the percent of cellulose digestion observed

79
in traumatic gastritis animals. This would suggest the activities
of the rumen microorganisms from.traumstic gastritis animals were
lower than those from the normal animals. There is no other evidence
available to confirm this. After 8 hour of fermentation the percent
of cellulose digested in 0.1 percent citric acid or dextrose fermentation
was similar in both normal and traumatic gastritis animals when alfalfa
leaf meal was used as substrate. This indicates that the microorganisms
of the traumatic gastritis animals were equally as active as those of
the normal animals after 8 hours of fermentation. After 24 hours the
percent cellulose digestion in both 0.1 percent citric acid and dextrose
fermentations by rumen inocula from normal and traumatic gastritis
-animals were essentially the same. .This.indicates that the 0.1 percent
citric acid was effective only in the early phase of cellulose digestion
by-rumenrmicroorganisms.

When Solka-Floc was used as a substrate the percent of cellulose
digested after addition.of citric acid was-not affected_in the traumatic
gastritis animals at the 4 hour fermentation period. Since pure cellu-
lose is attacked at a slower rate, according to the report by Huhtanen

and Elliott (1956), it is possible that the citric acid was attacked
.instead of cellulose by the rumen microorganisms during_the 0-4 hour
period. By the 8 hour period the increment in cellulose digestion in
the 0.1 percent citric acid sample-shows that the rumen microorganisms
‘were adapted to the Solka-Floc-substrate. _The cellulolytic activity
-of the rumen microorganisms in traumatic gastritis cases was found to
be less.

During the 16-24 hour period of fermentation the percent of the

~cellulose digested with 0.1 percent citric acid or dextrose added was

80
essentially the.same with inocula from normal and traumatic gastritis
animals when alfalfa leaf meal was used as a substrate.

Although the increase in the percent of cellulose digestion_in
both substrates with 0.1 percent citric acid was demonstrated in cer-
tain cases further work is required to claim the definite superiority
of citric acid over dextrose as an adjuvant in cellulose digestion by
rumen microorganisms. It could be fruitful in the improvement of
cellulose digestion by traumatic gastritis animals which exhibit anorexic
conditions and it may aid the recovery of traumatic gastritis animals

after the foreign body has been removed.

Vgé production _a_s_ avcriterion of ill 3355.9. cellulose digestion

Gray, Pilgrim-and weller (1951), Warner (1956) and Barnett and
Reid (1957) have reported volatile fatty acids as’the end products of
cellulose fermentation and VFA production as an index to measure
cellulose digestion to their i§_vit£g.studies.

The precise role of microflora and the importance of VFA pro-
duction is controversial. One school of thought stresses the nutri-
tional importance of VFA found in the rumen. This view is typified by
the work of Phillipson and McAnally (1942), and Barcoft _£__l, (1944).
The Opposing view is that the VFA are of little nutritional value to
the animal and that it is the microflora itself which is important,
serving as a source of carbohydrate, protein and B-complex vitamins.
This later point is favored by Baker (1942) who-stated that it is
the substances synthesized such as microbial protein and poly—

saccharides, rather than the initial products of decomposition such as

organic acids that are utilized by the host animals. It is assumed by

81
Baker (1942) that the organisms convert the starch and fiber of the diet
rinto bacterial starch. These organisms laden with starch pass through
the forestomachs into the small intestine where the starch is hydrolysed
to glucose by the pancreatic and intestinal amylases.

Pearson and Smith (1943) have-shown that because of the increased
amount of material available for digestion, a higher percent of VFA was
produced in in xi££2_fermentation experiments and absorption of VFA
was absent. Therefore one may expect a slightly higher proportion of
acid to be present in vitrg than i2_!i!2_(Elsden 1946, and Davey g£_al.,
1960).

Gray and Pilgrim (1952) reported that as the concentration of
total VFA in the rumen increased there was a decrease in the ratio of
acetic to prepionic acid. A similar difference was shown in the experi-
ments of Elshazly (1952) between samples taken before and after feeding
but not in the experiments of Shambye-and Phillipson (1949) where
high concentrations of lactic acid were observed and the ratio of acetic
to propionic acid was unusually low. Gray 23 _l. (1951) have reported
'that fatty acids produced could be taken to represent those actually
found in the rumen. They stated that the fatty acids are removed con-
tinuously by absorption through the rumen wall and failure to reproduce
this phenomenon i2_vi££g may have some influence on the rate of fer-
mentation and the prOportions in which the acid products are found.

Barnett and Reid (1957), Sijpestejn (1951), Sijpestejn and Elsden
(1952) and Hungate (1950) suggest that certain microorganisms, for
example Ruminococcus flavifaciens and Bacteriodessuccinogenes, break-
}down cellulose to succinic acid which in turn is rapidly converted to

prepionic acid presumably.by decarboxylation. -These findings have been

82
confirmed by inVXEXQDstudiesand it is significant that succinic acid
does not accumulate in the rumen. Elsden (1946) has shown that lactic
acid is an intermediate in short‘chain.VFA production in the rumen.

He found that prepionic acid is the acid produced in the greatest
amount; however, Gray and Pilgrim (1952), Gutierrez (1953) and Davey
gt a}, (1960) found acetic acid in the greatest amount. Elsden,
Volcani, Gilchrist and Lewis (1956) found that cellulose was rapidly
fermented with the production of VFA and considerable quantities of gas.
They found that glucose used in the fermentation was exhausted within
24 hours, and acetic acid predominated i2_vizg_fermentation whereas
prOpionic was the main product in_gi££g,

The results obtained_in these studies agree in part with the
results of Elsden 25.31, (1956) but in contrast to his results acetic
acid predominated as can be seen from Figure 6.

The production of acetic acid with 0.1 percent citric acid added
was-observed to be considerably higher after 16 hours of fermentation
when alfalfa leaf meal was used as a substrate. This increase could
be due to the acetic acid produced from.the citric acid, or to the
utilization of butyric acid and propionic acid. Acetate accumulation
was reported by Doestch, Robinson, and Shaw (1952) as a result of
rapid oxidation of maltose and glucose in in zitrg fermentation. After
24 hours-of fermentation the production of VFA was in close agreement
with the percent cellulose digested in both 0.1 percent dextrose and
0.1 percent citric acid.

When Solka-Floc was used as a substrate similar results were

obtained as with alfalfa leaf meal as substrate, with 0.1 percent added

83
dextrose or citric acid. The production of acetic acid was not markedly
higher at 16 hours of fermentation but the increase was evident during
the 0-16 hour period of fermentation as can be seen.from Figure 7. The
amount of each volatile fatty acid produced was similar in both sub-
strates. These results are in agreement with the report by Church and
Peterson (1960) that VFA production was dependent upon the amount of
cellulose in the fermentation.

Figure 8 shows the production of VFA by inocula taken from
animals with traumatic gastritis. A great deal of variation was en-
countered in the fermentation at different time intervals. The total
VFA produced with alfalfa leaf meal as substrate in 0.1 percent dextrose
or citric acid was produced in 4 hours. At 16 hours the total VFA pro-
duced was higher in the fermentation containing citric acid. At 24
hours no difference in total VFA was observed.

When Solka-Floc was used the results were similar until 16 hours
of fermentation. After 24 hours the total VFA was lower with added
citric acid but the difference was not statistically significant.

It can be seen from Table 7 that in the traumatic gastritis
'cases the total VFA produced with alfalfa leaf meal and Solka-Floc as
‘substrates was lower when 0.1 percent citric acid was added during the
0-4 hour period of fermentation while at the 16 hour period of total
VFA was slightly higher in citric acid fermentation. This suggests
that the activity of microorganism was greater during 4-16 hours of
fermentation due to a delayed utilization of citric acid. However,
the difference in total VFA production with 0.1 percent dextrose and
0.1 percent citric acid was not significant with both Solka-Floc and

alfalfa leaf meal substrates. This is in agreement with the results

84

obtained.for the percent cellulose digestion with the two substrates.

This is also in agreement with the result obtained by Jacobson
(1958) who reported that animals showing anorexia had an essentially
normal fatty acid ratios in the rumen; however, the corresponding fatty
acid production in zi££2_wasquite unusual. He suggested that even .
though total fatty acid production in the rumen was altered, the molar
percentage of the fatty acids present in the rumen may remain within
the normal range.

The total VFA fluctuation during the course of the experiments
with inocula from traumatic gastritis animals may be due to variable
diets, the degree of sickness of the animals and the interval of

feeding time.

Citric acid disappearance

The disappearance of citric acid from the in zi££2_rumen fermen-
tation was followed in order to gain knowledge whether citric acid was
~metabolized by the rumen microorganisms. The time and magnitude of dis-
appearance was thought to be in accordance with the cellulose digestion
and VFA production. .The utilization.of citric acid by rumen micro-
organism has not been demonstrated. However, dissimilation of compounds
-of the citric acid cycle, such as succinate degradation, has been studied
by Sijpestejn and Elsden (1952), Johns (1948) and Doetsch, Robinson,
Brown, and Shaw (1953). Pyruvate, fumarate, malate, and oxaloacetate
are degraded by rumen microorganisms with the production of gases and
volatile fatty acids (Doetsch 25.31,, 1953). They established that

lactate is not attacked with the production of detectable VFA and

fumarate yields solely acetate.

85

The results obtained in the experiments in which the disappearance
of citric acid from the fermentation mixture was followed showed that 75
percent of the citric acid disappeared during the 0-4 hour period of
fermentation when the inocula were obtained from normal animals and
alfalfa leaf meal was the substrate. When Solka-Floc was used as a
substrate 68 percent of citric acid disappeared from the fermentation
mixture (Table 8). This shows the relatively slow activity of rumen
microorganisms with Solka-Floc as compared to alfalfa leaf meal. This
is in agreement with the work reported by Salsbury (1955) and Huhtanen
and Elliott (1956) who reported that with Solka-Floc as substrate the
in zi££2_activity of rumen microorganisms was slower during the first
period of fermentation.

Doestch g£__l, (1953) using pyruvate as substrate found that
pyruvate was always vigorously attacked and was not detectable in the
cell substrate mixture after 5 hours of fermentation. They indicated
that pyruvate must be utilized as rapidly as it is formed and that the
anaerobic mechanism of the rumen process is one involving low energy
yielding anaerobic dissimilations.

When inocula were from traumatic gastritis animals the citric
acid disappearance at 4 hours of fermentation with both substrates was
very slow. This supports the idea that the activity of rumen micro-
organisms from animals suffering from traumatic gastritis is lower as
compared to normal animals. Jacobson _£._l: (1958) reported that
animals off-feed may have an abnormal bacterial metabolism though it
is not clear as to whether the change would proceed or follow the

development of inappetance.

After 16 hours of fermentation using inocula from traumatic

86
gastritis animals 93 percent of the citric acid disappeared when alfalfa
leaf meal was used as a substrate and 68 percent disappearance was
observed with Solka-Floc. This again shows the relative slow activity
of rumen microorganisms with Solka-Floc as a substrate. At 24 hour all
of the citric had disappeared with alfalfa leaf meal as a substrate and

93 percent of the citric acid disappeared with Solka-Floc as a substrate.

SUMMARY

£n_!i££g_cellulose digestion studies were undertaken to test the
effect of citric acid concentrations of 0.2 percent, 0.1 percent, 0.05
percent and 0.01 percent, on cellulolytic activity of rumen micro-
organisms obtained from normal fistulated animals. Alfalfa leaf meal,
Solka-Floc and timothy hay were used as sources of cellulose. A11 con—
trol fermentations contained equivalent amounts of dextrose as well as
the cellulose source. The percent of cellulose digestion was deter-
mined at O, 4, 8, 12, 16 and 24 hours of fermentation. The difference
in percent cellulose digestion between the dextrose and citric acid
fermentations was found not to be statistically_significant at the 5
percent level (t .975, )» 0.05 percent). The 0.1 percent citric acid
concentration was found suitable to work with during further
experiments.

Rumen flora obtained from nine animals with traumatic gastritis
were used as sources of inocula to observe the affect of 0.1 percent
citric acid when added to the Solka-Floc and alfalfa leaf meal. No
‘significant difference was found in percent cellulose digestion by
adding 0.1 percent citric acid in the test fermentations. Wide vari-
ability in the results was postulated to be due to the condition of the
inocula obtained from the different animals.

Volatile fatty acids were determined on Celite 535 columns at
0, 4, 16 and 24 hours of fermentations when inocula from normal and

traumatic gastritis animals were used. .The kinds of VFA produced

88
were acetic acid, propionic acid, and butyric acid. No significant
differences in the VFA production between the fermentations with 0.1
percent dextrose and 0.1 percent citric acid were found in normal and
traumatic gastritis animals. Variability in VFA production with
inocula from traumatic gastritis animals was observed.

Citric acid disappearance was determined from the fermenters
at 0, 4, l6 and 24 hour periods. It was found that about two-thirds of
the total citric acid disappeared in the initial 4 hour fermentation
period when alfalfa leaf meal and Solka-Floc were used as substrates
and the inocula were obtained from normal animals. Only about one-
sixth of the total citric acid disappeared when the source of inocula
was traumatic gastritis animals. After 16 hours of fermentation most
.of the citric acid had disappeared in both situations. Factors causing
variability in the percent cellulose digestion and VFA production are
discussed.

It is concluded that under the experimental conditions used in
these studies there was no statistically significant effect of adding
,0-1 percent citric acid on the percent cellulose digestion and VFA
production when inocula were obtained from normal and traumatic gastritis

animals.

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