INVES‘e‘I’IGATIQN OF THE OXIDATION-REQUCTION
PQTENIIAL OF RUMEN CGNTENTS

Thesis {or “we Degree of M. 5.
MICHIGAN STATE UNIVERSITY

Ransom L. Baldwin
1958

THESIS

INVESTIGATION OF THE OXIDATION-REDUCTION
POTENTIAL OF RUMEN CONTENTS

By

Ransom L. Baldwin

A THESIS

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

MASTER OF SCIENCE

Department of Dairy

1958

 

 

‘
~i.
. .
,

c
o
H»,

 

 

 

 

 

ACKNOWLEDGEMENTS

The author expresses his sincere appreciation to Dr.
R. S. Emery, Dairy Department, for his guidance and assist-
ance throughout this work and for his critical reading of
this manuscript.

Thanks are extended to T. R. Lewis, Dairy Department,
for his helpful criticisms and aid in various phases of the
investigation.

The writer wishes especially to acknowledge his grati-
tude and indebtedness to his wife, Mary Ellen, whose sacri-

fices and encouragement made this study possible.

 

 

1|Il ,..L.\.U.u1. l

INVESTIGATION OF THE OXIDATION-REDUCTION
POTENTIAL OF RUMEN CONTENTS

By

Ransom L. Baldwin

AN ABSTRACT

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

MASTER OF SCIENCE

Department of Dairy

1958

, .7) / ‘7

,. J -
Approved an, 7- ', (.J7W/7’7 .
* I
/,.

ABSTRACT Ransom L. Baldwin

The oxidation-reduction potential of rumen contents
removed from several cows at three times during the day in-
dicates that a close relationship exists between Eh and
metabolic rate. Titration of samples, from which the large
feed particles had been removed by centrifugation, with acid
and a strong oxidizing agent, respectively, indicated that a
linear relationship exists between Eh and pH and that the
metabolic rate of the microflora is reflected by the amounts
of reducing substances found. Very little of the reducing
capacity of acidified rumen contents was found to be associ-
ated with the bacterial cells indicating that extreme care
must be taken to maintain the prOper redox potential when
the rumen microflora are separated from their natural en-
vironment if a normal inocula is desired. The titration
curve for rumen contents indicates that there is no large
amount of any one reductive substance present that is re-
sponsible for the potential observed in rumen contents.

The only rumen function that seemed to be affected by
alteration of the reductive characteristics of rumen fluid
was gas production. Additives employed were DPPD, methylene
blue, cysteine and thioglycollate. The explanation sug-

gested for decreases in the per cent of methane produced was

ABSTRACT Ransom L. Baldwin

that the potential may have been stabilized at a level higher
than the Optimum level for methane producing bacteria. Addi-
tion of oxygen affected neither the redox potential nor the
gas, volatile fatty acid or ammonia production of rumen con-
tents incubated ig gitgg. Methylene blue did not affect gas
production in cultures of rumen flora that were stabilized

with formate as their sole carbon source.

TABLE OF CONTENTS

INTRODUCTION 0 C O O O O O O O O O O O O O O O O 0

REVIEW OF LITERATURE O O O O O O O O O O O O O O 0

PART

PART

Explanation of Oxidation-Reduction Potentials

Some BiOIOgically Active Oxidation-Reduction
Systems . . . . . . . . . . . . . . . . . .

Pathways of Carbohydrate Metabolism in the
Rum en 0 O O O O O O O O O O O O O O O O O O

lE.I$E£2 Techniques . . . . . . . . . . . . .
Redox Potentials in Bacterial Systems °.° . .
I. REDOX POTENTIAL OF BOVINE RUMEN CONTENTS.
Procedure . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . .

II. STABILITY AND IMPORTANCE OF THE REDOX
POTENTIAL IN RUMEN CONTENTS . . . . . . . . .

Procedure . . . . . . . . . . . . . . . . . .

Results 0 O O O O O O O O O O O O O O O O O 0

DISCUSSION 0 O O O O O O O O O O O O O O O O O O C

SUMMARY 0 O O O O O O O O O O O O O O O O O O O O

APPEN.DIX O O O O O O O O O O O O O O O O O O O O 0

LITERATURE CITED . . . . . . . . . . . . . . . . .

18
24
28
55

55
56

41
41

SO
58
6O
66

Table

LIST OF TABLES

Relationship Between Eh and pH . . . . . .

Proportion of Reducing Substances Present
in the Various Fractions of Rumen Fluid .

Potentiometric Titrations of Fraction B .
Eh and pH of Fraction A . . . . . . . . .

Effect of Thioglycollate and Cysteine Upon

Eh O O O O O O O O O O O O O O O O O O O 0

Effect of Thioglycollate and Cysteine Upon
Gas Production . . . . . . . . . . . . . .

Effect of Methylene Blue and Ascorbic Acid
Upon Eh and Gas Production . . . . . . . .

Effect of Methylene Blue and Oxygen Upon
In Vitro Fermentations

LIST OF APPENDIX TABLES

Potentiometric Titrations of Fraction B .

Effect of DPPD Upon End Products . . . . .

.Effect of Thioglycollate and Cysteine Upon

Production of the Volatile Fatty Acids and
Ammonia o o o o o o o o o o o o o o o o 0

Effect of Methylene Blue and Ascorbic Acid
Upon Volatile Fatty Acids and Ammonia . .

Changes in Gas Composition During Culture
Stabilization . . . . . . . . . . . . . .

Page
56

37
58
4O

45

46

47

48

61
62

65

65

INTRODUCTION

Oxidation-reduction systems play an intimate and essen-
tial part in the processes of life. In fact, life could be
defined as a continuous oxidation-reduction reaction. The
end products of rumen fermentation arise as a result of the
interaction of many oxidation-reduction systems. Very little
is known about these systems and the environment in which they
act.

The purpose of this investigation is to study the
oxidation-reduction potential which exists in the rumen, the
stability of this potential and the effect alterations of

the potential have upon several rumen end products.

REVIEW OF LITERATURE
Explanation of Oxidation-Reduction Potentials

Taken in the general sense, oxidation occurs whenever
an atom loses electrons, while reduction takes place when an
atom.gains electrons. The oxidation-reduction (redox) poten-
tial measures the tendency to take on or lose electrons on a
relative scale. The basis of the scale is the standard hydro-
gen electrode which is arbitrarily assigned a value of zero.
Biological convention assigns a positive value to compounds
that are oxidized by hydrogen (Hewitt, 1950). Giese (1957)
indicates that there are four variables affecting the meas-
ured potential (Eh): the ratio of the concentrations of the
oxidized and reduced forms of a compound, the hydrogen ion
concentration, the valence change involved and the tempera-

ture. This is summarized in Peter's equation (West and Todd,

1957):

RT [oxidan RT +
Eh = E0 + E In Qeductanéi + F ln [H I

where: R the gas constant (8.515 joules per degree)

T = the absolute temperature
n a valency change involved
F a the Faraday (96,000 coulombs)

E = normal potential when [oxidanfl = [reductanfl and

Eh: observed potential
- 2 -

at 50° when n a l
oxidant-IT

Pardee (1954) states that every compound may be con-

 

sidered to have a definite amount of free energy (F) stored
in it under any set of conditions, mostly as the energy of
bonds holding the atoms together. He further defines the
change in free energy (AF) as the difference between the
free energies of one mole of reactant and its products under
standard conditions. Eh is proportional to the maximum work
potential of each electron transferred between the hydrogen
electrode and the redox system under study (Clark, 1948).
Thus, it is possible to estimate the work available from the
reaction of one mole by subtracting the Eh value of the re-
actant from the Eh value of the products and converting to
AF according to the equation:

11F . -nF (Ehp - Ehr)

where:

AF :- change in free energy or the work available from
the reaction

n a no. of equivalents per mole
F a Farraday (25,068 cal. per volt equivalent)
E

hp Eh of the product

E

hr F$ of reactants

In order for a reaction to go spontaneouslyAF must be

negative; i.e. the products must possess less free energy

4

than the reactants. WhenJAF of a reaction is zero the reac-
tion tends to go equally in both directions and the reactants
and products are at equilibrium. 13F and subsequently Eh can
also be calculated from thermodynamic data (Pardee, 1954).

The most direct method of determining the normal poten-
tials (E0) of redox systems is the electrometric measurement
of equimolar mixtures of the two components (oxidant and re-
ductant), while the more convenient method and in some cases
the only feasible one is afforded by potentiometric titra-
tion of the reduced component (Ryklan and Schmidt, 1944).
Some systems have so far resisted attempts to make them
amenable to electrical study while others can only be
studied by means of mediators that are electromotively
active (Clark, 1948; West and Todd, 1957).

The standard hydrogen electrode is difficult to main-
tain. Therefore, in practice a reference electrode that has
a known potential relative to the hydrogen electrode is em-
ployed (Clark, 1948). Some commonly used reference elec-
trodes are the calomel, the quinhydrone, and the silver:
silver chloride (Hewitt, 1950). The potential of the calo-
mel electrode varies with the concentration of KCl used in
its manufacture. Hewitt (1950) finds the 5.5 normal KCl
electrode most satisfactory for measurements in biological

systems because of its low temperature coefficient and

stability. The quinhydrone electrode is very sensitive to
pH changes and must be prepared fresh daily (Clark, 1948).
The silver: silver chloride electrode is becoming popular
and has a lower potential than the calomel (Hewitt, 1950).

The electromotive force between the reference electrode
and a oxidation-reduction system can be measured with any
volt meter with sufficient sensitivity. An inert electrode
is necessary to carry electrons from the redox system to
the volt meter (Clark, 1948). Hewitt (1950) recommends the
bright platinum electrode for measurements in biological
systems since it is readily prepared and cleaned, and yields
steady, reproducible readings. Longsworth and MacInnes (1955)
prefer the gold electrode for investigations of bacterial
cultures. Lepper and Martin (1951) report that the pure
graphite electrode is unsuitable for continual use and that
electrodes of Iriduim can be used successfully.

The electrical circuit between the reference electrode
and the solution of reactants must be completed without per-
mitting the two systems to mix. The liquid: liquid junc-
tion typified by the salt bridge made of KCl saturated water
and 2% agar performed very satisfactorily for Clark (1928)
and Hewitt (1950).

Complete descriptions of apparatus suitable for the
continual measurement of Eh in bacterial cultures and of E0
in organic redox systems appear in Hewitt (1950, 1950),
Clark (1928) and Ryklan and Schmidt (1944).

Compounds that change color when they are transformed
from the oxidized to the reduced state can be used as Eh
indicators in much the same manner in which weak acids and
bases that change color when they become ionized can be em-
ployed as pH indicators. Clark and his collaborators (1928)
have characterized a considerable number of dyes which behave
as oxidation-reduction indicators. This data has been com-
piled in tabular form by Hewitt (1950). With the help of
Clark's data it is possible to measure the Eh of biological
systems providing, of course, that the color change is ob-
servable in the system under investigation. Dyes as indi-
cators of Eh are not in widespread use due to difficulty in
the interpretation of data obtained.

Ball and Chen warned in 1955 that: "Results obtained
by means of oxidation-reduction indicators on unstable sys-
tems or on biological material where such systems are present,
must be interpreted with great care." Hewitt (1950) attaches
significance to the poising or capacity factor in biological
systems; i.e. the concentrations of oxidants and reductants
that make up the observed potential. Indicators measure the
existing Eh and not the capacity of a system to remain at that
Eh' Biological systems are under constant change and an in-
dicator that equilibrates slowly makes it nearly impossible
to determine electrode potentials at different periods

(Hewitt, 1950). The indicator dyes are reversible oxidation-

reduction systems (Clark, 1928) and as a result of this
property along with other prOperties such as basicity are
able to act as catalysts (Barron and Hoffman, 1950; Schott
and Borsook, 1955; Green 23 a1., 1954; Greville and Stern,
1935; Young, 1957) and as inhibitors (Perdrau and Todd, 1955;
Quastel and Wheatley, 1951; Chambers gt al., 1951).

Some Biologically Active O-R Systems

In this chapter attention is directed chiefly toward a
discussion of the potentiometric behavior and biological
significance of the sulfhydryl compounds, coenzyme I, the
flav0proteins, ascorbic acid, the tocOpherals, methylene
blue and several commercial antioxidants.

Although a considerable amount of experimental work
has been done on the estimation of the oxidation potentials
of sulphydryl compounds the subject is still in a state of
controversy (Ryklan and Schmidt, 1944). Dixon and Quastel
(1925) measured the potentials of solutions of cysteine and
glutathione with their corresponding disulfides. They found
that the potentials were independent of the relative concen-
trations calculated from the relation 1r 'Fo - A}; 1n [RSH] +
%? 1n [Hf] where "'13 the observed potentia1,fflb is the nor-
mal reduction potential and the other notations have their

usual significance. This relation does not describe a system

that is reversible in the thermodynamic sense but suggests

a...

.3 W

 

S

e
V;

a change in free energy without a corresponding change in the
state or prOperties of the system (Ryklan and Schmidt, 1944).

Kendall and Nord (1926) reported that in the presence
of small amounts of oxygen an active addition product was
formed with the sulfhydryl compound whereby the system acquires
the power to reduce indigo carmen. They concluded that in
the presence of the hypothetical addition compound the system
was reversible. It has been noted that Kendall and Nord
(1926) failed to report a stoichiometric relationship to sup-
port their conclusion (Dixon and Tunnicliffe, 1927; Michaelis
and Flexner, 1928).

Potentiometric titration of cysteine with iodine, potas—
sium dichromate and potassium iodate yielded variable results
but the titration curves resembled those of reversible redox
systems. This evidence led Williams and Drissen (1950) to
postulate that the potentials observed depended not only upon
the presence of cysteine and hydrogen ions but also upon the
presence of an intermediate oxidation product. This view was
supported by the results of Fischer (1950). Other workers
who investigated the behavior of the sulfhydryl: disulfide
couple employed dyes, different electrodes and thermal data
with divergent results (Ghosh 33 al., 1952; Green, 1955;
Ghosh and Gangula, 1954; and Borsook gt al., 1957b). Ryklan
and Schmidt (1944) pointed out that Dixon and Quastel (1925),
Kendall and Ford (1926), and Dixon and Tunnicliffe (1927)

failed to set up the correct conditions for a reversible
couple and that the methods of calculation employed by
Williams and Drissen (1950) were erroneous. Ryklan and
Schmidt (1944) found that the potential was dependent upon
the concentration of cysteine, cystine and hydrogen ion.
During potentiometric titrations, addition of cysteic acid
caused a large increase in potential thus excluding the pos—
sibility that this compound might be the intermediate oxida-
tion product postulated by Williams and Drissen (1950).
Mixtures of cysteine and cystine did not establish equili-
brium at the platinum electrode when pure solutions were
employed but equilibrium was quickly reached when potassium
iodide was used as a mediator (Ryklan and Schmidt, 1944).
The normal redox potentials (E0) reported by Ryklan and
Schmidt (1944) are:

 

RSH Component Ea in volts
thioglycollic acid .27
cysteine .27
thiolhistidine .52
monothioethyleneglycol .55
ergothioneine .56
glutathione .45

These workers suggested that the sulfhydryl groups of pro-
teins probably have a potential equal to or larger than that
of glutathione, in view of the large potential of glutathione

10

in relation to the simpler compounds and the fact that cys-
teine activates protein sulfhydryl groups.

The importance of active sulfhydryl groups, present in
the enzymes themselves or belonging to substances associated
with the substrates has long been recognized (Lugg, 1945).

An extensive list of enzyme systems that require an active
sulfhydryl group for activity was compiled by Elliot (1946).
Many of the systems on the list were investigated by Barron
and Singer (1945). These workers noted that enzyme systems
inactivated by p-chloromercuribenzoate and iodoacetamide were
reactivated by glutathione. This led these workers to suggest
that the main function of glutathione is the continuous reac-
tivation of sulfhydryl enzymes; a view supported by Elliot
(1946) but contrary to the results reported by Ames and
Elvehjem (1945). A more complete discussion of the biologi-
cal significance of glutathione is given by Colowick (1954).

The f1avoproteins are complex proteins composed of one
of the isoalloxazine nucleotides derived from the vitamin,
riboflavin, in combination with a specific protein (Heller-
man, 1947). Early workers noted the accumulation of an inter-
mediate oxidation product that was red in acid solutions and
green in neutral solutions of lactoflavin held at low tempera-
tures. These workers concluded from the characteristics of
the titration curve of this flav0protein that the colored

form was a semiquinone (Kuhn and Wagner-Jauregg, 1934;

1111

0X

ll

Michaelis gt gt., 1956; and Klemperer gt g1., 1957). The
electrode potential of riboflavin in neutral solutions was
reported to be -0.208 millivolts (Michaelis gt gl., 1956).
Kuhn and Boulanger (1956) found that phosphorylation of ribo-
flavin did not affect the potential appreciably but that when
the phospho-riboflavin-protein complex was formed the poten-
tial rose to -0.060 volts at pH 7.

Measurement of the redox potential of diphosphOpyridine
nucleotide (DPN or Coenzyme I) with various dyes indicated
the potential was -0.26 volts at pH 7.2 (Ball, 1959). A dif-
ferent value was calculated from thermal and free energy data
(Borsook, 1940). Burton and Wilson (1955) calculated that
the potential of DPN was -0.520 volts at pH 7. Their calcu—
lations were justified by Rodkey (1955) who reported that
within the pH range from 6.5 to 10.5 the potential of solu-
tions of coenzyme I are described by the relation:

Eh a -0.107l + 0.0501 log %%%%%I.- 0.0501 pH
The stoichiometry of the titrations carried out by Rodkey
(1955) indicated the following reaction for the DPN couple:
DPNH:=3 DPN+ + 26 + HI. He reported that the potential of
the system did not change in the presence of increasing levels
of xanthine oxidase.

The significance of the flavins and phosphOpyridine
nucleotides as components of the general scheme of aerobic

oxidation is well rec0gnized. Excellent reviews of recent

12

advances in the study of biological oxidations appear an-
nually in "Annual Review of Biochemistry." Clark (1948)
presents a comprehensive list of the oxidation-reduction
potentials of many thermodynamically reversible systems
illustrating the possibilities for the stepwise release of
energy through the progressive increase in the oxidation
level of electrons removed from metabolites. Burton and
Wilson (1955) calculated the changes of free energy asso-
ciated with the exchange of hydrogen between several known
hydrogen carriers.

Despite the mass of data relating to the effects of
ascorbic acid in enzyme systems and intact organisms, no-
thing certain is known of the exact function of this vitamin
aside from the effects of its reducing prOperties (Snell
and Metzler, 1956). Early attempts to measure the redox
potential of ascorbic acid were largely unsuccessful. Green
(1955) reported that the potential was due to the concentra-
tion of the reductant alone. Borsook (1955) noted that in
solutions with a pH higher than 5.75 the oxidized form was
unstable, while Fruton (1954) reported that the potential
of ascorbic acid in neutral solutions was -0.081 volts. The
oxidation of ascorbic acid in three separate steps, the first
step being reversible when the system was measured colori-
metrically, was reported by Borsook (1957). Employing methy—

lene blue, indigotetrasulfonate and thionine as mediators,

cr
3]
ac

de

15

Ball (1957) succeeded in characterizing the electromotively
sluggish ascorbic acid system. He reported that the normal
potential was + 0.590 volts; that the lepe of the Eh/pH
curve changed from 0.06 to 0.05 at pH 4 remaining at 0.05
slope up to pH 8.6; and that the half-life of dehydroascorbic
acid at pH 7.25 and 58° C. was 2 minutes. He concluded that
dehydroascorbic acid could not exist in tissue. The function
of ascorbic acid in plant growth was investigated by Virtanen
and Hansen (1949). These workers found that growth was stunted
in the presence of too little or too much vitamin C and that
normal growth occured when this vitamin function of ascorbic
acid in plants was to regulate the redox potential at an
Optimum level. Koser and Thomas (1955) noted that the re-
quirement for cysteine was eliminated in certain strains of
Lactobaccili when ascorbic acid was added to the media.
The stimulatory effect that ascorbic acid had upon the action
of the enzyme homogentisicase was traced to protection of the
ferrous ion required by the enzyme (LaDu, 1952; Schepartz,
1955; Greenberg and Rinehart, 1955; Crandall, 1955; and LaDu
and Greenberg, 1957). Ascorbic acid also protects or aids
in the mobilization of the ferrous ion required by aconitase
and is therefore, necessary for the efficient Operation of
the Krebs cycle (Takeda and Hara, 1955).

Antioxidants have been defined as substances which in-
hibit the autoxidation of fats in contact with molecular

oxygen (Dam, 1957). Clark and Geissman (1949) discussed the

In

14

practical applications Of physiological antioxidants and
suggested several mechanisms by which these compounds may
act. Many antioxidants act as simple hydrogen donors. That
is, they prevent undesirable oxidations because they are pre-
ferentially oxidized (Weissberger, 1948; Greenberg and Rine-
hart, 1955; Crandall, 1955; Hickman and Harris, 1946; Green-
berg gt g1., 1957). Some antioxidants interfere with the
mechanisms of fat oxidation (Michaelis, 1948; Weissberger,
1948), while others inhibit "undesirable“ enzymes (Seifter,
1948; Fieser, 1948). Seifter (1948) and Nason and Lehman
(1955) have reported cases where antioxidants promote de-
sirable oxidations.

The tocOpherals (vitamin E) and redox substances which
have similar physiological prOperties have been the subject
of extensive research. The remainder of this chapter will
be concerned with a discussion of the characteristics of
these physiological antioxidants, and their mode of action.

Michaelis (1948) suggested that the antioxidant activity
of hydroquinones depended not only upon reversible oxidation
to their respective quinone but also upon the reversible
formation of an intermediate semi-quinone. Evidence that the
rate of semi-quinone formation depended upon the partial
pressure of oxygen and the hydrogen ion concentration was
presented by Weissberger (1948). He also noted that cysteine,

ascorbic acid and thioglycollate depressed the oxidation of

I:

An.

15

hydroquinone until they themselves were completely oxidized.
Michaelis and Wollman (1949) observed the free radical or
semiquinone form of vitamin E at the temperature of liquid
air (indicated by the color development). A reversible
quinone-semiquinone-hydroquinone couple was proposed by
Mackenzie (1949). Seifter (1948) found that no correlation
existed between the chemical prOperties of hydroquinones or
diamines and their toxicity. Investigation of the antimalarial
action Of hydroquinones indicated that their effectiveness
was due to the ability of their side chains to combine with
proteins rather than their redox characteristics (Fieser,
1948). Ames and Harris (1948) reported that alpha-tocOpheral
phosphate inhibited all the enzymes it was tested on and

does not act as meantioxidant.

The effectiveness of certain non-tocopheral antioxi-
dants in preventing or delaying the appearance of the signs
of vitamin E deficiency in various species have been demon-
strated. The development of exudative diathesis in chicks
was prevented by the administration of nordihydroguaiaretic
acid (NDGA), disulfram (tetraethylthiuram disulfide), methy-
1ene blue, and to some extent by ascorbic acid (Dam, 1957).
Encephalomalacia did not deveIOp in E-deficient chicks when
NDGA; ascorbic acid; methylene blue; diphenyl-p-phenylenediamine
(DPPD); 2, 6-di-t-butyl—4-methoxy phenol (BHT); or 1, 2-
dihydro-2, 2, 4-trimethyl-6—ethoxyquinoline (Santoquin) was

l6

administered (Dam, 1957; Singsen, 1955). Several commercial
antioxidants and redox dyes prevented reabsorbtion gestation
and muscular dystrOphy in the rat (Dam, 1957; Moore gt gt.,
1954). Blaxter gt gt. (1955) reported that methylene blue
protected against muscular dystrophy in calves induced by
feeding cod liver oil but noted that the effect may have
been due to a direct protective action of methylene blue
against the toxicity of polyethenoic acids of cod liver oil
rather than a relationship with vitamin E. Booker gt gt.
(1954) prOposed that the increased uptake Of oxygen observed
when animals treated with antioxidants were subjected to
stress was due to protection of the sulfhydryl groups of
enzymes necessary for normal metabolism. Vitamin E defi-
cient rats had less resistance to the effects of oxygen under
high pressure than did rats treated with alpha-tocOpheral,
glutathione or methylene blue (Taylor, 1958). DPPD failed
to prevent exudative diathesis in chicks fed on diets essen-
tially devoid Of vitamin E. Scott gt gt. (1955) concluded
from this data that the effect of DPPD was due to protection
of the vitamin E existing in the tissue. Shull gt gt. (1957)
observed that the protective action of antioxidants against
muscular dystrophy in guinea-pigs disappeared during long
term experiments. These workers concluded that antioxidants
are only effective while there is some vitamin E present in

the tissue. Draper gt gt. (1958) studied the effectiveness

17

of DPPD, BHT, and methylene blue in preventing and curing
reabsorbtion-gestation in rats fed E-deficient diets for
prolonged lengths of time. They found that DPPD and methy-
lene blue were effective but BHT was not. They suggested
that DPPD either participated directly in some physiological
function essential to reproduction and normally assumed by
vitamin E or activated residual amounts of E that were other-
wise inadequate. ‘Greenberg gt gt. (1957) and Tucker gt gt.
(1957) reported that tocopheral, ascorbic acid and DPPD enhanced
the therapeutic value of iron in the treatment of anemia in
milk fed rats. Although not identifying a toc0pherol as a
participant in the oxidative transport chain, Nason and Leh-
man (1955) did implicate a tocOpheral or some closely related
lipid soluble co-factor as a participant somewhere between
DPN and cytochrome C. Several antioxidants including DPPD
did not have this activity. Limited work on the effect of
the level of vitamin E in the diet of dairy cattle indicated
that increasing levels of vitamin E in the diet result in
increasing levels of vitamin A in milk (Worker and McGillivray,
1957; Hickman and Harris, 1946). Several antioxidants fed

at low levels increased vitamin A and carotene retention in
dairy calves but higher levels had the Opposite effect
(Pirchner gt gt., 1957; DeLuca gt gt., 1956).

vie

tel

pat

fat

18
Pathways of Carbohydrate Metabolism in the Rumen

The fermentative and oxidative pathways leading to
pyruvate, acetaldelyde, triosethXMate and other intermedi-
ates of carbohydrate metabolism in microorganisms were re-
viewed by Gunsalus, Horecker and Wood (1955). In this chap-
ter attention is directed chiefly toward a discussion of the
pathways leading from metabolic intermediates to volatile
fatty acids, 002 and methane.

Werkman and Wood (1942) proposed two mechanisms for the

formation of prOpionic acid from pyruvic acid:

 

 

 

A. HOOCOCHB c.1393: HOOCCOCH2COOH .I—f—i—e HOOCCHOHCH2COOH
pyruvic acid oxalacetic acid malic acid
-H20 4- 2H - CO2 ‘
_,. HOOCCH=CHCOOH _v____.———__.-bHOOCCH20H2000H ;: —,
fumaric acid succinic acid
3000011ch5 + co2

prOpionic acid

+ H — H O H
B. Hooccocn5 ———2-=-. HOOOCHOHCHag—‘Z—e Homes-ca2 __2__..

pyruvic acid lactic acid acrylate

HOOCCH2CH5 -——-) HOOCCH3 + CO2

prOpionic acid acetic acid
Elsden (1945) reported the conversion of lactic acid to ace-
tic acid, prOpionic acid and CO2 by a non-cellulytic PrOpioni-
bacterium isolated from the rumen of sheep. A similar organism
isolated from enrichment cultures Of rumen bacteria by Johns
(1949) seemed to form prOpionic acid by a reaction sequence

similar to (A) prOposed by Werkman and Wood (1942). The

 

"

19

Optimum pH for the decarboxylation of succinic acid by this
organism was 5-5.2 while at pH 6.8 the reaction proceeded
very slowly. Delwiche (1948), Johns (1951a) and Johns (1951b)
demonstrated the quantitative decarboxylation of succinic
acid by PrOpionibacterium pentosaceum and Veillonella
ggzogenes. Washed suspensions of rumen bacteria formed suc-
cinic acid as an intermediate during cellulose digestion and
decarboxylated added succinate with quantitative yields of
prOpionate (Elsden and Sijpesteign, 1950; Sijpesteign and
Elsden, 1952). Delwiche gt gt. (1956) investigated the
mechanism of succinate decarboxylation while Wood gt gt.
(1956) demonstrated the formation of acetic acid from pro-
pionate.

Cardon and Barker (1947) reported that Clostridium
progionicum converted lactate, pyruvate, and acrylate to
acetic and prOpionic acid. Although he was unable to find
acrylate in solution, Johns (1952) postulated that gt.
propionicum produced prOpionic acid by the reaction sequence
B prOposed by Werkman and Wood (1942) on the basis that
washed suspensions of this organism did not attack either
succinate, malate, or fumarate. Cell free extracts from the
rumen organism LC-l converted mixtures of acrylate and lac-
tate to hydrogen, carbon dioxide, acetate, and prOpionate.
The reaction was not as simple as reaction B since the coupled

oxidation-reduction involving lactate and acrylate yielded

20

some pyruvate which extracts from this same organism were
able to convert to hydrogen, carbon dioxide and acetate
(Walker and Todd, 1958; Todd and Walker, 1958).

The condensation of two acetate molecules to form suc-
cinic acid and hydrogen by Aerobacter indologenes 2. pggtg-
saceum, and E. ggtt was demonstrated (Slade and Werkman,
1945; Carson, 1948; Kalnitsky gt gt., 1945). Krampitz 22.§l-
(1945) reported the enzymatic fixation of carbon dioxide and
pyruvate to form oxalacetate. The phosphoroclastic split of
pyruvate to acetate and formate was demonstrated by Utter gt
al- (1945).

The large number of prOpionibacterium found in the rumen
led Gutierrez (1955) to conclude that they are important
in acetate and prOpionate formation. Barker and Lipmann
(1944) investigated the dependence of the prOpionic acid
fermentation upon the hydrogen balance or oxidative state
of the substrate and found that glycerol, the reduced sub-
strate, yielded a higher prOportion of prOpionate than did
pyruvate, the highly oxidized substrate. Gall gt gt. (1955)
reported increased levels of lactic acid in the rumen of
cows fed a high grain diet. A high correlation was found
between the amountsof lactate and prOpionate found in the
rumen (Waldo and Schultz, 1956). Barnet and Dow (1957)
found that pyruvate yielded more acetate than prOpionate

whereas prOpionate was the chief acid derived from lactate.

21

Alpha-ketoglutarate was utilized slowly to form acetate.
These workers suggested that the following reactions occur

in the rumen:

DPN j>laifate

DPNH pyruvate -—-? acetate
DPNHJ) oxalacetate -—-’ acetate + formate —--)CH4 and/or CO2
DPN mayate If oxalacetate

fumarate citrate

glyoxylic cycle

suciinate /9uccinate

prOpionate

[Kornberg and Krebs (1957)

Hershberger gt gt.(l956) postulated that an acetate to
prOpionate ratio of 2 to 1 was necessary to maintain a pro-
per hydrogen balance. This postulate did not account for
other fermentation end products such as carbon dioxide and
methane.

Clostridium kluyveri derives its energy solely from the
conversion of 2-carbon compounds to 4 or 6—carbon compounds.
Enzyme preparations from this organism carry out the reaction

by this mechanism:

 

+ acetate + 4H
ethanol-)acet 1 00A ’1 C4 ————-)butyrate
Ir
acetate

(Barker gt gt., 1945; Stadtman and Barker, 1949; Stadtman,

1952). Rumen organisms synthesize valerate from prOpionate

22

and acetate while butyrate and caproate arise from acetate
(Gray gt gt., 1951; Gray gt gt., 1952). LC, an organism
isolated from the sheep's rumen, carried out the following
reactions:

COA _
pyruvate W acetyl COA + 2H+ + 29
2

+ _ acetate -——-+Vbutyrate
acetyl COA + 4H + 43 +

prOpionate-——§valerate
butyrate ___—9 capro ate

+ COA + Hg)

(Elsden and Lewis, 1955).

Gray and Pilgrim (1952) demonstrated the production of
butyric acid from proteins. El-Shazly (1952a) found a high
cOrrelation between ammonia production and increased levels
of isobutyric, isovaleric, and higher fatty acids formed
during rumen fermentation and concluded that the volatile
fatty acids arise from protein as well as from carbohydrate.
Further experiments carried out with casein hydrolysate as
substrate for washed suspensions of rumen organisms substan-
tiated this conclusion (El-Shazly, 1952b). Sirotnak gt gt.
(1954) described the reductive deamination of aspartic acid
to form succinic acid and the subsequent decarboxylation of
succinate to form prOpionate and carbon dioxide. The conver-
sion of leucine to isovalerate was investigated by Otakaki
22 2.1.. (1955).

Considerable progress has been made in the study of

rumen methanogenesis over the past few years. Mylroie and

23

Hungate (1954) isolated Methanobacterium formicicum from
sewage sludge. This organism utilized no carbon source other
than formate, or hydrogen and carbon dioxide. This same
group of workers later reported that rumen microorganisms
produced carbon dioxide, hydrogen and methane from formate

14 labeled

and methane from carbon dioxide and hydrogen. C
formate was quantitatively converted to carbon dioxide which
in turn could be reduced to methane:

HCOOH --—-?CO2 + H2

CC + 2H2-'§ CH4

2
(Carrol and Hungate, 1955). Washed suspensions of rumen
organisms have the ability to reduce carbon dioxide to
methane (McNeill and Doetsch, 1956; McNeill and Jacobson,
1955) as does Methanobaccillus omelianski (Pine, 1958).
Stabilized cultures of rumen bacteria were established that
were able to utilize formate as their sole source of energy
(Nelson gt gt., 1956). These cultures produced carbon diox-
ide and methane in theoretical amounts according to the
equation:

4HCOOH -—)CH4 + 5002 + 2H20

Cultures stabilized to formate adapted very slowly to changes
in substrate (Opperman gt gt., 1957a).
Pine and Barker (1956) investigated the pathway of hydro-

gen in the acetate fermentation and found that the methyl

24

carbon goes to methane with its hydrogens intact:

X + CO

2
+2H\b CH4 + HX

(where X is an unknown compound) Opperman gt gt. (1957b)

CH COOH + HXJEEL, CH

5 5

established stabilized cultures which could utilize acetic,
butyric and valeric acid but could not establish a culture
which could utilize prOpionic acid. The ratios of gas pro-

duced from these substrates indicated the following reactions:

CH COOH --9CH4 + CO

5 2

2CH3(CH + 2H O-——-94CH

2 2 5

COOH + 4H2O—92CH5CH2COOH + 2CH3COOH + 4H2

Cultures stabilized to butyrate or valerate utilized acetate

COOH + CO COOH + CH4

2)2
20H5(CH2)5
readily. PrOpionate produced in the valerate cultures was

not utilized but remained as an end product (Nelson gt gt.,

1957).

In Vitro Techniques

Many tg gtttg techniques for the study of the biochemis-
try and microbiology of the rumen have been develOped in re-
cent years. The principal methods used by most investigators
may be roughly classified into:

(a) Pure culture studies

(b) Centrifuged cells

25

(c) Whole rumen liquor diluted or undiluted (artificial
rumen)

(d) Stabilized cultures.

Pure cultures allow one to study the metabolic activi-
ties of the predominant bacterial species of the rumen (Anony-
mous, 1958). Hungate (1957) investigated changes in the micro-
bial pOpulation in the rumen Of cattle fed a constant ration.
He made direct microscOpic and culture counts on samples ob-
tained by stomach tube and identified Streptococcus bovis,

Ruminococcus albus, R. flavefaciens, gt. lochheadii, Bacter-

 

gtggg succinoggnes, and Qt. Longisporum. Samples withdrawn
with the stomach tube were mixed and diluted 1:10 with a

salt solution in equilibrium with 5% carbon dioxide in a
screw tOp bottle. Samples were taken to the laboratory where
air was again displaced with a N2 - CO2 mixture. Further di-
lutions were made and innoculated into rumen fluid cellulose
agar and feed extract agar. §ttgp. tgttg were observed after
incubation at 59° C. for 24 hours. Total colony counts were
made 10 days after innoculation and cellulytic colonies in
rumen cellulose agar were examined after 20 days. Sttgg.
tgttg was isolated by picking colonies and subculturing in
an agar medium containing inorganic salts, rumen fluid and
starch. Other colonies were subcultured in tubes of rumen
fluid cellulose agar. Great care was taken to maintain

anaerobiosis. The fermentative characteristics of the various

26

isolates were investigated by innoculation into tubes con-
taining various fermentable carbohydrates. Other workers
have investigated the nutritional requirements of organisms
in pure culture (Bryant and Burkey, 1955; Bryant and Doetsch,
1954; Chu and Bryant, 1958; Hungate, 1947; Hungate, 1950).
The fermentative mechanisms of pure cultures have also been
investigated (Elsden, 1945; Johns, 1952; Delwiche gt gt.,
1956; Walker and Todd, 1958; Todd and Walker, 1958).

The washed suspension technique was develOped as a
means for studying the whole rumen pOpulation in a simplified
system. The first worker to utilize this system employed a
technique which might more correctly be called the centrifuged
unwashed cell technique (Marston, 1948). I

The desire to study the metabolism of the whole rumen
flora in a system uncomplicated by presence of a myriad of
substrates and end products led to the develOpment of the
centrifuged cell technique which has been modified by various
workers to suit their particular needs. Marston (1948) re-
moved the coarse feed particles from cheese-cloth strained
rumen fluid by slow centrifugation. The microorganisms were
separated from the resulting supernatant in a high speed
centrifuge and suspended in a phosphate buffer. The suspended
cells were then put into a 5.5 liter hermatically sealed
glass pot containing 2.5 L. of a salt solution and a source

of cellulose. Anaerobiosis was maintained by passing a

27

continuous stream of nitrogen into the fermentation vessel.
The pH was adjusted back to 6.8 at regular intervals by add-
ing 0.5 M Sodium Carbonate. Elsden and Sijpesteijn (1950),
Sijpesteijn and Elsden (1952) and Bentley gt gt., (1954) used
unwashed cells prepared by similar techniques to investigate
the end products of cellulose digestion and the requirements
of the rumen flora. Considerable amounts of Substrate and
unidentified factors stimulatory to cellulose digestion were
carried over into the fermentation vessel when unwashed cen-
trifuged cells were used. Cheng gt gt. (1955) reduced the
amount of carry-over by washing the centrifuged cells before
adding them to the fermentation flask. Doetsch gt gt. (1955)
modified the washed cell method still further by preincuba-
tion to deplete reserves within the cells so that little or
no activity would occur in the absence of added substrate.
Three main types of artificial rumen systems have been
described: (1) Undiluted or slightly diluted rumen liquor
incubated in all-glass impermeable systems (Pearson and
Smith, 1945); (2) Whole rumen fluid diluted to about half
strength with a mineral solution (Burroughs gt gt., 1950);
and (5) Whole undiluted rumen liquor incubated with substrate
in a semipermeable container surrounded by a mineral solution
(Louw gt gt., 1949). Pearson and Smith (1945) who studied
protein metabolism in whole rumen fluid claimed that the

microflora remained normal for 2 - 4 hours in their impermeable

28

glass system. Burroughs gt gt. (1950) attempted to prevent
alteration in the microflora due to accumulation Of end
products by diluting rumen fluid before and during the fer-
mentation while Louw gt gt. (1949) allowed the end products
to diffuse out of his semipermeable fermentation vessel, into
a salt solution. The system of Louw gt gt. (1949) was modi-
fied by Wasserman gt gt. (1952) and Huhtanen gt gt. (1954).
Several biological and chemical criteria for establishing the
validity of artificial rumen studies have been prOposed by
Warner (1956).

A method for establishing stabilized enrichment cultures
of rumen bacteria was develOped by Opperman, Nelson, and
Brown (1957) for studying methane and carbon dioxide forma- ~
tion from various substrates. Cheese-cloth strained rumen
fluid was added to an equal volume of salt solution. Formats,
acetate, butyrate or valerate was added to the fermentation
vessel daily as the sole source of carbon. Only organisms
which could utilize this substrate (directly or symbiotically)
survived after a period of several weeks. When the yield and
constitution of the gas produced remained consistent for

several days the cultures were considered stabilized.

Redox Potentials in Bacterial Systems

Perhaps the greatest difficulty in dealing with biologi-

cal systems generally and with bacterial pOpulations in

29

particular arises from the fact that we do not have rigidly
defined static systems possessing fixed characteristics.
The redox potentials Observed in bacterial pOpulations re-
flect to some extent the dynamic aspects of this phenomenon
we call life. This chapter deals with the significance of
redox potentials observed in bacteriological systems.
According to Gillespie (1920) measurements of the redox
potential in bacterial cultures gave very characteristic curves
representing the lag, lOgarithmic growth, and death phases
in the culture. Coulter and Issacs (1929) PrOposed that the
rapid drOp in potential observed during the logarithmic growth
phase was due to the disappearance of oxygen from the media
and the stability of this low potential during the death phase
resulted from the release of reductive substances from the
dead cells. This explanation was discredited by Hewitt (1950)
as an attempt to explain a cause by listing the symptoms.
The oxygen deficit arises as a result of the metabolic activi-
ties of the multiplying organisms. In the absence of oxygen
all available hydrogen acceptors are utilized by the organ-
isms in order that metabolism may continue. Thus the reduc-
tion of intercellular oxidizing systems and the surrounding
media which results in low redox potentials are symptoms of
the dynamic equilibrium that exists between cellular enzymes

and medium constituents (Hewitt, 1950).

50

The oxidation-reduction potential observed in bacterial
cultures is due to the presence of many electromotively active
compounds in various states of oxidation. When these com-
pounds are present in minute quantities or are predominantly
in the oxidized or reduced state the potential is usually
unstable. When large quantities of these compounds are pre-
sent, considerable oxidation or reduction must take place
before the observed potential will vary. As a general rule,
the potentials Of bacterial cultures tend to vary over a
range Of 3 5O millivolts (Hewitt, 1950). The reducing proper-
ties Of aerobes generally range from about +0.4 volts to -O.2
volts while anaerobes are capable of reducing the potential
from about +0.05 volts to about -O.4 volts. These reducing
properties are a manifestation of the characteristics of the
enzyme systems that the various organisms possess. That is,
aerobes are capable of metabolizing in a highly oxidative
media because they possess a cytochrome system while anaerobes
depend largely upon enzymes which cannot metabolize normally
until the media is partially reduced. (Aubel, Rosenberg, and
Grunberg, 1946; Hewitt, 1950).

Very little information is available concerning the redox
potential of rumen contents. The potential observed in the
12.1i332 system of Marston (1948) was in the vicinity of
-o. 580 volts at pH 7 with rapid fluctuations of i 30 milli-

volts. The potential Of rumen contents treated with various

51

levels of aureomycin and pennicilin ranged from -0.195 volts
to -0.288 volts (Horn, Snapp and Gall, 1955). Broberg (1957a,
b,c and 1958) reported his measurements of rumen potential
as rH in an attempt to simplify his data. Hewitt (1950)
severly criticized the use of the term rH when referring to
biological systems ”Since these are systems of unknown
constitution and incompletely studied properties, the rela-
tion between Eh and pH cannot be defined and hence no term
containing both factors can be obtained in our present state
of knowledge, whilst the use of rH makes a tactic assumption
that there is a known simple relationship between the Eh and
pH of the system. For this and other reasons, Clark rightly
insists that the use of the term rH, originally introduced
by himself, should be discontinued." (Hewitt, 1950). The
relationship between rH, pH and E11 at 50° C. is represented
by the equation: Eh . .05(rH - 2pH) (Hewitt, 1950).
Broberg's results were converted to Eh by inserting his
rH and pH values into the above formula. This conversion was
made in order to facilitate a comparison Of his experimental
results with those reported by other investigators. Measure-
ments of the potential in untreated rumen contents taken from
eight healthy bovines while they were in the stable, after
they had been on pasture for two days and after three months
on pasture were not significantly different (Broberg, 1957a).

Samples taken from fistulated sheep on a normal ration twenty

52

to thirty minutes after concentrate feeding had an rH of

6.9 1 0.09 (E11 of -0.14V at pH 5.75) while samples taken from
these same sheep later in the day had an rH Of 8.5 i 0.06

(Eh of -0.15V at pH 6.7) (Broberg, 1957a). Acidification Of
rumen contents under both oxygen and carbon dioxide atmos-
pheres showed a characteristic curve until the pH drOpped be-
low five. Thereafter the ability of rumen contents to main-
tain a constant redox potential was lost and a continuous
change toward higher values could be Observed in the presence
of oxygen (Broberg, 19570). The redox potential of rumen
contents from sick animals was very stabile except in a few
caSes of complete anorexia for extended periods, due to acute
overeating Of grain and in a number of bloat cases (Broberg,
1957b). The author suggested, in view of the wide varia-
tions in potential observed in the bloat cases, that enzymatic
processes other than the normal govern the course of events
in a number of bloat cases. Large amounts Of oxygen bubbled
through rumen contents failed to cause more than a temporary
rise in potential tg tttg. NO correlation was noted between
carbon dioxide production and oxygen consumption tg ttttg

(Broberg, 1958).

PART I
THE REDOX POTENTIAL OF BOVINE RUMEN CONTENTS

Procedure

Three cows, T5, T25 and K159, fitted with the screw cap,
plastic fistula plugs described by Hentschl gt gt. (1954)
were used in this experiment. T5 and K159, respectively,
were maintained on hay rations Of 20 pounds of timothy daily
and 20 pounds of first cutting alfalfa daily. T25 was fed
10 pounds of first cutting alfalfa hay and 10 pounds of a
grain mixture which was 77.5% ground shelled corn, 20% soy-
bean oil meal, 1% calcium carbonate, 1% dicalcium phosphate,
0.5% trace mineral salt and contained 10,000,000 international
units of vitamin A and 90,000 international units of vitamin
D per ton. The grain mixture was 15.8% crude protein. All
animals received 50 grams of trace mineral salt daily. The
experimental rations were fed once daily at 7 A.M. for at
least three weeks before the investigation was initiated.

The animals had free access to water at all times.

Samples of rumen fluid were collected from each animal
at 6:00 A.M., 10:50 A.M. and 6:00 P.M. No animal was sampled
more than once daily in order to eliminate the possibility
of a carry over effect in the second sample. Samples were
strained through a double layer of cheese cloth into an

Erlenmeyer flask that had been flushed with nitrogen beforehand.

- 35 -

54

In order to facilitate the determinations the samples were
separated into several fractions by centrifugation. The
large feed particles (fraction C) were separated from strained
rumen fluid (fraction A) in an International Centrifuge run-
ning at 200 x G for 5 minutes. The resulting supernatant
fluid (fraction B) was then subjected to a force of 27,600
x G for 15 minutes in a Servall centrifuge type SS-l. The
supernatant from this treatment was designated as fraction
D and the residual as fraction E.

The measurements of the initial Eh and pH were made on
a Beckman, Model K, automatic titrator. A glass electrode
and a fiber junction saturated calomel electrode were used
to determine the pH, while a bright platinum electrode and
a liquid junction saturated calomel electrode connected to
the sample through a salt bridge made of KCl saturated water
and 2 per cent agar were used for measurements of electro-
motive force. All electrodes were checked daily against
standard solutions. The use of the salt bridge was deemed
necessary due to the marked instability of the calomel elec-
trode when placed in rumen fluid for the prolonged lengths
of time required for measurements of electromotive force.
All measurements were conducted under a continuous flow Of
nitrOgen and one drOp of 0.002 per cent methylene blue was
added to each sample to act as a mediator. The rumen fluid

was constantly stirred to insure a measurement of the whole

55

system. Potentiometric titrations were carried out on the
same equipment under the same circumstances with the excep-
tion that fraction B was used instead of fraction A. Ceric
Sulphate was used as the oxidizing agent for most of the
determinations of reducing capacity. Similar values were
obtained when potassium dichromate was used but the titra-
tions took much longer. In view Of the fact an actively
metabolizing system such as rumen fluid continually produces
reducing substances, it was necessary to kill the organisms
in order to determine the reducing capacity at a given instant.
When ceric sulphate was used as titrant this was accomplished
by adjusting the pH to approximately 1, the pH of the eerie
sulphate solution. In cases where dichromate was used the
system was killed by the addition of 2 milliliters of saturated
mercuric chloride solution. The relationship between pH and
Eh was determined by using 50 per cent sulphuric acid as the
titrant and measuring the subsequent changes in the pH and
Eh of the sample. All determinations were made in duplicate.
While the first titration was being carried out the duplicate
was kept in a water bath at 40° C. The hay-grain extract

was prepared by adding 50 grams of ground alfalfa hay and

10 grams of cornmeal to 1000 ml. of water and allowing the

mixture to stand overnight in a refrigerator.

Results

56

The data from the titrations carried out with sulphuric

acid as titrant appear in table 1.

setting the initial pH and E
-10 millivolts respectively and adjusting the other values

 

h

Table 1

Relationship Between Eh and pH

 

This data was coded by

of each titration at 0.2 and

 

 

 

Titrations J:

5 4
pH EMFa pH EMF pH EMF pH EMF pH EMF pH EMF
6.2 -260 6.8 -500 6.6 -280 7.2 -580 7.0 -500 7.2 -270
5.0 -200 6.0 -250 5.5 -200 5.9 ~500 5.8 -250 6.5 4200
4.5 -150 5.1 -200 4.5 -150 5.1 -250 5.1 -200 5.2 -150
5.4 -100 4.2 -150 5.7 ~100 4.2 -200 4.2 -l50 5.0 -100
2.4 - 40 5.6 -100 2.6 - 50 5.5 -150 5.4 -100 4.0 - 50
2.2 + 50 2.6 - 50 1.8 0 2.5 -100 2.4 - 50 2.8 0

1.9 0 1.8 - 50 1.5 o
1.0 0

 

Regression coefficient b a 0.0595 volts
Standard error a 10.0061 volts

aEMF expressed in millivolts potential between calomel electrode
and rumen fluid.

57

accordingly. The regression coefficient, b, calculated by
the method of least squares was -0.06 volts and the standard
error (deviation about the regression line) calculated by the
method of Snedecor (1946) was i 0.006 volts. The relationship
between the pH and Eh of rumen fluid can be expressed by the
relation Eh a Eo - 0.06pH where Eh is the potential in volts

at any pH; E0 is the potential in volts and 0.06 is the regres-
sion coefficient.

The Eh of the water extracts of alfalfa hay and cornmeal
was + 0.250 volts at pH 7. Data on the titrations of the
various fractions of rumen fluid with ceric sulphate appear
in table 2. In view of the potential observed in the water

extracts of hay and cornmeal all titrations reported were

Table 2

Proportion of Reducing Substances Present in
The Various Fractions of Rumen Fluid at 10:50 A.M.

 

 

 

 

Fraction A B C D E
Average milliliters
per fraction 50 56 14 54 2
Milliequivalents
reducing substance
per fraction 1. 1.95 0.90 1.00 0.70 0.15
2. 2.20 1.10 1.00 0.90 0.20
5. 2.40 1.10 1.20 0.80 0.15
4. 2.00 1.05 .90 0.80 0.25
Mean 2.14 1.04 1.05 0.80 0.19
Per cent of total 100.0 47.6 47.2 57.5 8.9

 

58

carried only up to a potential Of + 0.200 volts at pH 7 to
eliminate the possibility that the titration values would be
influenced by the amount of water soluble feed constituents
present. Fraction A was taken as representing the total amount
of reducing substances present and set at 100 per cent. Frac~
tions B, C, D, and E contained 47.6, 47.2, 57.5 and 8.9 per
cent Of the reducing substances, respectively.

The amounts Of reducing compounds found in fraction B at
three different times of day in cows on three different ra-
tions are presented in table 5. The values here represent the
amounts of reducing substances with a potential below + 0.225
volts at pH 7.

Table 5

Potentiometric Titrations of Fraction B

 

 

 

Time
Cows
6:00 A. M. 10:50 A. M. 6:00 P. M.
T-25 1.55a 2.65 1.64
T-5 1.25 2.55 1.75
K159 1.78 2.21 2.01

 

aMean of 4 values presented in Appendix Table 1
expressed as milliequiv. reducing substance/50
ml. fraction B.

The method for analysis of variance in multiple classifi-

cations presented by Snedecor (1946) was applied to this data.

59

Ration, time, and the ration by time interaction were de-
signated as sources of variance. The differences in titra-
tion values with time of day were highly significant at the
0.01 level of confidence, the amount of reducing substance
being highest at 10:50 A.M. The interaction between ration
and time was also significant (P - 0.01) due to the rela-
tively high amounts of reducing components present in samples
from T25 at 10:50 A.M. and the continual presence of moderate
amounts of reducing compounds in the hay fed cows.

The values in table 4 represent the Eh and pH of strained
rumen fluid collected at three times of day from the same
three cows. The corrected values presented were adjusted
for pH according to the formula: Eh = Eo - 0.06pH.

This data was analyzed in the same manner that was
described for the titrations presented earlier. The effect
of time of day upon the uncorrected Eh values approached
significance at the 0.05 confidence level. When the Eh values
were corrected to pH 7 the effect of time of day upon the Eh
was significant at the 0.01 level of confidence. The Eh was

always lowest at 10:50 A.M. Ration did not influence the Eh'

4O

 

 

 

 

 

 

 

Table 4
Eh and pH Of fraction A
Time 6 A.M. 10:50 A.M. 6 P.M.
Cow Eha pH Ehb 'Eha pH Ehb Eha pH Ehb
T5 -l25 7.5 —107 -l55 6.4 -191 - 75 7.0 - 75
- 65 7.1 - 59 -l45 6.5 -187 -125 7.5 -107
T25 -125 7.2 -115 -l55 6.4 -l7l - 95 7.2 - 85
-l55 7.1 ~129 -l55 6.7 -l75 - 15 7-1 - 9
K159 -105 7.4 - 81 -165 6.8 -l77 - 95 6.2 -145
- 55 7.2 - 25 - 85 6.2 -155 -155 7.4 -l51
aUncorrected Eh'
b
Eh corrected to pH 7.0.

PART II

STABILITY AND IMPORTANCE OF THE REDOX POTENTIAL
IN RUMEN CONTENTS

Procedure

 

Inoculum from the same cows described in part I were
used for the tg ttttg fermentations in this phase of the
eXperiment. The cows were sampled in the same manner as
was previously described. Measurements of Eh and pH and
the titrations were carried out as described in part I.

The methylene blue and DPPD used as additives in these
fermentations were in the oxidized form while the cysteine,
thioglycolate and ascorbic acid were used in their reduced
forms.

The short term fermentations were carried out in 500
milliliter Erlenmeyer flasks in a water bath held at 40° C.
The flasks were closed with rubber stOppers with two outlets.
The first outlet was connected to a calibrated gas burette
while the second outlet was kept closed except in the fer-
mentations to which oxygen was added. In this case the
second outlet was attached to the oxygen source. In the
fermentations where DPPD was the additive, 5 grams of corn-
mean and 5 grams of ground alfalfa hay were added to the

flask before the fermentation was started. The other

_ 41 _

42

fermentations were carried out without added substrate. The
fermentation systems were thoroughly flushed with nitrogen
before 250 milliliters of fresh rumen fluid were added and
then the system was flushed again. The first 25 milliliters
of gas produced were discarded. All additives used in these
fermentations were added beforehand with the exception of oxy-
gen gas which was added in small amounts throughout the fer-
mentation time. The fermentation mixtures were shaken every
twenty minutes, with the exception that during the runs where
Oxygen was added all flasks were shaken almost continually.
At the end of each fermentation, the gas collecting system
was closed off and the ferment subjected to a force of

2,700 x g in a centrifuge for 5 minutes. Fifty milliliters
of the resulting supernatant were refrigerated in a bottle
containing 1 milliliter of 50 per cent (V/V) sulphuric acid
for further determinations. The gas collected was analyzed
for carbon dioxide, methane and oxygen as quickly as possi-
ble in a Burrell portable type gas Analyzer, model 59. Cul-
tures of rumen organisms were stabilized to formic acid

using the technique outlined by Opperman gt gt. (1957). Gas
from cultures stabilized late in the experimental period was
analyzed for carbon dioxide in the Burrel analyzer previously
mentioned while amounts of methane and hydrOgen were determined
in the Cenco Vapor Phase Analyzer with a silica gel column.

Nitrogen flowing at 26.6 milliliters per minute was used as

45

the carrier gas. The temperature was 100° C. Hydrogen

and methane peaks were identified from their retention times.
The amounts of methane per sample were determined from a
standard curve and hydrogen by difference.

Volatile fatty acids were determined by the direct
chromatographic technique described by Keeney (1955) with
the exception that petroleum ether (B.P. 65-110) was used
instead Of hexane and the valeric acid was not separated
from the higher acids.

Ammonia was determined by the permutite method described
by Hawk gt gt. (1954) with modifications. Four ml. of rumen
fluid were added to a 100 ml. volumetric flask containing 2
grams of amberlite lR-l2OH. The mixture was allowed to
stand several minutes before the rumen fluid was decanted
from the flask and the resin washed with distilled water.

Two ml. of 10 per cent sodium hydroxide solution were added
and the flask was allowed to stand again. After the addition
of 75 m1. of water to the mixture, 10 m1. of Nessler's reagent
were added and five minutes allowed for color development
before diluting to 100 ml. Two drops of Gum Ghatti solution
were added before the Nessler's reagent to prevent clouding.
The optical density of the samples was then determined at

490 mu in a Beckman B SpectrOphotometer. A standard curve
was prepared in a similar manner using aqueous ammonium sul-

fate solutions.

44

Statistical analysis of data obtained in this phase of
the experiment was carried out according to the methods sug-
gested by Snedecor (1946) for the analysis of variance in
experiments with multiple classifications. The only sources
of variance Considered were treatment differences and differ-

ences due to day to day variation in the innoculum. T
Results

DPPD (diphenyl-para-phenylenediamine) lowered methane

 

production tg ttttg but the effect was not significant at the _ \ E_
0.05 level of confidence (Appendix Table 2). No effect upon
volatile fatty acid or ammonia production was noted. Al-
though the addition of cysteine and thioglycollate seemed

to result in a lower potential at the end of the fermenta-
tion, the prObability of such an effect only approached
significance at the 0.05 confidence level (table 5). These
additives did lower methane production significantly (P = 0.05)
(table 6). The effect was most notable when cysteine was
added. The difference between thioglycollate and cysteine

was not significant (P = 0.05). It should be noted that

some cysteine was utilized by the organisms as shown by the
lower titration values at the end of the fermentation. No
effect upon volatile fatty acid and ammonia production was

noted (Appendix Table 5).

45

Table 5
Effect of Thioglycollate and Cysteine Upon Eh

 

Control Thiqglycolate Cysteine__
Run #1 Run #2 Run #1 Run #2 Run #1 Run #2

 

an at zero time —180a -200 -200 ~29o -200 ~520
pH at zero time 6.8 6.4 6.7 6.4 6.7 6.4

Reducing substance b
at zero time 5.48 5.52 6.60 6.64 12.4 12.6
Eh at end of fer-
mentation + 10 ~210 -100 -500 -220 -510

pH at end of fer-
mentation 5.5 6.5 5.2 6.5 5.5 6.4

Reducing substan—
ces at end of
fermentation 5.12 5.52 6.68 6.60 11.5 12.0

 

aMillivolts.
bMilliequivalents/5o ml. fraction B.

Table 6

46

Effect of Thioglycollate and Cysteine Upon Gas Production

 

 

 

 

Ml. cog__ Ml. CH4 Total Gas % 0025-==

Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 Run 1 Run 2

Control: 1 70.5 145.5 54.5 69.7 125 215 56.4 67.6

2 75.1 144.1 56.9 65.9 150 210 56.2 68.6
Thioglycollate:

1 78.8 147.0 51.2 65.0 150 210 60.6 70.0

2 76.7 162.9 55.5 62.1 150 225 59.0 72.4

3 75.4 159.6 54.6 70.4 150 250 58.0 69.4

Cysteine: 1 79.6 164.7 50.4 60.5 150 225 61.2 75.2

2 86.8 165.6 55.2 64.4 140 250 62.0 72.0

5 79.2 152.5 50.8 57.7 150 210 60.9 72.5

 

The data in table 7 indicate

that both methylene blue

and ascorbic acid caused slight increases in the potential.

Very little of the ascorbic acid remained at the end of the

fermentation (table 7).

Methylene blue caused a very sig-

nificant increase in the per cent of carbon dioxide produced

(table 7) but did not alter volatile fatty acid or ammonia

production (Appendix Table 4).

Results from fermentations

to which catalytic amounts of methylene blue and oxygen were

added are presented in table 8.

None of these treatments

caused a significant change in potential although the values

47

rillii! ‘INU ‘11 I . w»

.ueaoeaaaass

 

6.6a 6.6s s.a6 0.66 6.ss a.6s 6.a6 0.66 mmw a
4.66 0.Hs 0.6m 4.6H 6.6H 0.6H 4.66 H.mm 460 .as
6.60 0.00s 6.66 4.06 0.66 6.66 6.64 6.64 600 .H2
0 0 0 6.6 6.6 6.6 0 0 meanness mmMMWM”mmas
6.6 6.6 6.6 6.6 6.6 6.6 4.6 4.6 me can
66- 66- . 66- 6s- 66- 66- 6aa- 6aa- as 666
0.6 0.0 0.6 6.6 6.6 6.6 0 0 ac .mwwwwmmmas
6.6 6.6 6.6 6.6 6.6 6.6 0.6 0.6 60 66666
66- 66- 66- 66- 66- 66- 66a- 666a- as 66666

 

6 .H666 6 .H066 H .Hacm 6 .aoom 6 .H066 a .Hsom m .Hacm H .Haem

 

OHnuooms ms maonpqOo

 

 

 

 

doapoSdonm 660 was mm womb
sacs enhances 006 60H6 masseuse: no 666666
6 cases

48

Table 8
Effect of Methylene Blue and Oxygen

Upon tg Vitro Fermentations

 

Control MBa MBa + 0 0

2 2
Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 Run 1 Run 2

 

~55 ~65 ~55 ~65 ~55 ~65 ~55
pH at zero time 6.8 6.7 6.8 6.7 6.8 6.7 6.8 6.7

Eh at end of
fermentation ~85

Eh at zero time -65

-105 ~55 ~75 ~85 ~85 ~55 ~105

pH at end Of fer-
mentation 6.2 6.2 6.1 6.2 6.2 6.2 6.1 6.1

155.5 215.5 152.1 251.1 169.7 251.5 172.2 258.8
72.1 87.5 72.0 85.5 45.5 78.0 51.4 98.1

ml. CO2 produced
m1. CH4 produced

Total m1. gas -

. _- u- —.6

 

produced 225.6 505.0 224.1 516.6 215.2 509.5 225.6 556.9
% 002 68.0 71.1 67.9 75.0 79.0 74.8 77.0 75.6
ml. 02 utilized —- —- -- -- 56.0 28.0 51.8 50.5
5-02 in gas 1.5 1.5 1.0 1.0 9.0 15.0 9.0 15.7
Acetic acid° 7.57° 7.00 7.54 7.09 6.95 7.24 7.16 7.45
PrOpionic acid 1.92 1.65 1.95 1.60 1.92 1.62 1.79 1.64
Butyric acid 1.70 1.77 1.71 1.85 1.75 1.62 1.76 1.76
Higher acids .57 .58 .55 .59 .55 .54 .65 .55
Ammonia nitrogend 41.2 58.4 40.8 59.1 40.2 58.0 41.0 42.0

 

a5 milligrams/250 m1. rumen fluid.

bMillivolts.

cMillimoles acid/100 ml. rumen fluid.

d

Milligrams ammonia nitrogen/100 ml. rumen fluid.

49

did seem lower in the controls than in the flasks to which
methylene blue was added in the absence of oxygen. Despite
the rather notable increase in carbon dioxide production
when oxygen was present the difference was not statistically
significant (P =(0.05) in any case.

Cultures stabilized to formate produced theoretical
amounts of carbon dioxide and methane (5C02 to lCH4) whether
methylene blue was present or not (Appendix Table 5). Con-
siderable amounts of hydrogen (up to 5 per cent of total gas)
were produced by two of the cultures during the second week
of the stabilization period but this could not be attributed
to methylene blue since only one of the cultures contained
the compound. Early attempts to stabilize cultures to for—
mate were unsuccessful in the respect that a l to 1 ratio of
carbon dioxide to methane production persisted for prolonged
lengths of time and the ratio was very stable (Appendix Table
5). The techniques used to stabilize all cultures were iden-
tical except that the cultures producing a l to 1 ratio were
from samples of rumen contents collected at 10:50 A.M. while
the cultures which produced theoretical yields were from

samples collected at 6:00 P.M.

DISCUSSION

The linear relationship between Eh and pH shown in
table 1 is not in agreement with the results of Broberg
(1957a) who found no consistent relationship between Eh and
pH when he acidified rumen contents either by stages or
abruptly. A possible explanation for the difference is that

Broberg worked with a living pOpulation while dead rumen bac-

 

teria were used in this experiment. Broberg reported that F
when the pH was within the physiological range 10 to 15 min—
utes passed before the system came to equilibrium. When the ‘WJ
organisms are dead equilibrium is reached very quickly.

These facts indicate that the effect of pH upon Eh is modi-

fied by the living organisms. The equation reported above

is known to be true only in the case of dead rumen contents

and may not rigorously hold in the case of a living system.

In most cases, therefore, results, in this experiment, were

only adjusted according to this equation when dead rumen

contents were under investigation. The one exception, in

table 4, when this author took the liberty of applying the

correction to living rumen contents is, perhaps, not justi-

fied but the correction was very convenient for the purposes

of discussion.

The results of the titration of the various centrifugally

separated fractions of rumen fluid reported in table 2 show

_ 5o -

51

that only 8.9 per cent of the reductive substances found in
rumen fluid are directly associated with the bacterial cells.
This result indicates the terrific ability that microorgan-
isms have to affect their environment. The significant in-
crease in the amounts of reductive substances found in
fraction B several hours after feeding (table 5) and the
significant decrease in potential at the same time, illus-
trate the capacity of the rapidly metabolizing rumen popu-
lation to utilize the hydrogen acceptors in their environ-
ment. These results indicate that the potentiometric char-
acteristics of rumen contentscan be used as an indicator of
metabolic rate in the same manner that pH measurements are
now used.

The significant interaction between ration and time
(table 5) correlates very well with the accepted fact that '
concentrates are digested more quickly than roughages.o The
cow on a ration of concentrate and roughage had a high level
of reduced component several hours after feeding with com-
paratively low amounts later in the day. Conversely, the
cows that received roughage alone maintained intermediate
amounts of reductive component throughout the day.

The low poising capacity associated with the bacterial
cells (fraction D) indicates that extreme care must be taken
to maintain reducing conditions when the pure culture,

washed and centrifuged cell techniques are employed. Several

52

workers have already noted that some reactions proceed very
slowly or not at all when these techniques are used unless
proper reducing conditions are maintained (El-Shazly, 1952a;
Hungate, 1957; Gilroy and Heuter, 1957; Chu and Bryant, 1958).
Smith and Hungate (1958) reported that Methanobacterium
ruminantium cultured better at Eh levels below those produced
when cysteine is used as the reducing agent. Carson (1948)
has reported the dependence of the prOpionic acid fermenta-
tion upon the reductive state of the substrate.

The identities of the compounds which heretofore have
been referred to as reducing substances are mostly unknown.
The titration curve of rumen fluid from approximately -O.25O
volts to +0.2OO volts is almost a straight line indicating
that there is no large amount of any one substance present
that is responsible for the potential observed in rumen con-
tents but rather a large number of compounds present in vari-
ous states of oxidation whose normal potentials represent a
continual gradation from very low to moderately high values.
The following data taken from West and Todd (1957) presents

the normal potentials of many common biological redox systems.

 

System Eh in volts _pg
Cytochrome A +0.29 7.4
Cytochrome C +0.27 7.0
Cytochrome B -0.04 7.4

Flav0protein (yellow
enzyme) -0.059 7.0

55

 

System Eh in volts _pfi
Lactate-pyruvate system -O.186 7.0
Riboflavin -O.208 7.0
Cystine-cysteine —O.227 7.15
Glutathione —O.255 7.15
Coenzyme I -O.525 7.4

The Eh values reported in this table represent the po-
tentials of these compounds when the concentration of the
oxidized form equals the concentration of the reduced form.

Redox substances are analogous to pH buffers and must be sub—

 

jected to considerable oxidation or reduction before their
potentials vary substantially from the normal. The cytochromes
would exist in an almost totally reduced condition in the
rumen. In view of the function of the cytochrome system in
aerobic metabolism one would expect that any oxygen present
would be rapidly utilized. Flav0proteins, also, would be in

a highly reduced state in the rumen while the lactate-pyruvate,
cystine-cysteine, and glutathione systems could range from a
highly reduced to a highly oxidized state depending upon the
metabolic rate of the rumen contents. Coenzyme I would be

at least one-half oxidized most of the time indicating that
reactions requiring this coenzyme would not be impaired to

any great extent in the rumen even at the highest metabolic

rates.

54

The amounts of reducing substances found in rumen con-
tents are not very high, at least, not high enough to be
totally responsible for the stabile conditions that exist in
the rumen. For example, it would take only 5 equivalents of
a strong oxidizing agent to raise the potential of 175 pounds
of rumen contents to +0.2OO volts if the titration values were :4
the only stabilizing factor in rumen fluid. A potential of 1
+0.2OO volts is inconsistent with normal anaerobic metabolism.
The second part of this experiment was initiated to investigate
other factors that contribute to the stability of the rumen
environment. The several additives used in this phase of
the experiment were chosen because they represented various
potential ranges and the potentiometric behavior was known.

DPPD was chosen because its behavior resembles that of
the tocOpherols and it is or was commonly used as a stabilizer
for vitamin A and carotene. Although the exact normal poten-
tial of DPPD is unknown and attempts to measure it were unsuc-
cessful it was assumed that the potential falls between
+0.100V and +0.500V and that DPPD should act as a hydrogen
acceptor. Results obtained with DPPD as an additive were very
inconclusive, probably due to the low solubility of this com-
pound in water.

Cysteine and thioglycolate did not affect the potential
of the rumen contents substantially. This result was expected

since their normal potentials are approximately equal to the

55

Eh of untreated rumen contents (table 5). The objective in
adding these compounds was the stabilization of the potential
and alteration of the characteristics of the titration curve.
The only notable effect was a decrease in methane production
and an increase in carbon dioxide production. This result
is hard to rationalize since both were added in the reduced fa
state and therefore could not be acting simply as hydrogen 1
acceptors. A possible explanation is that these additives m
poised the system above the Optimum Eh level of Methanobac-
tgpium ruminantium. Smith and Hungate (1958) reported that -1“
this organism subcultured much better when the Eh of the
media was lower than the potential produced when cysteine
was used as reducing agent.
The choice of ascorbic acid as an additive was unfor-
tunate since according to Ball (1957) the life of this com-
pound in neutral solutions is very limited. This was the
case in rumen fluid since none of theascorbic acid remained
at the end of the fermentation. Since the ascorbic acid dis—
appeared and its exact fate or its length of life in the
rumen fluid is unknown, no explanation for the increase in
002 production can be offered.
Methylene blue is known to act as a hydrogen acceptor
in biological systems. The significant decrease in methane
production found was, therefore, expected when comparatively

large amounts of methylene blue were added (table 7). Even

56

though the hydrogen accepting capacity of these large amounts
of methylene blue were sufficient to account for the decrease
in methane production several factors indicated that this
may not have been the only effect of methylene blue. The
decrease in methane production, for example, was not large
enough to explain the increase in carbon dioxide formation. F4
The methylene blue was added in the oxidized state but was 9
reduced quickly and the total amount added could be accounted
for by titration differences almost immediately. After the
initial saturation it is doubtful whether or not the effect
could have been due simply to hydrogen acceptance. In view
of these observations further investigation of this additive
seemed advisable.

The effect of catalytic amounts of methylene blue alone
and in the presence of oxygen was investigated (table 8).
No significant differences in gas composition were found
despite the notable affect of oxygen upon CO2 production.
A very slight tendency for methylene blue to affect gas pro-
duction was still noticeable so the effect of this compound
upon the gas production of cultures stabilized to formate
was investigated. No apparent affect was noted. No explana-
tion for the cultures which produced carbon dioxide and
methane in a l to 1 ratio can be offered. The effect of
large amounts of methylene blue upon gas production may be

attributed to its hydrogen accepting capacity or to an

57

alteration in the conditions required by the methane pro-
ducing organism reported by Smith and Hungate (1958).
Neither of these affects would occur in stabilized cultures
due to the long lengths of time involved.

The failure of substantial amounts of oxygen to alter
the redox potential of rumen samples and the minor effect
of this gas upon the end products of fermentation (Table 8,
and Broberg, 1957c) lends support to the hypothesis that the
potential of rumen contents is very staUU3. Although the ru-
men fermentation is mainly anaerobic in nature, it is com-
monly agreed that many facultative organisms are present.
It is upon the presence of these organisms thatan explana-
tion for the stability of the potential in the presence of
a strong oxidizing agent such as oxygen must be based. The
presence of oxygen in microbiological media will inhibit the
growth of anaerobes but if a facultative organism such as E.
93;; is added along with the anaerobe both organisms grow
well. The E. 221; utilize the oxygen in the media and lower
the potential to a level where the anaerobe can also meta-
bolize. The rumen is a somewhat more complex case than the
one considered above but the situation is the same when the
rumen contents become saturated with oxygen. This function
which is fulfilled by the facultative organisms is a symptom
of the dynamic equilibrium which exists in the rumen and the

capacity of the rumen pOpulation to control its environment.

SUMMARY

The oxidation-reduction potential of rumen contents
removed from several cows at three times during the day in-
dicates that a close relationship exists between Eh and
metabolic rate. Titration of samples, from which the large
feed particles had been removed by centrifugation, with acid
and a strong oxidizing agent, respectively, indicated that
a linear relationship exists between Eh and pH and that the
metabolic rate of the microflora is reflected by the amounts
of reducing substances found. Very little of the reducing
capacity of dead rumen contents was found to be associated
with the bacterial cells indicating that extreme care must
be taken to maintain the prOper redox potential when the
rumen microflora are separated from their natural environ-
ment if a normal innocula is desired. The titration curve
for rumen contents indicates that there is no large amount
of any one reductive substance present that is responsible
for the potential observed in rumen contents.

The only rumen function that seemed to be affected by
alteration of the reductive characteristics of rumen fluid
was gas production. Additives employed were DPPD, methylene
blue, cysteine and thioglycollate. The explanation sug-
gested for decreases in the per cent of methane produced was
that the potential may have been stabilized at a level higher

- 58 -

59

than the optimum level for methane producing bacteria.
Addition of oxygen affected neither the redox potential nor
the gas, volatile fatty acid or ammonia production of rumen
contents incubated ip vitpq. Methylene blue did not affect
gas production in cultures of rumen bacteria that were

stabilized with formate as their sole carbon source.

APPENDIX

-60-

61

.m defipowam .Ha om\moonwpmnsm wqwodcon Ho manoaw>fldwowaaasw

 

 

 

mm.a oo.m om.m ms.m mm.a om.a m

mo.m mo.m mm.m mm.m mm.a oo.m H mmaum

ss.a on.a oe.m mm.m mm.H om.H m

us.a ms.a om.m mm.m ma.a om.a H mus

mm.a mm.a ou.m mo.m mm.a mm.a m

Hu.H ms.a os.m om.m ma.a mam.H a mmua
.oao .Hasm .oap .Haoq .pap .Haom .Haom goo
.s.m cone .2.« omuos .s.< 0016 “mass

 

 

H oases ascooad4

m soapomsw mo macapwnpfip oanpoaowpnopom

#6605948 emdwo m

wwwoOd ow wwww tech 089 bHoQfiodm

 

 

 

 

eonmw mmm <0Hmdwwo weddw bowamo
wuoasodwobm R com & omp zms bowdwo meow. wcdwuwo <mHmHHo mHmuoH
UWWU H wmm mO.H Hw.m wn.m HN.H w.Hm m.#o o.mo 0.0w
m wmm mo.w H©.w w©.m HH.mw w.mm m.mm O.wo 0.0w
w wmw qu.o mw.o wm.w HH.H@ w.0¢ H.mm H.0H 0.0#
# wwm mm.m Hw.¢ ww.¢ Hm.Hm w.mw H.©© m.nw 0.0m
m wwo mm.H Hw.© #H.m IIIII IIII IIII IIII IIII
m 860 m~.m Hm.u oo.p IIIII IIII IIII IIII IIII
6 mmo 6m.m mc.m IIII IIIII IIII IIII IIII IIII
OodeOHH wmm QV.@ mm.H wn.m Hm.OH w.mo m.mw O.wfl 0.0m
m w#o mo.o H©.H #O.H HH.mm w.mw m.mm O.#m 0.0m
w wmw um.m mw.m wm.© HH.OO w.oo m.#w $.00 0.0m
a wow mm.m Hn.m ww.m Hm.wo w.mm m.ww o.mm o.ow
w mom ,um.m ms.m cw.o IIIII IIII IIII IIII IIII
0 wow u©.m mo.m wo.o IIIII IIII IIII IIII IIII
o moo on.m mm.» IIII IIIII IIII IIII IIII IIII
waswppaosm\spmmw.

EHHHHmeBm mBBome bwdflomob\poo SH. Hcaob szwa.
asappssopom moaoxpoo 8H. seems spoon.

Z9

.' '51 t

,c‘

.7"-\

65

.0H5HH amass .Ha ooa\domonpaq deoaam mawnwwaafls

 

 

 

m

.0H8HH ocean .Hs 00H\0Hoo mcHosHHHHsH
0.Hc m.am III III IIII IIII IIII IIII IIII IIII m
s.He 0.06 00. m0. Ha.H HH.m m0.H ao.m 00.0 mm.m m

0.m4 H.0m mm. as. 00.H 00.m 00.H m0.m 00.0 0m.m H ooHopmao
m.H¢ 0.5m III IIII IIII IIII IIII IIII IIII IIII m
m.Hc 6.06 mm. mm. 00.H 00.m 08.H m0.m HH.u 00.0 m

opwHHoo

0.0a n.0m Ha. m0. 00.H m0.m mm.H 00.m 00.5 Hm.m H IsHmons
m.He 0.06 00. _H0. mm.H H0.m Hm.H mo.m m0.0 mm.m m

H.m¢ H.0m 00. 08. 0m.H H0.m H0.H 00.m Hm.s 0m.m H Hoapooo

womoHpHn Honmfin OHHNpSQ .Qonm oHpoom
-waqoaad mUHo< hppwm o and o>

qOHposvonm moms onwopmho was opmaaoohawoanp no poomwm

manoaad dam mowed hppdh oHHpsHob 0:» mo

m oHome xHoqoaaa

.UHSHH amass .Ha ooa\dfiom moaoawaafian
.0HDHH amass .Ha ooa\nomonufiq manoaam mawnwwaawsm

 

III am. on. IIII mm. mm. 00. 00. soomHm
III m0.H m8.H IIII 00.H so.H mm.H 00.H oHaspom
III 00.H sm.H IIII m0.H 00.H 00.H as.H .aoam
III 4m.0 0a.0 IIII ma.o Ha.c 60.0 08.0 ooaaoca

0.mm o.mm m.mm H.06 0.4m 0.mm 0.46 o.mm «a aHooaaa

 

m N H m N a N a
0Ho< OHQHoomd moan oqmahgpms mHoHpnoo

 

IJ‘

mHnoaad use mowed hppwm oawpmao>
womb 0Ho4 oHQHoomd 0am oSHm onoflhdpos Ho uoouwm

4 0H089 «chomaq

65

N

.oononomwwv hp Umpdadoamoo
.moHdpado can now oSawb some mpqomonmon mow prop Ho pnoo mom

 

 

 

 

.oonzom nophwo oHom onwahomm
N mm mm m SN 05 mm
m mN mm d mN mm mm
d mN mm d 5N mm mN
m on mm m 0m mm Hm
5N so mN mo ma
Ho #N om mm NH HH HwHHa
Nm no m:
cm dd ow
om mm am
we ma ma H amass.
m smo N00 6mm emu omoo Amadev
swan owoahnpoa maonpnoo noprndodH
moapflmomaoo
snowprHHHpopm

oHSpHSO quHdQ noHpHmomaoo mow aw momnmno

m oHnma anooagq

LITERATURE CITED

Ames, S. R. and C. A. Elvehjem. Enzymatic oxidation of
1945 Glutathione. J. Biol. Chem., 159:549.

Ames, S. R. and P. L. Harris. Recent develOpments in the
1948 chemistry and metabolism of vitamin E. Biologi-
cal Antioxidants, Trans. Third Conf., Josiah
Macy Jr. Foundation. New York.

Anonymous. Rumen MicrobiOIOgy, Agradata, 2:5. Chas. Pfizer
1958 and Co., Inc. Terre Haute, Indiana.

Aubel, E., A. G. Rosenberg and M. Grunberg. Study of oxida-
tion-reduction potential. Limit of growth of the
anaerobic bacteria. Helv. Chem. Acta. 29:1267
(from Hewitt, 1950).

Ball, E. G. and T. T. Chen. Studies on oxidation-reduction.
1955 XX Epinephrine and related compounds. J. Biol.
Chem., 102:691.

Ball, E. G. Studies on oxidation-reduction. XXIII Ascorbic
1957 acid. J. Biol. Chem., 118:219.

Ball, E. G. The catalytic action of milk flav0protein in the
1959 oxidation of reduced diphosphOpyridine nucleo-
tide (cozymase). J. Biol. Chem., 151:767.

Ball, E. G. Energy relationships of oxidative enzymes.
1944 Ann. of the New York Acad. of Sci., 45:565.

Barker, H. A. and F. Lipmann. On lactic acid metabolism in
1944 prOpionic acid bacteria and the problems of
oxido-reduction in the system fatty-hydroxy-
keto acid. Arch. Biochem., 4:561.

Barker, H. A., M. D. Kamen and B. T. Barnstein. The synthesis
1945 of butyric and caproic acids from ethanol and
acetic acid by Clostridium kluyverii. Proc.
Nat. Acad. Sci., Wash., 51:575.

Barnett, A. J. G. and M. G. C. M. Dow. Utilization of carbo-

1957 hydrate metabolites by rumen micro-organisms.
Nature, 180:548.

-66...

67

Barron, E. S. G. and Z. A. Hoffman. Catalytic effect of dyes
1950 on oxygen consumption of living cells. J. Gen.
Physiol., 15:485.

Barron, E. S. G. and T. P. Singer. Studies on biological
1945 oxidations. XIX Sulfhydryl enzymes in carbo-
hydrate metabolism. J. Biol. Chem., 152:221.

Barron, E. S. G. and T. P. Singer. Studies on biological
1945 oxidations. XX. Sulfhydryl enzymes in fat and
protein metabolism. J. Biol. Chem., 152:241.

Bentley, 0. G., R. R. Johnson, S. Vanecko and C. H. Hunt.

1954 Studies on factors needed by rumen microorganisms
for cellulose digestion 1g vitro. J. An. Sci.
15:581.
Blaxter, K. L., F. Brown, W. A. Wood and A. M. MacDonald. The
1955 nutrition of the young ayrshire calf. 14. Some

effects of natural and synthetic antioxidants on
the incidence of muscular dystrOphy induced by
cod liver oil. Brit. J. Nutr., 7:557.

Booker, W. M., J. R. Tureman, F. Da-Costa, J. Poulson and
1954 J. Mitchell. Oxygen uptake of whole body and
certain tissues in intact and adrenalectomized
mice subjected to cold stress. Fed. Proc., 15:15.

Borsook, H. and G. Keighly. Oxidation-reduction potential of

1955 ascorbic acid (vitamin C). Proc. Nat. Acad. Sci.,
19:875.
Borsook, H., H. W. Davenport, G. E. P. Jeffreys and R. C.
1957a Warner. Oxidation of ascorbic acid and its reduc-

tion. J. Biol. Chem., 112:257.

Borsook, H., E. L. Ellis, and H. M. Huffman. Sulfhydryl
1957b oxidation-reduction potentials derived from
thermal data. J. Biol. Chem., 112:281.

Borsook, H. The oxidation-reduction potential of coenzyme I.
1940 J. Biol. Chem., 155:629.

Broberg, G. Measurements of the redox potential in rumen
1957a contents. I 1n vitro measurements on healthy
animals. Nord. Vet.-Med., 9:918.

Broberg, G. Measurements of the redox potential in rumen
1957b contents. II 1p vitro measurements on sick
animals. Nord. Vet.-Med., 9:951.

68

Broberg, G. Measurements of the redox potential in rumen
1957c contents. III Investigations into the effect
of oxygen on the redox potential and quantita-
tive 1p vitro determinations of the capacity
of rumen contents to consume oxygen. Nord. Vet.—
Med., 9:942.

Broberg, G. Measurements of the redox potential in rumen
1958 contents. IV 1; vivo measurements. Nord. Vet.-
Med., 19:265.

Bryant, M. P. and L. A. Burkey. Cultural methods and some
1955 characteristics of some of the more numerous
groups of bacteria in the bovine rumen. J.
Dairy Sci., 55:205.

Bryant, M. P. and R. N. Doetsch. Factors necessary for the

 

1954 growth of Bacteriodes succinogenes in the vola-
tile fatty acid fraction of rumen fluid. Science,
120:944.

Burroughs, W., N. A. Franke, P. Gerlaugh, and R. M. Bethke.
1950 Preliminary observations upon factors influenc-
ing cellulose digestion by rumen microorganisms.
J. Nutr., 99:9.

Burton, K. and T. H. Wilson. The free energy changes for
1955 the reduction of diphosphOpyridine nucleotide
and the dehydrOgenation of L-Malate and L—
Glycerol l-phosphate. Biochem. J., 53:86.

Cardon, B. P. and H. A. Barker. Amino-acid fermentation
1947 by Clastridium prOpionicum and Diplococcus
glycin0phi1us. Arch. Biochem., 19:165.

Carrol, E. J. and R. E. Hungate. Formate dissimilation and
1955 methane production in bovine rumen contents.
Arch. Biochem. and BiOphysics, 55:525.

Carson, S. F. Design and interpretation of carbon isotOpe
1948 experiments in bacterial metabolism. Cold Spr.
Harb. Sym. Quant. Biol., 15:75.

Chambers, R., B. Cohen and H. Pollack. Intracellular oxida-
1951 tion studies. III. Permeability of Echinoderm
ova to indicators. Brit. J. Exp. Biol., 5:229.

Cheng, E. W., G. Hall and W. A. Burroughs. Method for the
1955 study of cellulose digestion by washed suspen-
sions of rumen microorganisms. J. Dairy Sci.,

5§:l225.

69

Chu, H. and M. P. Bryant. The B-vitamin and nitrogen require-

 

1958 ments of Bacteroides succinogenes on important
ruminal cellulolytic bacterium. J. Dairy Sci.,
91:625. _

Clark, W. M., B. Cohen, H. D. Gibbs and R. Cannon. Studies
1928 on oxidation-reduction. I-X. Hygienic Labora-
tory Bul. No. 151.

Clark, W. M. Topics in physical chemistry. Williams and
1948 Wilkins 00., Baltimore, Maryland.

Clark, W. G. and T. A. Geissman. Practical application of
1949 physiological antioxidants. Biological anti-
oxidants, Trans. Fourth Conf., Josiah Macy, Jr.
Foundation, New York, New York.

Colowick, S. Glutathione, a symposium. Academic Press, Inc.,
1954 New York, New York.

Coulter, C. B. and M. L. Issacs. Oxidation-reduction equili-

1929 bria in biological systems. II Potentials of
aerobic cultures of g. Typhosus. J. Exp. Med.,
fl9:7ll.

Crandall, D. I. Homogentisic Acid Oxidase. II Pr0perties

1955 of the crude enzyme in rat liver. J. Biol. Chem.,
gig:5650 '

Dam, H. Influence of antioxidants and redox substances on
1957 4 signs of vitamin E deficiency. Pharmacological
Reviews, 9:1.

DeLuca, A. P., R. Teichman, J. E. Rousseau, Jr. and R. E.
1955 ' Johnson. Relative effectiveness of various
antioxidants fed to lactating dairy cows on in-
cidence of COpper induced oxidized milk flavor
and on carotene and toc0pherol utilization. J.
Dairy Sci., 5§:695.

Delwiche, E. A. The mechanism of prOpionic acid formation

 

 

 

 

1948 by Propionibactepium pentosaceum. J. Bact.,5§:811.
Delwiche, E. A., E. F. Phares and S. F. Carson. Succinic acid
1956 decarboxylation system in Propionibacterium
pentosaceum and Veillonella Gazogenes. II.
Evidence for an active "Cf complex. J. Bact.,

21:604.

I

70

Dixon, M. and J. H. Quastel. A new type of oxidation-
1925 reduction system. I Cysteine and glutathione.
J. Chem. Soc., 125:2945.

Dixon, M. and H. E. Tunnicliffe. On the reducing power of
1927 glutathione and cysteine. Biochem. J., g1z844.

Doetsch, R. N., J. C. Shaw , J. J. McNeill and P. Jurtshuk.
1955 Observations on the use of mixed suspensions
of bovine rumen bacteria as a technique of the
rumen microbiologist. Univ. of Maryland Misc.
Publ. No. 258.

Draper, H. H., S. Goodyear, K. D. Barbee and B. C. Johnson.
1958 A study of the nutritional role of anti-oxidants
in the diet of the rat. Brit. J. Nutr., 1g:89.

Elliot, K. A. C. Biological oxidations and reductions. Ann.
1946. Rev. Biochem., 15:1.

Elsden, S. R. The fermentation of carbohydrates in the rumen
1945 of the sheep. J. Exptl. Biol., gg:5l.

Elsden, S. R. and A. K. Sijpesteijn. The decarboxylation of
1950 succinic acid by washed suspensions of rumen
bacteria. J. Gen. Microb. 4:XI.

Elsden, S. R. and D. Lewis. The production of fatty acids

1955 by a gram negative coccus. Biochem. J., 55:185.
El-Shazly, K. Degradation of protein in the rumen of the
1952a sheep. I. Some volatile fatty acids, including
branched-chain isomera found 1g vivo. Biochem.
J. 51:640.

El-Shazly, K. Degradation of protein in the rumen of the
1952b sheep. 2. The action of rumen microorganisms
on amino acids. Biochem. J., 51:647.

Fieser L. F. Napthoquinones as antimalarials and inhibitors
1948 of respiration. Biological Antioxidants. Trans.
Third. Conf. Josiah Macy, Jr. Foundation. New
York, N. Y.

Fischer, E. K. Oxidation-reduction potentials of certain sul-
1950 fhydryl compounds. J. Biol. Chem., 59:755.

Fruton, J. S. Oxidation-reduction potentials of ascorbic
1954 acid. J. Biol. Chem., 105:79.

71

Gall L. S., C. W. Huhtanen, R. Saunders and W. Schmidt.

1955 Comparison of rumen flora and environment in
roughage vs. grain fed animals. (abs.) J.
Dairy Sci., 55:587.

Ghosh, J. G., S. N. Raychandhuri and S. C. Ganguli. Oxidation-
1952 reduction potential. 1. cystine 2. thiogly-
collic and thioacetic acids. J. Ind. Chem.
$00., 9:45.

Ghosh, J. C. and S. C. Ganguli. A note on the reduction po-
1954 tential of cysteine-cystine mixtures. Biochem.
J., 28:581.

Giese, A. C. Cell Physiology. First Edition. W. B. Saunders

1957 Company, Philadelphia and London, p. 268-512.
Gillespie, L. J. Reduction potentials of bacterial cultures
1920 and of water logged soils. Soil Sci., 9:199.
Gilroy, J. J. and F. G. Hueter. Nutritional requirements of
1957 rumen bacteria. Univ. of Maryland Misc. Publ.

No. 291.

Gray, F. V., A. F. Pilgrim, H. J. Rodda and R. A. Weller.
1951 Volatile fatty acids in the rumen of sheep.
Nature, Land., 162:954.

Gray, F. V., A. F. Pilgrim, H. J. Rodda and R. A. Weller.
1952 Fermentation in the rumen of the sheep. IV.
Nature and origin of the volatile fatty acids
in the rumen of sheep. J. EXP. Biol., 59:57.

.Green, D. E. The reduction potentials of cysteine, gluta-
1955 thione and glycylcysteine. Biochem- J., 52:678.

Green, D. E. The reduction potentials of ascorbic acid.
1955 Biochem. J., 52:1044.

Green, D. E., L. H. Stickland and H. L. A. Tarr. Studies on
1954 reversible dehydrogenase systems. III Carrier-
1inked reactions between isolated dehydrogenoses.
Biochem. J., 55:1812.

Greenberg, L. D. and J. F. Rinehart. 'Serum iron levels in
1955 Rhesus Monkeys with chronic vitamin C deficiency.
Proc. Soc. Exptl. Biol. Med., 55:525.

Greenberg, S. M. R. G. Tucker, A. E. Heming and J. K. Mathews.
1957 Iron absorbtion and metabolism. I. Interre-
lationship of ascorbic acid and vitamin E. J.

Nutr., 55:19.

72

Greville, G. D. and K. G. Stern. The reduction of nitro-
1955 phenols by redox indicators and enzymes. Bio-
chem. J., 59:485.

Gunsalus, I. C., B. L. Horecker and W. A. Wood. Pathways
1955 of carbohydrate metabolism in microorganisms.
Bact. Rev., 19:79.

Gutierrez, J. Numbers and characteristics of lactate utiliz-
1955 ing organisms in the rumen of cattle. J. Bact.,
55:125.

Hawk, D. B., B. L. Oser and W. H. Summerson. Practical Physi-
1954 ological Chemistry. Thirteenth edition, McGraw-
Hill Book Company, Inc., New York.

Hellerman, L. Some aspects of interference with oxidation in
1947 the cell with reference to the suppression of
flavoenzyme catalysis, and the application of
the sulfhydryl hypothesis. Biological Antioxi-
dants, Trans. Second Conf., Josiah Macy Jr. Fond.,
New York.

Hentschl, A. F., R. N. Berry and C. F. Huffman. A plastic
1954 plug for use in the bovine rumen fistula.
M. S. C. Veterinarian, 15:77.

Hershberger, T. V., O. G. Bentley, J. H. Cline and W. J.
1956 Tyznik. Formation of short-chain fatty acids
from cellulose, starch, metabolic intermediates
by ovine and bovine rumen organisms. Agr. and
Food Chem., 5:952.

Hewitt, L. F. Oxidation-reduction potentials of cultures of
1950 haemolytic Streptococci. Biochem. J., 55:512.

Hewitt, L. F. Oxidation-reduction potentials in bacteriology
1950 and biochemistry. Second Edition. The Macmillan
Company, New York, N. Y.

Hickman, K. and P. L. Harris. Toc0pheral interrelationships.
1946 Adv. Enxymol., 5:469.

Horn, L. H., Jr., R. R. Snapp and L. S. Gall. The effect of
1955 antibiotics upon the digestion of feed nutrients
by yearling steers with bacteriological data.
J. An. Sci., 15:245.

75

Huhtanen, C. N., R. K. Saunder and L. S. Gall. Fiber diges-
1954 tion using the miniature artificial rumen.
J. Dairy Sci., 52:528.

Hungate, R. E. Studies on cellulose fermentation. III.
1947 The culture and isolation of cellulose-decom-
posing bacteria from the rumen of cattle. J.
Bact., 55:651.

Hungate, R. E. The anaerobic meSOphilic cellulytic bacteria.
1950 Bact. Revs. 15:1.

Hungate, R. E. ‘Microorganisms in the rumen of cattle fed a

 

 

1957 constant ration. Can. J. Microbiol., 5:289.
Johns, A. T. Mechanism of Pr0pionic Acid formation in bac-
1949 terial fermentation. Nature,l64:620.
Johns, A. T. The mechanism of prOpionic acid formation by
1951a ' Veillonella gaz0genes. J. Gen. Microbiol.,
5:526.
Johns,-A. T. The mechanism of propionic acid formation by
1951b the ErOpionibacteria. J. Gen. Microbiol.,
5:557. ‘
Johns, A. T. Mechanism of prOpionic acid formation by clos«
1952 tridium propionicum. J. Gen. Microbiol., 5:125.

 

Kalnitsky, G., H. G. Wood, and C. H. Werkman. CO -fixation
1945 and succinic acid formation by a cell.2- free
enzyme preparation of Esherichia Coli.-Arch.
Biochem., 5:269.

Keeney, M. Direct chromatOgraphic determination of C to
1955 C fatty acids in rumen fluid. Univ. of Maryland
m so. publ. No. 258.

Kendall, E. C. and F. F. Nord. Reversible oxidation-reduction
1926' of cysteine-cystine and reduced and oxidized
glutathione. J. Biol. Chem., 59:295.

Klemperer, F., O. A. Bessy and A. B. Hastings; Oxidation-
1957 reduction potentials of certain synthetic
flavins. Proc. Soc. Exptl. Biol. Med., 52:114.

Kornberg, H. L. and H. A. Krebs. Synthesis of cell consti-
1957 tuents from Cg-units by a modified tricarboxylic
acid cycle. Nature, 129:988.

75

McNeill, J. J. and O. R. Jacobson. Studies on the methane
1955 and hydrogen metabolism of bovine rumen bacteria.
Univ. of Maryland Misc. Publ. No. 258.

McNeill, J. J. and R. N. Doetsch. Mechanism of methane
1956 formation by bovine rumen bacteria and the effect
of hydrogen acceptors and donors on methane
production. J. Dairy Sci., 59:941.

Michaelis, L. and L. Flexner. Oxidation—reduction systems of
1928 biological significance. I The reduction
potential of cysteine. J. Biol. Chem., 29:689.

Michaelis, L., M. P. Schubert and C. V. Smythe. Potentio-
1956 metric study of the flavins. J. Biol. Chem.,
116:587.

Michaelis, L. The mechanism of autooxidation and the action
1948 of an antioxidant. Biological antioxidants,
Trans. Third Conf. Josiah Macy Jr. Found.,
New York, New York.

Michaelis, L. and Wollman, S. H.- The semiquinone radical of

1949 t000pherol. Science, 109:515.
Moore, T., J. M. Sharman and R. J. Ward. The partial vitamin
1954 E activity of certain redox dyes. Biochem. J.,
55:VII.

Mylroie, R. L. and R. E. Hungate. Experiments on the methane
1954 bacteria in sludge. Can. J. Microbiol. 1:55.

Nason, A. and I. R. Lehman. Toc0pheral as an activator of
1955 cytochrome C Reductase. Science, 122:19.

Nelson, W. 0., R. A. Oppermann and R. E. Brown. 15 vitro
1956 ~studies on methanogenic rumen bacteria. J.

Dairy Sci., 59:940.

Nelson, W. 0., R. A. Opperman and R. E. Brown. 15 vitro
1957 studies on methanogenic rumen bacteria. II.
Fermentation of butyric and valeric acid. J.

Dairy Sci., 51:545.

Opperman, R. A., W. 0. Nelson and.R. E. Brown. 15 vitro
1957a studies on methanogenic rumen bacteria. J.
Dairy Sci., 55:779. ~

Opperman, R. A., W. 0. Nelson and R. E. Brown. 15 vitro _
1957b studies on methanogenic rumen bacteria. Utill-
zation of valeric acid and butyric acids. J.
Dairy Sci., 55:652.

74

Koser, S. A. and J. L. Thomas. Effect of ascorbic acid on
1955 cysteine requirements of lactobacilli. Proc.
Soc. Exptl. Biol. Med., 95:454.

Krampitz, L. D., H. G. Wood and C. H. Werkman. Enzymic
1945 fixation of carbon dioxide in oxilacetate. J.
Biol. Chem., 142:245.

Kuhn, R. and T. Wagner-Jauregg. Oxidation-reduction rela-
1954 tions and a colour reaction for lactoflavin.
Ber., 62B:56l. (from Hewitt, 1950).

Kuhn, R. and P. Boulanger. Relationship of oxidation-reduction
1956 potential and chemical constitution of flavin.
Ber., 69B:1557. (from Hewitt, 1950).

LaDu, B. N. Jr. Effect of ascorbic acid on oxidation of
1952 tyrasine. Fed. Proc., 11:244.

LaDu, B. N. Jr. and D. M. Greenberg. Ascorbic acid and the
1957 oxidation of tyrosine. Science, 112:111.

Lepper, E. and C. J. Martin. On the behavior of'dndifferent"
1951 electrodes when used for the determination of
oxidation-reduction potential in the presence
of hydrogen. Biochem. J., 55:45.

Longsworth, L. G. and D. A. MacInnes. Bacterial growth with
1955 automatic pH control. (A) An apparatus. (B)
Some tests on the acid production of Lactobac-
cillus acidophilus. J. Bact., 59:595.

Louvr, J. G., H. H. Williams and L. A. Maynard. A new method
1949 for the study 15 vitro rumen digestion. Science,
110:478.

Lugg, J. W. H. The chemistry and metabolism of the compounds
1945 of Sulphur. Annual Rev. of Biochem., 15:265.

Mackenzie, J. B. The vitamin E activity of toc0pheryl-
1949 hydroquinone and toc0pheryequinone. Biological
antioxidants, Trans. Fourth Conf. Josiah Macy
Jr. Found., New York, N. Y.

Marston, H. R. The fermentation of cellulose 15 vitro by
1948 organisms from the rumen of sheep. Biochem. J.
55:564. .

76

Otagaki, K. K., A. L. Black, H. G088 and M. Kleiber. 15
1955 vitro studies with rumen microorganisms using
carbon-14-1abeled casein, glutamic acid,
leucine and carbonate. Agr. and Food Chem.

5:948.
Pardee, A. B. Chemical pathways of metabolism. Vol. 1.
1954 pp. 1-25. Ed. David H. Greenberg. Academic

Press. New York, N. Y.

Pearson, R. M. and J. A. B. Smith. The utilization of urea
1945 in the bovine rumen. III. Synthesis and
breakdown of protein in rumen ingesta. Biochem.

J., 51:155.

Perdrau, J. R. and C. Todd. The photodynamic action of
1955 methylene blue on bacteriOphage. The photodyna-
mic action of methylene blue on certain viruses.
Proc. Roy. Soc., Land. B., 115:277.

Pine, M. J. and H. A. Barker. Studies on the methane fer-
1956 mentation. XVII The pathway of hydrogen in
acetate fermentation. J. Bact., 71:644.

Pine, M. J. Methane fermentation of formate by methanobacillus
1958 omebanski. J. Bact., 75:556.

Pirchner, F., R. S. Allen and N. L. Jacobson. Effect of
1957 diphenyl-p-phenylenediamine on the utilization
of carotene and vitamin A by the calf. J. Dairy
Sci., 55:1448.

Quastel, J. H. and A. H. M. Wheatley. The action of dyestuffs

1951 on enzymes. Biochem. J., 55:629.
Rodkey, F. L. Oxidation-reduction potentials of the diphos—
1955 phopyridine nucleotide system. J. Bi01. Chem.,
215:777.
Ryklan, L. R. and C. L. A. Schmidt. The oxidation potentials
1944 , of cystine—cysteine and related systems. Univ.

of Cal. Publ. in Phys., 5:257.

Schepartz, B. Inhibition and activation of the oxidation of
1955 homogentisic acid. J. Biol. Chem., 205:185.

Schott, H. F. and H. Borsook. Coupled reactions in biolo-
1955 gical systems. Science, 22:589.

77

Scott, M. L., F. W. Hill, L. C. Norris, D. C. Dobson and
1955 T. S. Nelson. Studies on vitamin E in poultry
nutrition. J. Nutr., 56:587.

Seifter, J. General pharmacology and toxicology of quinones
1948 and hydroquinones. Biological antioxidants,
Trans. Third Conf., Josiah Macy, Jr., Found.
New York, N. Y. .

Shull, R., R. B. Alfin-Slater, H. J. Devel, Jr., and B. H.

1957 Ershoff. Comparative effects of alpha-tOCOpheral,
DPPD and other antioxidants on muscular dys-
trOphy in guinea pig. Proc. Soc. Exp. Biol.
and Med., 95:265. '

Sijpesthn, A. K. and S. R. Elsden. The metabolism of suc-
1952 cinic acid in the rumen of sheep. Biochem. J.,
22:41.

Singsen, E. P., R. H. Bunnel, L. O. Matterson, A. Kozeff and
1955 E. L. Jungherr. Studies on encephalomalcia in
the chick. 2. The protective action of diphenyl-
p-phenylenediamine against encephalomalcia.
Poult. Sci., 55:262.

Sirotnak, F. M., R. N. Doetsch, R. Q. Rebinson and J. C. Shaw.
1954 Aspartate dissimilation reactions of rumen bac-
teria. J. Dairy Sci., 52:551.

Slade, H. D. and c. H. Werkman. Assimilation of acetic and
1945 succinic acids containing heavy carbon by
Aerobacter indologenes. Arch. Biochem., 5:97.

Smith, P. H. and R. E. Hungate. Isolation and characteriza-
1958 tion of Methanobacterium Ruminantium. N. Sp.
. J. Bact. 25:715.

Snedecor, G. W. Statistical Methods. 4th edition. Iowa
1946 State College Press, Ames, Iowa.

Snell, E. E. and.D. E. Metzler. Water soluble vitamins. Ann.
1956 Rev. Biochem., 55:455.

Stadtman, E. R. and H. A. Barker. Fatty acid synthesis by
1949 enzyme preparations of Clostridium kluyveri
I. Preparation of cell free extracts that catalyze
the conversion of ethanol and acetate to butyrate
and caproate. J. Biol. Chem., 155:1085.

78

Stadtman, E. R. The purification and prOperties of phospho-
1952 transacetylase. J. Biol. Chem., 196:527.

Takeda, V. and M. Hara. Significance of ferrous ion and
1955 ascorbic acid in the Operation of the tricar-
boxylic acid cycle. J. Biol. Chem., 214:657.

Taylor, 0. W. Effects of t000pherols, methylene blue and
1958 glutathione on the manifestations of oxygen
poisoning in vitamin E deficient rats. J.
Physiol., 155:57.

Todd, J. W. and D. J. Walker. The fermentation of lactate
1958 and acrylate by cell-free extracts of rumen
organism LC-l. Biochem. J., 69:29P.

Tucker, R. G., S. M. Greenberg, A. E. Heming and J. K. Mathews.
1957 Iron absorbtion and metabolism. 11. Substitu-
tion of N, Nl-diphenyl-p-phenylenediamine (DPPD)
for vitamin E. J. Nutr., 55:55.

Utter, M. B., F. Lipman and C. H. Werkman. Reversibility of
1945 the phosphoroclasticsplit of pyruvate. J. Biol.
Chem., 158:521.

Virtanen, A. I. and S. S-V. Hausen. Role of substances formed
1949 during germination in the growth of plants.
Nature, 165:482.

Waldo, D. R. and L. H. Schultz. Lactic acid production in
1956 the rumen. J. Dairy Sci., 55:1455.

Walker, D. J. and J. W. Todd. A coupled oxidation-reduction
1958 reaction involving lactate and acrylate.
Biochem. J., 69: 29P.

Warner, A. C. I. Criteria for establishing the validity of
1956 15 vitro studies with rumen microorganisms in
so-called artificial rumen systems. J. Gen.
Microbiol., 15:755.

Wasserman, R. H., C. W. Duncan, E. S. Churchill and C. F.
1952 Huffman. The effect of antibiotics on 15 vitro
cellulose digestion by rumen microorganisms.
J. Dairy Sci., 55:571.

Weissberger, W. Autoxidation of alpha-ketols, enediols, and
1948 hydroquinones: quinone catalysis and inhibitian.
Biological antioxidants, Trans. Third Conf.,
Josiah Macy, Jr., Found., New York, N. Y.

79

Werkman, C. H. and H. G. Wood. HeterotrOphic assimilation

1942 of carbon dioxide. Adv. in Enzymology, 5:155.
West, E. S. and W. R. Todd. Textbook of Biochemistry.
1957 Second Edition. The Macmillan Co., New York,
N. Y.

Williams, J. W. and E. M. Drissen. Oxidation-reduction
1950 potentials of certain sulphydryl compounds. J.
Biol. Chem., 52:441.

Wood, H. G., R. Stjernhohm and F. W. Leaver. The role of
1956 succinate as a precurser of prOpionate in the
prOpionic acid fermentation. J. of Bact., 25:142.

Worker, N. A. and W. A. McGillivray. Factors influencing
1957 the vitamin A content of milk fat. II. The
influence of botanical composition of the
pasture., J. Dairy Res., 55:85.

Young, L. The effect of pyocyanine on the metabolism of
1957 cerebral cortex. J. Biol. Chem., 119:659.

'Iv
van

Uu! "
- um:

;|VV'.¥I T
”,1" ' f a: a 7
. J
. f .. ”if:
(I9_‘I,I ’ I 13’
k. ,h ‘171 'I
"' (2 j" " NI
I'- 5 ‘ '
d’ ‘ ‘
A‘il‘vI}. " I 7‘. ‘
~i‘ ‘ '. .7-
.“JJA Ltd-‘31.}. (Ann,7
944'
0
"#3 I.

 

 

 

""‘II III‘IIIIIII‘II 111111111111 III“