Microbial Rumen Fermentation
JAMES B. RUSSELL 1"4 and R O B E R T B. HESPELL 2'3
INTRODUCTION
With the ruminant animal, we have essen-
tially two ecosystems, namely, the microbial
ecosystem within the rumen and the animal's
external environment. In dealing with the mi-
crobial ecosystem, we have made significant
strides in the last few decades toward accom-
plishing the overall goal of ecology, which is
to understand the relationships of organisms to
their environments. As pointed out by Hungate
(47), a complete ecological analysis of any
natural habitat requires an elaboration of:
1 ) kinds and numbers of organisms, 2) activities
of the organisms, and 3) extent to which their
activities are expressed. Within the last 25 yr,
much information has been gained in the first
two aspects of the ecological analysis. Currently
we are just beginning to address the third
aspect.
The rumen is an ideal fermentation site. In
most ruminant species, the rumen is approxi-
mately one-seventh of the mass of the animal,
is maintained at a relatively constant tempera-
ture (39°C), is buffered well by salivary secre-
tions, and compared to many other microbial
ecosystems is well supplied with nutrients.
End-products of the fermentation (e.g., volatile
fatty acids), which can be toxic to microbial
metabolism, are removed across the rumen wail.
The microflora inhabiting the rumen is dense
and contains approximately 10 l° to 1011
bacterial and 106 protozoal cells per milliliter.
Diversity within this population is extensive,
and approximately 200 species of bacteria and
20 species of protozoa have been isolated (13).
Although a few of these bacteria may be
"casual passengers brought in with the food"
and thus not be "authentic rumen bacteria",
Received August 29, 1980.
1 Department of Animal Science.
2 Department of Dairy Science.
3 To whom reprint requests should be addressed.
4 Department of Animal Science, Cornell Univer-
sity, Ithaca, NY 14850.
1981 J Dairy Sci 64:1153-1169 1153
Departments of Animal Science and Dairy Science
University of Illinois
Urbana 61801
the complexity of ruminal bacteria is great
(13).
During rumen fermentation, short chain
fatty acids and microbial ceils are formed from
feedstuffs, and these products serve as sources
of energy and protein, respectively, to the
animal. Methane, heat, and ammonia are
evolved as well, and these products can repre-
sent a loss of energy and nitrogen for the
animal. The efficiency of nutrient utilization by
ruminants is determined largely by the balance
of these fermentation products, and this
balance ultimately is controlled by the types of
microorganisms in the rumen.
C U L T I V A T I O N OF R U M I N A L M I C R O O R G A N I S M S
The rumen environment is one of extreme
anaerobiosis, and, as expected, inhabiting
microorganisms are strict anaerobes sensitive to
oxygen. It was not until development by
Hungate (46) of techniques that prevented
exposure to oxygen at all times that definitive
studies on ruminal bacteria could be begun in
earnest. Although basic concepts and manip-
ulations remain the same, these anaerobic
techniques have been modified (15, 40, 49) and
must be employed in any meaningful studies
involving ruminal microorganisms. Within the
last decade, a major development in cultivating
strict anaerobes has been the refinement and
usage of plastic anaerobic gloveboxes and
serum-capped vessels. These newer techniques
have not supplanted but rather are used in
conjunction with the Hungate techniques and
allow one to do such manipulations as routine
Petri dish plating or replica plating (41, 56) and
cultivation of methanogens on a large scale (5).
Beginning in the 1950's and extending
through the 1960's, many successful studies
were undertaken to develop appropriate culti-
vation media and to isolate, enumerate, and
characterize various bacterial species. Through
the perseverance of these researchers, primarily
M. P. Bryant and his colleagues, we now know
1154 RUSSELL AND HESPELL
many (if not most) of the major bacterial
species and have a reasonable understanding of
their general functional roles in the rumen
(Table 1 ).
The majority of ruminal bacterial species can
be grown on relatively simple media that in-
cludes one or more carbohydrates (e.g., cellu-
lose, cellobiose, starch, xylan, glucose), ammo-
nia and Trypticase, b-vitamins, heme, vitamin K
derivatives, mineral salts, and a reducing agent
such as sodium sulfide and L-cysteine. Many
species also require (or their growth is stimu-
lated by) short straight- or branch-chained
volatile fatty acids and carbon dioxide. For
nonselective isolation and enumeration,
clarified rumen fluid often is included in the
media as a source of nonspecific factors (19);
however, rumen fluid can be replaced by other
components (21). Within the last several
years, media have been developed that have
been selective for enumeration of carbohydrate-
utilizing subgroups (but not to the species
level) from ruminal contents (3, 34, 56).
Specific details on the nutrition and t a x o n o m y
of ruminal bacteria have been discussed else-
where (13, 26, 48).
With respect to cultivation and studies with
ruminal protozoa, microbiological effort has
not been as successful as with bacteria, proba-
bly because of a combination of factors,
including the inability of investigators to grow
protozoa in the absence of bacteria or other
protozoal species, that protozoa are not essen-
tial to the host animal, and the general scarcity
of protozoologists competent in anaerobic
techniques. Given these limitations, however,
accomplishments have been significant since
the early cultivation attempts by Margolin (62)
and Hungate (45). There have been a number of
studies, primarily by G. S. Coleman and his
colleagues, in which various species of ciliate
protozoa have been cultivated with bacteria in
vitro for as long as 5 yr (28). Cultivation media
have included a complex mixture of basal
salts, clarified rumen fluid, and carbohydrate
energy sources such as whole-grain flour, dried
grass, rice starch, or washed bran. The use of
these particulate carbohydrates along with
inclusion of one or two antibiotics in the media
allows for proliferation of protozoa without
having overgrowth of bacteria. Because of the
undefined nature of cultivation media and
presence of bacteria, we do not have accurate
Journal of Dairy Science Vol. 64, No. 6, 1981
information on nutritional requirements of
ruminal protozoa. As shown by numerous
studies (28, 29, 82), some protozoa prefer to
use soluble carbohydrates whereas others
engulf particulate carbohydrates. Engulfed
bacterial cells can serve as the major nitrogen
source for most species, but other nitrogenous
sources such as particulate proteins, amino
acids, peptides, and ammonia also are utilized
to varying extent, depending upon the parti-
cular species. Bacterial cells also serve as a
source of precursors for synthesis of protozoal
nucleic acids (30 and references therein), and
it is logical to assume they serve as sources of
many other nutritional factors such as vitamins,
minerals, etc.
The ruminal protozoal population is pre-
dominated by ciliates, although a few species
of flagellates can be present. Flagellates are
often numerous in calves prior to development
of the ciliate population. Shortly after feeding
in the adult animal, large increases in the
flagellate Neocallirnastix frontalis can take
place (74). The ciliate ruminal protozoa are
composed of 20 or so species that can be
divided into two groups - the holotrichs and
oligotrichs. The holotrichs possess a simple
morphology, superficially resemble paramecia,
and are members of the genera Isotricba or
Dasytricha. In contrast, the oligotrichs are
morphologically complex, with various bands
of cilia, skeletal plates, and surface projections
such as spines. Species of Entodinum,
Epidinium, Diplodium, and Opbyroscolex are
in varying numbers in the rumen. An extensive
description of the ruminal protozoa and their
morphologies may be found in Hungate's book
(48).
BIOCHEMICAL SIGNIFICANCES
OF R U M I N A L M I C R O O R G A N I S M S
Overall Fermentation
Within the rumen, an intensive microbial
degradation of foodstuffs takes place. Plants
primarily are composed of carbohydrate
polymers, and these are hydrolyzed to small
saccharides that in turn are fermented to
numerous products (Figure 1). Although
numerous intermediates may be formed, the
final fermentation products that accumulate
within the rumen are acetate, propionate,
 TABLE 1. Ruminat bacteria and their fermentative properties.

Fermentation c
Species Animal diets a Functionality b products

Bacteroides succinogenes Many C, A F, A, S, --C

Z
Ruminococcus albus Many C, X F, A, E, H, C
Ruminococcus flaveJ?tciens Many C, X F, A, S, H, --C
Butyrivibrio fibrisolvens Many C, X, PR F, A, L, B, E, H, C
Clostridium lockbeadii Coarse hay C, PR F, A, B, E, H, C Z
Streptococcus bovis High grain A, S, SS, PR L, A, F (?) ,q
Bacteroides amylopbilus High grain A, P, PR F, A, S, --C >
~q
Bacteroides ruminicola Many A, X, P, PR F, A, P, S, - C
Succinimonas amylolytica Forage-grain A, D A, S, - C ~z
Selenomonas ruminantium Many; grain A, SS, GU, LU, PR A, L, P, H, C I
Lacbnospira multiparus Legume pasture P, PR, A F, A, E, L, H, C
Succinivibrio dextrinosolvens High grain P, D F, A, L, S, - C
m
Metbanobrevibacter ruminantium Many M, H M >
Metbanosarcina barkeri Many;molasses M, H M, C Z
Spirochete species Many P, SS F, A, L, S z
c <
Megasphaera elsdenii High grain SS, LU A, P, B, V, CP, H, C
Lactobacillus vitulinus Lush pasture; high grain SS L
Anaerovibrio lipolytica Forage; high lipids L A, P, S, - C >
Eubacterium ruminantium Forage SS F, A, B, C
~a
Vibrio succinogenes ... H S
,<

alt is doubtful any one species is completely absent from any rumen, but given diets indicate where the organism is more numerous.
b c = cellulolytic, X = xylanolytic, A = amylolytic, D = dextrinolytic, P = pectinolytic, PR = proteolytic, L = lipolytic, M = methanogenic, GU = glycerol-utilizing,
< LU = lactate-utilizing, SS = major soluble sugar fermentor, H = hydrogen utilizer.
o
ox CF = formate, A = acetate, E = ethanol, P = propionate, L = lactate, B = butyrate, S = succinate, V = valerate, CP = caproate, H = hydrogen, C = carbon dioxide.

z
0
ox

",O
 1156 RUSSELL AND HESPELL

CARBOHYDRATE POLYNERS (CELLULOSE, ST RCH PECT N

. . . . . . . . . . . . . ~. . . . . mation is known about this important process,
I
but some recent studies indicate both bacteria
~21 ~
S~;ALL SACCH~RIDES (CELLOBIOSE, MALTOSE,
XYLOBIOSE, HE×OSES, PENTOSES)
SUCROSE,

and protozoa are involved (4, 24). The second

stage, hydrolysis of released polymers to small

LACT#TE
~ BUTYRATE*
H2 + C0~" OXALOACETATE
CH 4 • SUCCINATE
FIG. 1. Generalized scheme for ruminal degrada-
tion and fermentation of carbohydrates. The products
marked by an asterisk are those which represent
terminal products and accumulate in the rumen.
butyrate, carbon dioxide, and methane. Ratios
of these products vary with diet and frequency
of feeding and are caused by changes in micro-
bial metabolism and species. Under abnormal
circumstances, such as either unusual feeding
practices or host animal sickness, other prod-
ucts like formate, lactate, or ethanol may
appear in the rumen. Proteins also are degraded
in the rumen, and ammonia, carbon dioxide,
and either short straight, branched-chain, or
aromatic fatty acids are formed (Figure 2).
Carbohydrate Metabolism
Degradation and fermentation of poly-
saccharides essentially can be conceived to
occur in three general stages (Figure 1). The
initial stage includes attachment of microor-
ganisms to plant particles and disassociation of
carbohydrate polymers from structural plant
cell matrices. Relatively little definitive infor-
DIETARY AND OTHER PROTEINS
i
POLYPEPTIDES
CAPROATE
saccharides, is catalyzed by numerous extra-
cellular enzymes of which "cellulose com-
plexes" are the predominating types. Here
again, our knowledge is scanty. Since the
~ PROPIONATE"
studies of King and his colleagues (54), re-
search on ruminal cellulases and other hydro-
lases has been relatively dormant. Part of this
stagnation can be attributed to the complex
number of organisms (Table 1) and enzymes
(see 37, 78). The final stage, the intracellular
fermentations of small saccharides, is relatively
well understood, primarily because of the use
of pure cultures in studies over the last two
decades.
Pure and mixed culture studies have estab-
lished that the major biochemical pathway
employed by ruminal bacteria for hexose
degradation is the Embden-Meyerhof-Parnas
(53, 111). For pentoses and deoxyhexoses,
there "is less information available, but the most
likely pathway is probably a combination of a
pentose cycle plus glycolysis (106). Pectins,
which are abundant in lush clovers and alfalfas,
are degraded rapidly and fermented in the
rumen. Several bacteria/ species are pectino-
lytic, and numerous other species can ferment
the breakdown products (103). Although the
extracellular degrading enzymes have been
characterized partially in recent years (103,
117), the specific intracellular enzymes in
fermentation of these uronic acids are not
known.
The major intracellular products formed
from hexose or pentose degradation are pyru-
vate and phosphoenolpyruvate. The compounds
are converted to an array of fermentation
products (Table 1) by various pathways;
however, some of these products (ethanol,
succinate, and lactate) rarely accumulate in

AMINO ACIDS +
i
SHORTPEPTIDES + NH3 + CO2
the rumen. The lack of some of these products
in rumen fluid can be explained partially by
the fact that the terminal fermentation product
I "CETATE
~[ " ISOBUT
RATF~ I "" MICROBIAL GROWTH
of one species may be catabolized further by
I--Im'~IETH YLBU TYR ATE " ~ I other species. For example, in the rumen most
~--~--ISOVALERATE I ] of the propionate is derived from succinate
(11), which is decarboxylated to propionate
"-NH 3 + CO2 ~ AMINO AC[DS by organisms such as Selenol~lonas ruminan-
FIG. 2. Generalized scheme for rurninal degrada- tium (96).
tion of proteins. Another major factor regulating fermenta-

Journal of Dairy Science Vol. 64, No. 6, 1981

 RUMEN FERMENTATION -- 75TH ANNIVERSARY ISSUE
tion products produced in vivo is interspecies
hydrogen transfer. Within the rumen the
partial pressure of hydrogen is low, but the
turnover of hydrogen through this pool is high
mainly because of its rapid utilization by
methane bacteria (48). Because the partial
pressure of hydrogen is extremely low, the
formation of hydrogen gas from reduced
pyridine nucleotides by non-methanogenic
species is thermodynamically feasible (116).
Thus, reduced pyridine nucleotides can be
oxidized directly with production of hydrogen
gas rather than by alternate means of oxidation
- such as formation of lactate, propionate,
succinate, or ethanol. These latter products
are produced by pure cultures, because hydro-
gen gas accumulates when methanogens are
absent. If methanogens are inhibited in vivo by
low pH or chlorinated hydrocarbons, hydrogen
accumulates and these more reduced fermenta-
tion products are formed. These concepts have
been demonstrated amply by Wolin, Bryant,
and their colleagues (55, 65,94, 96).
Nitrogen and Protein Metabolism
Proteins can be degraded extensively and
fermented in the rumen as shown by pathways
in Figure 2. The extent of ruminal protein
degradation can vary greatly, but studies di-
rected toward definitive information on factors
controlling proteolysis have not been per-
formed yet. Most studies have dealt with pre-
venting ruminal degradation and increasing the
flow of dietary proteins to the omasum by
chemical or physical treatments of dietary
proteins and alteration of rumen functions (22,
23). However, considerable knowledge has
accumulated in the last two decades concerning
nitrogen sources that can be utilized for bac-
terial growth. Considerably less in known about
nitrogen metabolism in ruminal protozoa; but
since engulfed bacteria (and particulate pro-
tein?) can serve as a major nitrogen source for
many species (29, 30, and references therein),
proteolysis is an important aspect of their
ecology.
Pure culture studies on nutritional require-
ments of ruminal bacteria have shown ammonia
to be a major nitrogen source for growth (20),
and in vivo studies using lS N have confirmed
these observations (63, 79). The ammonia is
derived either from bacterial urease action on
1157
urea (dietary and transferred blood urea) or
from microbial catabolism (Figure 2) of amino
acids and peptides (1, 80, 81). This latter
catabolism does not appear to produce suffi-
cient energy for growth in the absence of
carbohydrates of the various bacterial species
having these catabolic abilities. The amino acid
catabolizing enzymes in various species of
ruminal bacteria have not been characterized
biochemically.
Although ammonia is a major source of
nitrogen for bacterial growth, peptides and
amino acids are also important, particularly
with low quality diets (high fiber, low protein)
where up to 40% of the bacterial nitrogen does
not come from ammonia (72). As discussed
recently (40) insufficiency of these compounds
at certain times after feeding may be a major
factor causing energetic uncoupling, resulting in
continued production of fermentation products
without concomitant bacterial growth. Direct
evidence that this can take place in vivo was
shown over 10 yr ago by studies of Hume et
al. (44), which indicated that microbial cell
yields in the rumen are proportional to dietary
nitrogen. Finally, peptides and amino acids
are needed as precursors to produce the
branched-chain fatty acids that are growth
factors for a number of bacterial species,
particularly the cellulolytics (2). However,
definitive studies have not been done to ex-
plore whether dietary supplementation of
branched-chain fatty acids are efficacious in
promoting ruminal digestion beyond what can
be achieved by urea additions to poor quality
diets (e.g., high forage, low protein) as would
be encountered in range grazing or possibly
used in future years when grains may not be as
amply available.
Ruminal Protozoa
Much of the foregoing discussion on bio-
chemical aspects has centered on ruminal
bacteria because of the preponderance of
specific information that is available for them.
Furthermore, for any one biochemical process
in the rumen, such as production of a volatile
fatty acid, it is difficult to partition quantita-
tively contributions by protozoa and bacteria.
However, protozoa are less metabolically
active on a cell mass basis than bacteria simply
because of their larger cell volumes. Rumen
Journal of Dairy Science Vol. 64, No. 6, 1981
1158 RUSSELL AND HESPELL
protozoa are fermentative anaerobes, and their
fermentation products include acetate, butyr-
ate, lactate, carbon dioxide, and hydrogen.
Besides contributing to volatile fatty acid pro-
duction, protozoa aid in sequestering carbo-
hydrates from rapid bacterial attack by
engulfment of starch grains and other partic-
ulate carbohydrates. Without this, a significant
portion of the carbohydrates would be fer-
mented rapidly to lactate, and a lower ruminal
pH would result, both aspects of which are
detrimental to overall rumen functions. A
homologous situation may occur with partic-
ulate proteins, whereby engulfment would
allow for extended proteolysis, slower release
of products, and less catabolism of amino acids/
peptides to volatile fatty acids. However,
evidence for this latter role is scanty at this
time. Various interrelationships between the
protozoa, diets, and the host animal are dis-
cussed more fully in a recent article by
Coleman (29).
D E T E R M I N A N T S OF R U M E N E C O L O G Y
Since many microbial species inhabit the
rumen and have evolved together over millions
of years, one would expect numerous inter-
relationships among them. Within microbial
environments several types of ecological inter-
relationships are possible (67). If two species
have no effect on one another, one can say
that a state of "neutralism" exists. When the
growth of one species is promoted by the
presence of a second species, but the growth of
the second is unaffected by the presence of
the first, the relationship can be termed "com-
mensat". In a "mutualistic" relationship the
degree of interaction is even stronger, and the
growth of both species is enhanced by the
presence of the other. If two species are depen-
dent on the same limiting nutrient, a state of
" c o m p e t i t i o n " exists. When toxic products are
produced, "amensal" relationships are possible,
and if one species can consume another, "pre-
dation" can occur. Within the rumen eco-
system, there are examples of each of these
types of relationships. A particular microbial
species may be involved in several types of
interactions at a given time.
Realistic estimates of total microbial growth
as well as relative numbers of individual species
within the rumen is largely dependent on a
Journal of Dairy Science Vol. 64, No. 6, 1981
quantitative understanding of microbial inter-
relationships. These interrelationships are deter-
mined ultimately by the nutrition, biochemis-
try, and physiology of individual rumen micro-
bial species, and studies of their properties
have given us further insight into the dynamics
of rumen ecology. As will become evident in
the discussion below, this insight is still quali-
tative when one considers the true in vivo
situation.
Maximum Growth Rates
When soluble nutrients are plentiful, an
important determinant of relative microbial
success is maximum growth rate. At such times,
an organism with a higher maximum growth
rate is able to grow faster than an organism
with a lower maximum growth rate. When
maximum growth rates were compared among
several rumen bacteria, differences were large,
and maximum growth rates were dependent on
both energy source (85) and pH of incubation
medium (91).
Because bacteria grow exponentially, it is
impossible for them to maintain high rates of
growth for extended time. Streptococcus
boris is able to achieve a doubling time of 14
min (86). At such a growth rate, one S. boris
cell with a volume of approximately 1.2 times
10-13 cm 3 would be able to fill completely a
60-liter rumen in less than 14 h and would
equal the mass of the earth in approximately
34 h! Thus, other factors also must limit
bacterial growth in the rumen.
Substrate Affinities and Preferences
During much of the feeding cycle soluble
substrate concentrations are low in the rumen
(48, 97). At low concentrations of substrate,
increments of substrate will cause microbial
growth rate to increase, and this pattern follows
saturation kinetics typical of enzyme systems
(69). The affinity constant, K s , is defined as the
substrate concentration that will yield one-half
maximum growth rate. Since affinity constants
of most bacteria are low, affinity usually is
determined with chemostat cultures. Recent
studies have compared substrate affinities of
rumen bacteria and showed that affinity for
the same substrate can differ greatly among
species and that a species can have higher affini-
ties for some substrates than others (86). These
 RUMEN FERMENTATION - 75TH ANNIVERSARY ISSUE
data are consistent with some in vivo observa-
tions. F o r example, a strain of Butyrivibrio
fibrisolvens had high substrate affinities for a
variety of soluble carbohydrates, and this
species has been observed to proliferate in the
rumen on poor forage diets (13, 48).
In studies with nonrumen bacteria, orga-
nisms can utilize preferentially some substrates
to exclusion of others (59, 77, 83, 112). These
preferencing mechanisms allow bacteria to
select substrates that yield the highest growth
rates and to minimize the synthesis of degrada-
tire enzymes. When pure cultures of rumen
bacteria were grown in batch culture with a
mixture of fermentable carbohydrates, some
substrates were not utilized until others were
depleted, and the "more preferred" substrates
inhibited utilization of "less preferred" sub-
strates (85). Comparisons of organisms indicated
that they had different patterns of substrate
preference. Differences in substrate affinities
and preference patterns suggest 1) rumen bac-
teria have evolved different strategies of growth
and 2) these physiological factors may affect
competition among rumen bacteria.
Cell Yields and Maintenance
The early work of Bauchop and Elsden (8)
indicated that yield of bacterial cells in grams
per mole of ATP produced (YATP) was
10.5. However, theoretical calculations by
Stouthamer (100, 101) indicated that YATP
could be as high as 32.5 for growth of an orga-
nism in complex m e d i u m . The differences
between theoretical and observed yields are
presumably due to an organism's relative
efficiency of ATP utilization for growth.
Experimental determination of maximum
YATP with several rumen bacteria have indi-
cated that these organisms were efficient
utilizers of ATP for growth. Indeed, Seleno-
rnonas rurninantiurn exhibited a maximum
Y#.TP of approximately 28 (87), and as dis-
cussed recently (40), the rumen microbial
population as a whole has a potential YATP
of 22 to 26.
With the advent of continuous culture
techniques in the 1950's (70, 73), it became
apparent that microbial cell yields were lower
at slower growth rates (39). These observations
suggested that energy was used for both
growth-related functions and functions not
1159
directly related to growth (ion balance.
protein turnover, etc.). Nongrowth functions
have been termed maintenance energy expend-
itures and are analogous to maintenance energy
of animals. The underlying principle of maint-
enance is that growth only can occur after this
requirement is met. When growth rate is high,
maintenance makes up a relatively small
proportion of total energy utilization. How-
ever, when growth rates decrease, maintenance
becomes more significant, and all of the energy
is used for maintenance when growth ceases.
Within the rumen, bacterial numbers are high,
and average bacterial growth rate is low. Under
such conditions one would expect cell yields
to be lower than the maximum obtainable be-
cause of the increased involvement of mainte-
nance in total energy utilization, and this concept
is supported by data of Isaacson e t al. (51).
These maintenance expenditures are reflected
partly in heat of fermentation and represent a
loss of microbial protein (but not necessarily a
loss of volatile fatty acids) for the animal.
Although the maintenance energy of the
total rumen population is low as compared to
other bacteria (51), maintenance for individ-
ual rumen bacteria can vary greatly (87).
Butyrivibrio fibrisolvens and Sefenornonas
ruminantiurn had low maintenance expendi-
tures compared to other species. B. fibri-
solvens and S. rurninantium often are found in
high numbers in the rumens of animals fed poor
quality diets (13, 48) where microbial growth
rates would be expected to be slow. These
results are consistent with the idea that orga-
nisms with low maintenance energies would be
able to grow faster and dominate the popula-
tion when substrate availability and growth
rates are low in the rumen.
Tolerance to Low pH
Although the rumen is buffered by bi-
carbonate, phosphate, and proteins, produc-
tion of fermentation acids sometimes can
exceed the buffering capacity, and pH can
decline to the point where rumen function and,
hence, animal p e r f o r m a n c e is decreased. Low
rumen pH usually is associated with an accumu-
lation of lactic acid in the rumen, and during
this time Streptococcus boris and lactobacilli
increase in numbers (50, 58). One of the
factors responsible for this shift in the popula-
Journal of Dairy Science Vol. 64, No. 6, 1981
1160 RUSSELL AND HESPELL
tion appears to be tolerance of these orga-
nisms to low pH. When pure cultures of rumen
bacteria were grown in chemostats, S. boris
and Lactobacillus vitulinus were more tolerant
to low pH than cellulolytic organisms or the
lactate utilizer M. elsdenii (90). Furthermore,
S. boris produces more lactic acid at high
growth rates or at low pH values (89). Thus, the
combined effect of inhibition of growth by low
pH on many rumen bacteria along with contin-
ued lowering of rumen pH from lactate produc-
tion by the acid-tolerant S. boris are major
factors influencing onset of rumen acidosis
(89, 91).
Cell Lysis
Most studies with rumen bacteria have dealt
with growth of organisms, but considerable cell
death and lysis occur in the rumen as evidenced
by studies of nitrogen turnover (63, 72). Cell
death in most cases results from lack of nutri-
ent during part of the feeding cycle and causes
a decrease in potential growth capacity when
nutrients are available at other times. Pure
culture studies with rumen bacteria generally
have indicated that they are particularly sus-
ceptible to cell death and lysis. Rumen bacteria
differ from each other in their abilities to
maintain viability during times of nutrient
starvation (56, 66). Since ruminal bacteria have
limited survival abilities, death and lysis from
nutrient starvation are factors controlling the
total as well as species numbers of micro-
organisms in the rumen.
Cross'feeding
In addition to crossfeeding of succinate and
hydrogen, which was discussed earlier, other
types of crossfeeding between microbes may
occur in the rumen. When S. rurninanium was
cocultured with B. succinogenes on a cellu-
lose-containing medium, S. rurninantium was
able to grow even though it was unable to
degrade cellulose (96). Such findings suggest
that there could be a considerable crossfeeding
of cellulose degradation products in the rumen.
Hemicellulose is degraded primarily by cellulo-
lyric bacteria, but many noncellulolytic strains
are abld to utilize the xylans that are released.
Experiments by Dehority showed that B.
ruminicola and L. rnultiparus were able to
utilize the products of hemicellulose degrada-
Journal of Dairy Science Vol. 64, No. 6, 1981
tion when they were cocultured with cellulo-
lytic species (33).
A simultaneous crossfeeding of carbon and
nitrogen also can occur. For example, when
B. ruminicola and R. albus were cocuhured on
a medium containing cellulose and casein, the
proteolytic B. ruminicola was able to provide
ammonia and branched chain fatty acids
essential to the growth of R. albus. In turn,
cellulolytic R. albus provided the hexoses re-
quired by the noncellulolytic B ruminicola.
Many rumen bacteria have specific vitamin
requirements (13, 14, 16), and crossfeeding by
cell lysis or excretion probably occurs to a
large extent.
Predation by Protozoa
Under most natural conditions, the rumen
is inhabited by protozoa, and part of the
bacterial population is eaten by protozoa. Most
of our knowledge regarding protozoal engulf-
ment of bacteria has been derived in the last 15
yr from studies by G. S. Coleman and co-
workers (28, 29, 30, and references therein)
using in vitro incubations with in vitro grown
protozoa. In summary, and realising differences
between protozoal species, rates of engulfment
range from 130 to 21200 bacteria!~protozoan
per hour at bacterial densities of 10 cells/ml.
Intracellular digestion rates of bacteria range
from 345 to 1200 bacteria/protozoan per hour.
Ingestion of or availability of carbohydrates to
protozoa may influence digestion, but other
influencing factors have not been examined.
Depending on engulfment rate and for a high
protozoal concentration (106 cells/ml), any-
where from 2.4 to 45 g bacteria could be
digested protozoally per day in a sheep's
rumen.
Recent studies using in vivo grown protozoal
species have indicated that Entodinium and
larger species are selective in their engulfment
of bacteria, preferring mixed ruminal bacterial
suspensions (30). Entodina species engulfed
cellulolytic bacteria more rapidly than other
ruminal or nonruminal bacterial species. Opti-
mal engulfment occurred at pH 6, with drastic
declines in rates above or below this. As point-
ed out by the authors, these recent findings
may have important significances in vivo.
Namely, previous in vitro estimates of bacte-
rial engulfment and digestion may have been
 RUMEN FERMENTATION -- 75TH ANNIVERSARY ISSUE
too high. Additionally, selective engulfment
of bacterial species in vivo could lead to alter-
ed ruminal fermentation patterns.
Properties of the Feed
Bacteria possess transport systems capable
of taking up low molecular weight soluble
nutrients, but most feeds are composed of
large, relatively insoluble, complex polymers
(cellulose, hemicellulose, starch, pectin,
proteins, etc.). Thus, the" chemical, physical,
and structural properties of feedstuffs must be
important factors affecting rumen degradation.
Feedstuff polymers first must be degraded
to lower molecular weight compounds by
extracellular enzymes before they can be utilized
by rumen bacteria. As extracellular enzymes of
rumen bacteria must act on the surface of feed
particles, and as smaller particles have a greater
surface to mass ratio, particle size is an impor-
tant affector of fermentation rate (48). Within
the rumen, particle size can be determined
by feed treatment (pelleting, grinding, chop-
ping, cubing, flaking, etc.), rumination, and
digestion itself. Although reduction of particle
size would be expected to increase fermenta-
tion rate, small particles can pass unfermented
from the tureen. If this passage rate exceeds the
increase in fermentation rate, the overall extent
of rumen fermentation can be reduced (110),
and total animal digestion is then more depen-
dent upon the degrees of postruminal enzy-
matic and fermentative digestion.
Because intracellular enzymes come in
contact with feedstuffs through interactions of
water with feed, one would expect polymer
solubility to affect rumen fermentation rates.
Starch granules of most plants are inherently
insoluble and as such are resistant to the hydro-
lases of bacteria (37). This insolubility is par-
tially responsible for the rather slow fermenta-
tion rates and high passage rates of many native
starches (76, 105, 109). Heat and chemical
treatments have increased solubility and like-
wise increased both rate and magnitude of
starch digestion in the rumen (75).
Proteins also differ in solubility, and protein
solubility is determined largely by the distribu-
tion of hydrophobic and hydrophilic amino
acids on the periphery of the protein molecule.
All proteins whose tertiary structures are
known have many hydrophilic residues at the
surface, and hydrophobic residues usually are
1161
buried in the interior of the tertiary structure
(113). This arrangement increases solubility,
and more soluble proteins in some cases have
been degraded at a faster rate by rumen bacte-
ria. Denaturation by heat causes unfolding of
tertiary structure, greater exposure of hydro-
phobic amino acids to the surface, and
decrease in solubility. Heat treatment of feed-
stuffs can cause a reduction of protein fermen-
tation in vivo (25).
However, ovalbumin is a soluble protein;
y e t it is resistant to proteinases (61). In this
case, resistance appears to be from the cyclical
structure of the protein. Recent work by
Mahadevan et al. also has indicated that di-
sulfide bridges within a protein render it more
resistant to hydrolysis by rumen bacterial
proteinases (60). Soluble and insoluble soy-
bean proteins were degraded at nearly equal
rates, and such results suggest that factors
other than solubility must influence fermenta-
tion of proteins within the rumen.
Cellulose and hemicellulose fractions of
feeds are relatively insoluble and are degraded
slowly within the rumen (33, 107). However,
structural factors also influence their fermen-
tation rates. For example, within forage fibers,
lignin is in close association with cellulosic
materials, and it appears to present a physical
barrier to rumen bacterial cellulases. Deligni-
fication of fiber fractions by various chemical
treatments has increased the extent of cellulose
digestion in vitro and in vivo (108). Crystallin-
ity also appears to affect rate and types of
rumen bacteria digesting cellulose fibers. Cot-
ton fibers have a highly crystalline structure
and are more resistant to certain rumen bacte-
rial cellulases, t3. succinogenes is able to de-
grade cotton fibers at a rapid rate, but the rate
of cotton fiber digestion by R. albus and R.
flavefaciens is much slower (14). The signifi-
cance of crystallinity to in vivo forage diges-
tion has not been elucidated (107), but more
crystalline types of cellulose may select for
B. succinogenes.
Hemicellulose (xylan) is a complex polymer,
and its chemical and physical properties have
been studied in less detail. However, the
amount of hemicetlulose that could be digested
by R. flavefaciens and R. albus differed mark-
edly with the type of plant from which it was
isolated (33, 107). From these results it is
reasonable to assume that the arrangement of
Journal of Dairy Science Vol. 64, No. 6, 1981
 1162 RUSSELL AND HESPELL
various constituents within hemicellulose can
affect its rate of digestion. As mentioned earli-
er, hemicellulose degradation involves a symbio-
tic relationship between cellulolytic species that
are able to degrade the molecule and noncellu-
lolytic bacteria that utilize the breakdown
products. Thus, greater hemicellulose degrada-
tion can result from combined growth of cellu-
lolytic and noncellulolytic strains than growth
of cellulolytic strains alone (33).
The actual degradation of intact feedstuffs
by rumen microorganisms in vivo is complex,
but experiments suggest that chemical and
physical properties of feeds are not only im-
portant determinants of rate of fermentation
but also types of microorganisms. Yet, a
relatively clear understanding of how any one
plant polymer is degraded and by which orga-
nisms is simply not known. The dynamics of
rumen ecology will not be understood until
chemical and physical properties of feed-
stuffs are elaborated in more detail.
Rumen Dilution Rates
Because feed usually enters the rumen at
regular intervals and because digesta and
microorganisms are washed out of the rumen
to the lower gut, the rumen in many ways
operates as a continuous culture device. How-
ever, there are major differences between
kinetics of a laboratory chemostat and the
rumen. These differences sometimes have been
ignored by ruminant nutritionists.
Dilution rates in most laboratory chemostats
are controlled by addition of nutrient medium.
These nutrients are usually in a soluble, readily
utilized form, and a single nutrient limits
growth across all dilutions. In this case, if
dilution rate is less than the maximum growth
rate of the organisms, the growth rate of the
culture is proportional to dilution rate, because
increasing dilution rates are associated with an
increased delivery of the limiting nutrient.
Since soluble nutrients are provided, the system
operates under homogeneous, steady state
conditions, and all of the organisms in the
culture vessel are subjected to the same chances
of growth and washout.
In the rumen by contrast, most nutrients
are fed in an insoluble form, nutrient addition
is discontinuous (meals) rather than continu-
ous, and various nutrients can limit growth
Journal of Dairy Science Vol. 64, No. 6, 1981
during different phases of the feeding cycle.
Because there are both insoluble (feed particles)
and soluble fractions, the rumen does not oper-
ate as a homogeneous system, and there are at
least two major dilution rates - that of the
solids and that of the liquids. Feed particles
turn over at a slower rate (two to four times)
than the liquid fraction, and rumen microorgan-
isms can be associated with both the feed
particles and the liquid (48). As such, rumen
microbes can be subjected to different condi-
tions of growth and washout depending on
their position in the rumen. Thus, measure-
ments of solid or liquid dilution rates alone
cannot quantitate microbial cell yields. More-
over, even if both rates are determined, this
gives a net microbial growth rate less than the
true growth rate because of factors such as cell
lysis.
Inclusion of mineral salts in ruminant diets
will increase rumen liquid dilution rate, and
increased dilution rates have altered fermenta-
tion products (84 and references therein).
The most marked change in this fermentation
shift appears to be reduction in the molar quan-
tity of propionic acid. These fermentation
shifts also suggest that different microorga-
nisms (both bacteria and protozoa) may be se-
lected at different dilution rates within the
rumen. Microscopic examination of animals
supplemented with mineral salts have shown
that higher liquid dilution rates were associated
with increased numbers of cocci (104).
Since bacterial growth yields are greater at
higher growth rates (maintenance energy makes
up a smaller proportion of total energy utiliza-
tion), there has been an interest in increasing
delivery of microbial protein to the lower gut
by increasing tureen dilution rates. In some
cases increased rumen dilution rates have been
associated with an increased availability of
microbial protein to the animal (27, 38). How-
ever, it is difficult to say whether true microbial
growth rates actually were increased. Numerous
other possibilities exist, such as the increased
microbial protein was from washing microorga-
nisms out of the rumen before cell death and
lysis or a reduction in protozoal predation of
the bacteria.
SOME FUTURE DIRECTIONS
The bulk of our current knowledge on the
microbial ruminal fermentation has been gain-
 RUMEN FERMENTATION -- 75TH ANNIVERSARY ISSUE
ed within the last 25 yr. Although we have
made great achievements, our overall ecological
picture is still cloudy. Elucidation and integra-
tion of the myriad interactions and their
specifics between bacteria, protozoa, dietary
materials, and the host animal will give a
focused, detailed picture of the dynamic rumi-
nal fermentation and ecosystem, but this has
not been accomplished yet. It is in this context
that the goals of future research must be set to
gain the knowledge needed to predict accurate-
ly the dynamic changes in ruminal fermenta-
tions under various dietary conditions. This will
allow for fruitful manipulations of the entire
process. Based on our current status, some
possible directions for future research are
offered below.
Sampling/Enumeration
One aspect of many nutritional and micro-
biological studies that limits interpretation of
data has been the lack of good sampling techni-
ques or adequate descriptions of the sampling
methods used. The rumen ecosystem is dynam-
ic, but it is not completely homogenous as
exemplified by recent studies on ruminal
ammonia (115) that show distinct differences
in concentration with tureen location, time,
diet, and method of analysis. As suggested by
the classical studies of Smith et al. (97) similar
differences probably exist for many, if not all,
other biological components of the rumen eco-
system. Future nutritional and microbiological
studies must begin to give considerably more
attention to these differences. Further docu-
mentation that different diets yield different
degrees of ruminal digestion, volatile acid
production, or microbial cell yields is of rela-
tively small value. This is particularly true if
each aspect is determined in separate studies
with different animals and dietary materials.
The important point is to determine precisely
why these differences occur, and this can be
approached only if a more comprehensive
picture is obtained by measurement of numer-
ous parameters of rumen fermentation under
the same given set of dietary conditions.
In microbiological studies dealing with
mixed ruminal populations, times of animal
feeding and sampling can be and should be
controlled. The ruminal site of sampling must
be defined and the entry means (stomach tube,
1163
fistula, cannual, e t c . ) i n d i c a t e d . Future studies
must be directed towards a comprehensive
microbial analysis that should include as many
of the following aspects as possible (or as
applicable). First, delineation of not only the
bacteria but also the protozoa and their enu-
meration by speciation is necessary. The
microbial population that is obtained should be
separated with respect to whether it is "free in
the liquid" or "attached" to digesta particles.
Each of these subpopulations then could be
separated further by appropriate plating tech-
niques. Other measurements of the digesta
sample that should be made include pH, types
and amounts of fermentation acids, soluble
nutrient content (e.g., ammonia, soluble
sugars, peptides, and amino acids, etc.), and
complete (e.g., Van Soest-type) analysis of
digesta solids. Finally, but importantly, rumen
digesta samples should be taken over numerous
times to obtain diurnal changes or patterns.
Presently, the technical knowledge needed to
make many of the foregoing quantitative micro-
bial measurements is limited. However, a few
initial steps have been made. Some media and
techniques have been developed for separating
mixed ruminal populations into carbohydrate-
utilizing subgroups (3, 34, 57), and some
fractionation of attached and free bacteria has
been accomplished (e.g., see 68). Although
much has to be developed, the goals are not
unsurmountable.
Culture Techniques
In vitro incubations of rumen microorgan-
isms offer means of examining experimental
variables under more precisely controlled and
assayable conditions than are possible in vivo.
However, one must be careful in interpreting
results of such in vitro experiments. When
rumen inocula are used, extrapolation of the
data to in vivo situations only can be made with
confidence if both the resulting population of
microorganisms and fermentation products
approximate those of the rumen. Simple
batch culture techniques with high concen-
trations of soluble carbohydrate can select
for a lactate fermentation in as little as 3 h
(88) and is caused by the high maximum
growth rate in S. boris in comparison to other
rumen microorganisms. Selections of other
species also may occur if other substrates are
used. Thus, it seems that such simple batch
Journal of Dairy Science Vol. 64, No. 6, 1981
1164 RUSSELL AND HESPELL
incubations are likely to examine the effect
of experimental variables on selected organisms
rather than the total rumen microflora. How-
ever, less selection may occur with short term
limited-fed-batch systems that appear to
produce more normal fermentation products
and maintain the diversity of the rumen micro-
b i a l population (88). In these systems small
doses of carbohydrate can be fed throughout
the incubation period, and microbial growth
rates can be controlled. Since soluble carbo-
hydrates and microbial growth rates are kept
low, there is less selection of microbial types
during the incubation, and it seems that limit-
ed-fed-batch incubations are more apt to mimic
ruminal conditions.
Conventional chemostats offer an ideal
method of examining interactions between a
limited number of microbial species. Coculture
incubations in chemostats, as discussed earlier,
already have been used to demonstrate inter-
species transfer of hydrogen and several other
examples of crossfeeding. Cocontinuous cul-
tures also have demonstrated that relative
bacterial numbers might be predicted from the
physiological characteristics of organisms, and
it seems likely that future chemostat experi-
ments will allow rumen microbiologists to
build a more accurate model of relative microb-
ial growth in the rumen.
Although conventional chemostat methods
are helpful in elaborating the nutrition, bio-
chemistry, and physiology of individual species,
they are usually not suitable for incubations to
simulate the total mixed ruminal population.
Since one nutrient (usually in a soluble form)
must limit growth in chemostats, organisms
that are best suited (high affinity, lower maint-
enance energy, etc.) to the utilization of this
nutrient will be selected. Modified continuous
culture devices employing ground feed with
liquid and solid phases have been able to
maintain a diversity of microbial types (32,
43), but considerably more work has to be
done with them to validate this point. How-
ever, these latter chemostats may offer the
means to understand microbial degradation of
insoluble dietary substrates.
Nitrogen-Ammonia Assimilation
Ammonia is a major source of nitrogen for
ruminal microbial growth, yet little definitive
Journal of Dairy Science Vol. 64, No. 6, 1981
information is available on interactions between
growth rate, cell yields, ammonia assimilating
enzymes, and environmental ammonia and
other nitrogenous compounds. Affinities of
ruminal bacteria] species for ammonia range
from 5 to 45 /~molar (93) suggesting ammonia
uptake cannot occur by passive diffusion, and
it seems likely that specific transport mecha-
nisms are involved. However, nothing is known
about ammonia transport mechanism in any
ruminal bacterial or protozoal species. Research
is needed to ascertain whether these mecha-
nisms require energy and what factors regulate
their activity. Once within the cell ammonia
can be assimilated (or fixed) by either the
non-ATP requiring glutamate dehydrogenase or
by the ATP-requiring glutamine-synthetase
pathway, and both pathways were found in
Selemonas ruminantiurn (98). Whether similar
or other pathways exist in other ruminal
bacteria and which pathways are used in vivo
is totally unclear. Expression of glutamine
synthetase is needed for expression of urease
activity (99). Thus, in the rumen (and most
likely in the cecum and large intestine) an
interplay of ammonia assimilation pathways
probably takes place at various times after
feeding and with various dietary changes, but
the nature of these is not known.
Protein Degradation
Although it is well documented (42) that
a large fraction of dietary protein is degraded
by rumen microorganisms to ammonia and
excessive ammonia accumulation in the rumen
can cause a decrease in the nitrogen retention
of the animal (92), mechanisms of rumen pro-
teolysis have not been examined in detail. Early
work demonstrated that a variety of rumen
bacteria were able to degrade casein (10, 18),
but few actively proteolytic bacteria have been
identified (9, 10). These findings generally have
been interpreted as meaning that proteolysis in
the rumen is accomplished by the combined
action of many species, but the possibility
exists that shifts in the types of rumen micro-
organisms also could influence the rate of pro-
teolysis in vivo. Studies with nonrumen bacteria
have shown that extracellular proteinases are
subject to induction (12), repression (35, 64),
and catabolite repression (114). None of
these mechanisms has been demonstrated in
 RUMEN FERMENTATION -- 75TH ANNIVERSARY ISSUE
rumen bacteria, but their existence might allow
proteolysis to he manipulated by energy status
or chemical means.
The process of amino acid deamination also
has received little attention. Some studies have
noted amino acid utilization by several species
of rumen bacteria (95), but it is not clear
whether this utilization reflected actual de-
amination or a simple uptake for protein syn-
thesis. When various species of ruminal bacteria
were grown in glucose-limited chemostats with
ammonia and high Trypticase, the only species
that caused a net increase in the steady state
of ammonia was M. elsdenii (89). These results
suggest that growth conditions can influence
amino acid deamination.
Maintenance Energy
Many studies with bacteria have indicated
that maintenance is not a constant and varies
with growth conditions (71, 101, 102). For
example, when Klebsiella aerogenes was grown
in a chemostat at .1 h -1 under carbon, nitro-
gen, sulfate, and phosphate limitations, the
relative yields were 1.00, .40, .33, and .25,
respectively (71). When growth is limited by
nutrients other than carbon, bacterial cells will
uncouple growth and continue to metabolize
carbon in the absence of growth. The uncou-
pling of growth and energy utilization in rumen
bacteria has not been studied (40). Considering
that many rumen bacteria have specific re-
quirements (branch chain volatile fatty acids,
vitamins, amino acids, micro-minerals, etc.), bac-
terial growth in the rumen could be limited by
nutrients other than carbon during a portion of
the feeding cycle. If rumen bacteria uncouple
growth like other bacteria, such nutrient limita-
tions could have marked effects on the efficien-
cy of synthesis of microbial protein in vivo.
Computer Techniques
Fermentation of feedstuffs within the rumen
involves numerous complex interactions of the
nutritional, biochemical, and physiological
characteristics of the organisms, of the chemical
and physical properties of the feed, and of host
animal factors such as rumination and dilution
rates. Simple descriptions of the system under
one set of feeding conditions are not extrapo-
lated readily to other situations. This complex-
ity has confounded prediction of animal per-
formance from diets fed. One approach to
1165
understanding the system is to feed experi-
mentally all combinations of feedstuffs under
different levels of production. However, this
approach is impractical because there are
infinite numbers of these combinations. Anoth-
er approach is to develop a quantitative under-
standing of the basic variables that affect rumen
fermentations, and this latter approach seems
more promising and approachable. The develop-
ment of computer technology over the last two
decades had made it possible to quantitate and
integrate simultaneously a large number of
variables, and this technology has been adapted
readily in the chemical and physical sciences.
Baldwin and his colleagues have applied this
technology to digestion in the rumen and have
constructed several dynamic models of the
processes (6, 7, and references therein). Further
development of such computer models offers
the possibility of predicting animal perform-
ance from feedstuffs as well as expanding our
understanding of the rumen system in its
entirety.
Chemical Agents
As discussed recently by Chalupa (23),
rumen fermentations can be modified by chem-
ical additives. Amichloral, monensin, and
diphenyliodonium chloride are the chemical
agents that have been examined most thorough-
ly, but we are far from having a clear under-
standing of their effect on specific ruminal
microorganisms or on the true dynamics of
rumen fermentation. With most of the chemi-
cals the accumulated data is not sufficient to
provide an accurate explanation of the observed
effect on animal performance. Future re-
search must be shifted from the current de-
scriptive approach to one of an analytical
nature. Such a shift would result in new, more
logical approaches to predictable modification
of the rumen fermentation.
Interactions of Scientists
Regardless of the specified biological process
being examined, individual scientists must take
on an increasingly more holistic outlook in
their research. This stems directly from the tre-
mendous increase of scientific knowledge over
the last few decades that has brought into focus
how little we really know and how complex
biological processes are. However, a holistic
approach will require individual scientists to
Journal of Dairy Science Vol. 64, No. 6, 1981
1166 RUSSELL AND HESPELL
develop a greater interaction, understanding,
and even expertise in one or more additional
disciplines. For the rumen, there must be high
degrees of interactions between nutritionists
and microbiologists, and both scientists must
have some relative competence in each other's
field. Both also must begin to view ruminant
digestion from broader standpoints - namely,
from a comparative viewpoint encompassing
other species (horse, human, swine, etc.) and,
more importantly, the entire ruminant gastro-
intestinal tract. There are large indigenous
microbial populations in the cecum and large
intestine and numerically significant popula-
tions in the small intestine, yet our knowledge
of these fermentations in ruminants is virtually
nonexistent. The potentials of these ecosystems
must be examined, particularly if concepts such
as ruminal bypass of dietary materials are to be
exploited fully.
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