8
Rumen Microorganisms
and their Interactions
M.K. Theodorou
1
and J. France
2
1
BBSRC Institute for Grassland and Environmental Research, Aberystwyth,
Dyfed SY23 3EB, UK;
2
Centre for Nutrition Modelling, Department of
Animal & Poultry Science, University of Guelph, Guelph, Ontario N1G 2W1,
Canada
Introduction
Whilst herbivory is widespread in the animal kingdom, no vertebrates and few
invertebrates are capable of synthesizing cellulose- or hemicellulose-digesting
enzymes. Instead, herbivores have evolved symbiotic associations with micro-
organisms. Two main types of herbivory exist among mammals. The rumin-
ants, cloven-hoofed mammals of the Artiodactyla, are best equipped for
maximal digestion of plant biomass, which is achieved by prolonged retention
within the gastrointestinal (GI) tract. The second type of herbivory is exempli-
fied by members of the Equidae (horses) and Elephantidae (elephants), where
plant material is passed through the GI tract more rapidly at the expense of
maximal plant cell wall digestion. With this form of herbivory, a greater pro-
portion of the nutrient supply to the animal is obtained from plant-cell contents
than from cell-wall polymers.
Both types of herbivory are dependent upon microorganisms for the
degradation and fermentation of plant-cell contents, cellulose, hemicellulose
and pectin. Ruminants rely on a predominantly pre-gastric fermentation in the
rumen, whereas in horses and elephants the fermentation occurs in the hind-

grow in the presence of oxygen. Some facultative anaerobes are also present,
and these scavenge available oxygen that enters the rumen with the feed or
by diffusion across the rumen epithelium. Bacteria in rumen liquid are found
at concentrations of 10
9
À10
10
=ml, whereas protozoal populations range
from 10
5
to 10
6
=ml. The population density of rumen fungi (fungal
zoospores) appears to be within the range 10
3
À10
5
=ml. Bacteria are generally
believed to constitute most of the microbial biomass in the rumen, although
estimates of up to 40% have been recorded for protozoal biomass in some
animals. The amount of fungal biomass is thought to contribute less than 8% of
the total.
Over 200 species of rumen bacteria have been described since the pio-
neering work of R.E. Hungate began in the 1940s. All of the principal mor-
phological forms of small bacteria, including Gram-positive and Gram-negative
rods, cocci, crescents, vibrios and helices, occurring singly, in chains, tetrads
and clumps, are found in the rumen. Larger bacteria such as the distinctive
‘Quin’s and Eadie’s ovals’, notable from our inability to grow them in pure
culture, are also represented. The rumen also contains numerous species of
protozoa, most of which do not rely solely on plant nutrients for growth, but

rumen. The major species involved in cellulose degradation are Bacteroides
succinogenes, Ruminococcus albus, R. flavefaciens and Eubacterium cellu-
losolvens. These bacteria adhere closely to plant cell-wall surfaces forming
erosion pits as they degrade cellulosic substrates (Chesson and Forsberg,
1997). Recent molecular techniques allow an improved insight into the kinetics
of fibre attachment by rumen bacteria, demonstrating that degradation is not
necessarily synchronized with changes in attached bacterial biomass (Koike
et al., 2003). Hemicellulose is also degraded by some of the cellulolytic micro-
organisms, together with other bacteria such as Butyrivibrio fibrisolvens and
Bacteroides ruminicola (Hungate, 1966; Dehority and Scott, 1967). Fungi
and bacteria contribute most towards degradation of plant cell walls, the
protozoal contribution on the majority of diets being only some 5% to 20%
of total rumen NDF degradation (Dijkstra and Tamminga, 1995). The pectoly-
tic activities of the predominant pectin-degrading bacteria (e.g. B. fibrisolvens,
L. multiparus) and protozoa have been identified (Wojciechowicz et al., 1982;
Williams, 1986), though little has been published on their properties. In
contrast to rumen bacteria and protozoa, the anaerobic fungi exhibit little
hydrolytic activity towards pectin (Williams and Orpin, 1987).
Although absent from plant cell walls, starch is an important
component of many ruminant diets, especially those including grain.
Some cellulolytic bacteria, such as certain strains of B. succinogenes, are
also amylolytic. In general, however, the principal amylase-producing
bacteria, Bacteroides amylopilus, Selenomonas ruminantium and
Streptococcus bovis, have a limited ability to utilize other polysaccharides.
These microorganisms, together with soluble-sugar utilizers such as
Megasphaera elsdenii, occupy a distinct ecological niche in the rumen.
Although they are in competition with many other rumen microorganisms
Rumen Microorganisms and their Interactions 209
for these readily degradable substrates, they survive because of their faster
growth rates or greater substrate affinities (Hobson, 1971; Lin et al., 1985).

nantium and Methanosacina barkeri utilize either H
2
and CO
2
or formate,
acetate, methylamine and methanol for the production of methane. The in-
volvement of these bacteria in inter-species hydrogen transfer is an important
interaction that alters the fermentation balance and results in a shift of the
overall fermentation from less- to more-reduced end-products (Wolin, 1974).
Although fermentation pathways are well established, the prediction of the type
of volatile fatty acids (VFA) that is produced in the functioning rumen remains a
difficult task (Bannink et al., 2000).
Some of the bacteria that participate in degradation of structural polysac-
charides are unable to utilize all of the products liberated as a consequence of their
activity. Whereas R. flavefaciens produces both xylanase and pectinase, it can-
not utilize the end-products of xylan or pectin degradation (Pettipher and Latham,
1979a,b). Thus, these energy-rich compounds are made available as substrates
for growth of other rumen microorganisms. In a similar case, some of the energy-
rich products of hemicellulose degradation are not utilized by the anaerobic
fungus Neocallimastix hurleyensis that produces them (Lowe et al., 1987;
Theodorou et al., 1989). This apparently altruistic behaviour between rumen
microorganisms has been demonstrated on numerous occasions and is thought to
be related to cross-feeding interactions. In return for the provision of readily
210 M.K. Theodorou and J. France
utilizable substrates, the recipient microorganism provides the primary degrader
with an essential growth factor, such as a vitamin or cofactor. In another example,
the combination of a pectin-utilizing bacterium (B. ruminicola) increased the
degradation and utilization of lucerne pectin (Gradel and Dehority, 1972). In
this situation both organisms benefit from a mutualistic association.
Some microorganisms are able to coexist in the rumen without affecting

branched, tapering rhizoids (as in Neocallimastix spp., Piromyces spp. and
Orpinomyces spp.) or bulbous holdfasts (as in Caecomyces spp.). These pene-
trate plant substrates, both for anchorage and to obtain nutrients for growth.
Thus, due to their invasive habit, the anaerobic fungi may escape competition with
faster growing cellulolytic bacteria. Upon completion of the life cycle, the particle-
associated zoosporangium ruptures, liberating zoospores back into the rumen
liquid. These swimming cells have evolved a chemotrophic mechanism that assists
in the search for, attachment to and colonization of freshly ingested plant frag-
ments. The most likely role for rumen fungi is that they participate in primary
colonization of plant cell walls thereby increasing the accessibility of plant frag-
ments to invasion by other microorganisms (Bauchop, 1979a,b). Indeed, in
Rumen Microorganisms and their Interactions 211
co-cultures the fungal mode of attack reduces mechanical resistance of particles,
allowing increased bacterial attack on those damaged particles and possible
coexistence of fungi and bacteria (Dijkstra and France, 1997; Fonty et al.,
1999). In addition to degrading plant cell walls, these microorganisms can also
utilize certain soluble sugars, starch and proteins, but not pectin (Orpin and Joblin,
1988).
Although it is essential in rumen microbial ecology to obtain knowledge of
which species are present and of their activities, traditional methods have
limited applicability. Despite major improvements in isolation or cultivation
strategies, only a minority of the rumen microorganisms have been described
in pure culture. Total viable counts are usually much lower than total micro-
scopic counts (Zoetendal et al., 2003). The majority of microbial species
cannot be obtained in culture and have only been detected using molecular
detection methods (Amann et al., 1995), with an estimated culturability of
bacteria in the total GI tract of some 10–50%. To date, the majority of
molecular studies of microbial ecosystems have been focused on the character-
ization of the community structure or identifying the bacteria in the rumen.
More important, however, is the study of operation and interaction of different

i
Xt! L (8:2b)
where t (h) denotes time since inoculation, X (mg) is the amount of biomass at
time t and S
i
(mg) is the instantaneous quantity of substrate i, where
i ¼ 1, 2, ..., N. For constant m, integration of Eqs (8.1a) and (8.1b) gives:
X ¼ X
0
0 t < L (8:3a)
¼ X
0
e
m(tÀL)
t ! L (8:3b)
where X
0
is initial biomass, therefore biomass obeys the law of constant
exponential growth. Logarithmic transformation of Eq. (8.3b) yields:
ln X ¼ ln X
0
þ m(t À L) t ! L (8:4)
Thus the plot of log biomass against time (! L) is a straight line whose
slope equals the specific growth rate m. The growth lag L can also be deter-
mined graphically by extrapolating this straight line back to the initial biomass
level and reading off the intercept on the time axis. Values of m determined
in this way by Russell and Baldwin (1978) for rumen bacteria grown on a
single energy substrate in a defined medium are presented in Table 8.1.
Corresponding values for L appear to be in the range 0–2 h, mostly nearer
to 0 than 2 h.

M. elsdenii 0.45 0.55 0.14 0.21
Rumen Microorganisms and their Interactions 213
n ¼ [ln(X=X
0
)]= ln 2 (8:6)
The growth yield parameter provides a means of expressing the nutrient
requirement of a microorganism. Growth yield with respect to substrate i, Y
i
[mg biomass/(mg substrate i)], is defined by:
Y
i
¼ÀdX=dS (8:7)
For constant Y
i
, integration of Eq. (8.7) yields:
X ¼ X
0
þ Y
i
(S
i
,
0
À S
i
)(8:8)
where S
i
,
0

i
¼ m=Y
i
(8:10)
This can be shown by dividing Eqs (8.1b) by (8.2b) to give:
dX=dS
i
¼Àm=q
i
(8:11)
and comparing Eqs (8.7) and (8.11). Equation (8.10) can be used to estimate
the demands for substrates at different growth rates. For example, values of
q
glucose
for rumen bacteria obtained from Tables 8.1 and 8.2 range from 1 to
5.1 g/(g biomass)/h.
Table 8.2. Theoretical maximum growth yields on glucose for
rumen bacteria (derived from double reciprocal plots of yield
against dilution rate).
Species Yield (mg biomass per g glucose)
B. fibrisolvens 0.4
B. ruminicola 0.5
M. elsdenii 0.46
S. bovis 0.4
S. ruminantium 0.58
214 M.K. Theodorou and J. France