2005 Poultry Science Association, Inc.

Comparative Gut Microflora,

Metabolic Challenges,
and Potential Opportunities
J. Apajalahti1
Alimetrics Ltd, Hoylaamotie 14, FIN-00380 Helsinki, Finland

Primary Audience: Researchers, Nutritionists, Veterinarians

SUMMARY
Bacteria colonize practically every habitat in the nature. Bacterial community of the
gastrointestinal tract (GIT) is one of the major metabolic tissues of animals. Still, its species
composition is not known, because methods that have been previously used for bacterial analysis
do not capture the species present. The bacterial community within the GIT not only has
intracommunity interactions, but it also interacts with the host tissues. For the host, it is essential
to tolerate commensal bacteria and to recognize and fight intestinal pathogens. In addition to
recognized pathogens, there are many functional bacterial groups, which produce metabolites
considered harmful for the host. Such functional bacterial groups include protein-fermenting
bacteria that produce toxic end products in their amino acid metabolism, which is why a highprotein diet is often considered unhealthy. Carbohydrates are preferred substrates for
gastrointestinal bacteria, but in the distal colon, they may become depleted, and putrefaction
becomes the dominating type of fermentation. This largely accounts for the health effects of slowly
fermenting dietary fibers.
Microbial communities in the GIT of different animals have developed hand-in-hand with their
digestion strategy. In monogastric animals, bacterial fermentation has concentrated in the lower
GIT, whereas in ruminants bacteria are responsible for the initial digestion of the diet. Rumenlike sites with intense bacterial fermentation are found in most animals, but no other group of
animals totally depends on bacteria. The response of rumen bacteria to feed components can be
evaluated by an in vitro rumen simulation, which measures relevant fermentation parameters,
such as the yield of bacterial biomass (protein) and volatile fatty acids (energy). In monogastric
animals, it is essential that the host has measures to prevent bacterial competition for substrates
in regions in which absorption takes place; the use of prophylactic antibiotics for production
animals has aided this.
Availability of good methods for monitoring the total bacterial communities and their
metabolism is a prerequisite for informative studies in the future.
Key words: intestinal bacteria, chicken, rumen, human, putrefaction, bacterial metabolite,
antimicrobial growth promoter
2005 J. Appl. Poult. Res. 14:444453

To whom correspondence should be addressed: juha.apajalahti@alimetrics.com.

APAJALAHTI: INFORMAL NUTRITION SYMPOSIUM

DEVELOPMENT
OF GASTROINTESTINAL
MICROBIAL COMMUNITIES
Bacteria are spread everywhere in nature and
readily colonize a new habitat with available
nutrients and energy for growth and maintenance. Bacterial cells are carried by water, soil,
dust, aerosols, or simply by air. Indeed, there
are few bacteria-free zones in our environment.
Consequently, newborn animals are exposed to
bacteria from the very moment of birth or hatching. The gastrointestinal tract (GIT) of mammals
receive the first inoculum of bacteria from feces
and breast milk of the mother [1, 2], whereas
newly hatched chicks get bacteria from the surface of the eggshells [3, 4]. Very little information is available on the bacterial colonization of
the fish gut, but it seems logical that the original
inoculum for the fry comes from the surrounding
water. In chickens the GIT becomes rapidly colonized by bacteria, with the maximum bacterial
densities being reached within the first 5 d after
hatching [5]. During the following weeks, the
composition of microflora changes markedly
[6, 7].
The ingested bacteria from habitats other
than GIT mainly just pass through the intestine
unable to colonize any compartment of the tract.
However, some bacteria thrive under the gut
conditions and become members of the resident
microbial community. In such communities,
bacteria do not live independently of the other
species but interact and depend on each other
and their host in many ways. Bacterial communities are metabolically versatile mixtures of different bacteria whose relative abundance is regulated by environmental factors, such as substrate
flows, antibacterial compounds, and the structure and function of the host epithelium. As entities, bacterial communities are energetically
more efficient and metabolically more flexible
than representatives of any single bacterial species present or the host itself, because natural
selection inevitably forces out bacteria, which
do not have a unique role (ecological niche) in
the community. In addition, any strain becomes
replaced if a more efficient one appears. Following this general mechanism of bacterial community evolution, site- and diet-specific bacterial

445

communities evolve in the GIT compartments

of different animal species.
The GIT of an adult homeothermic animal
maintains bacteria in quantities exceeding the
total number of host cells and the metabolic
activity of any host tissue. Yet, the composition
and metabolic function of this bacterial tissue is
poorly known today, because most methods used
for bacterial analysis have been based on dilution
and cultivation; these methods separate the interdependent bacterial species from each other and
the host. Only a fraction of gastrointestinal bacteria grows independently as pure cultures with
the practices applied in most laboratories today
[8, 9, 10].
Intestinal lumen should be considered as a
part of the external world in which the animal
is at its closest vicinity to the bacteria of the
surrounding environment but still clearly separated by the intestinal epithelium. The bacterial
community residing within the GIT not only has
intracommunity interactions, but it also interacts
with the intestinal tissue of the host. A difficult
task of the defense mechanism of the intestinal
tissues is to allow close contact with the friendly
commensal bacterial community while recognizing and fighting pathogenic bacteria. Indeed,
well-directed immunological tolerance and defense mechanisms are important determinants of
intestinal health [11, 12]. While natural selection
drives the development of the bacterial community to the best possible fit for its habitat, a
similar selection drives the evolution of host
animals to accommodate a beneficial bacterial
community. Failure to tolerate commensal bacteria may be as detrimental as the failure to
recognize and successfully fight intestinal pathogens. Indeed, one of the challenges of todays
animal breeding is to select for animals, which
show both good performance and high resistance
against incidental intestinal pathogens.

DISTRIBUTION OF MICROBES
IS DETERMINED BY THE
DIGESTIVE SYSTEM OF THE HOST
Microbial communities in the GIT of different animal species have developed hand-in-hand
with their digestion strategy, because the structure and function of the digestive machinery
determines the sites of intestine in which both

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JAPR: Symposium

FIGURE 1. Relative intensity of bacterial fermentation in compartments of the GIT of various animals. Intensity of
bacterial metabolism in the intestinal compartments is indicated by the fill intensity in respective textboxes.

physicochemical and nutritional requirements

for bacterial growth are fulfilled. Still, there are
no sites in the GIT in which bacterial growth
would be entirely missing. The overall bacterial
activity may be modest in compartments, such
as an acidic stomach, but specialized bacteria
thrive under difficult conditions and benefit from
the reduced competition by other bacteria.
The nutritional diversity and digestion potential of bacterial communities clearly exceed
that of any host animals. Consequently, many
components in the diet cannot be attacked by
the host digestive enzymes but can be utilized
by intestinal bacteria. Coevolution of animal
species and their intestinal microflora has led
to a situation in which hot spots of bacterial
fermentation have been restricted to defined
parts of the GIT. In monogastric animals, bacterial fermentation has concentrated in the cecum
and colon (Figure 1). These parts of the GIT
receive dietary compounds that escape host digestion and absorption; therefore, bacteria do
not compete with the host when they ferment
the entering substrates. However, in the upper
GIT competition occurs for all the simple sugars
and amino acids utilized by the host, which are
also available for the bacteria. In poults, equines,
and rodents, the site for intense bacterial fermentation is a well-developed appendix or cecum.
In other monogastric animals, such as humans
and swine, the cecal appendix is diminished, and
the bacterial fermentation mainly occurs in the
colon (Figure 1).

In ruminants, bacteria are responsible for the

primary digestion of the diet. This strategy saves
energy, since the costs for feed digestion and
fighting against bacterial overgrowth are clearly
reduced. The drawback is that the metabolism of
the host depends on the end products of bacterial
metabolism, such as short-chain fatty acids,
which have only half of the energy content of
the carbohydrates. However, since the metabolic
potential and nutritional diversity of the rumen
bacterial community is far beyond that of the
host, ruminants can benefit from dietary components, such as cellulose and hemicellulose,
which are not available for monogastric animals.
Provided that feed intake is adequate, the microbial community of an adult cow with more than
1016 fully adapted bacteria is a high-capacity
fermentor capable of providing enough energy
for the host.
All the homeothermic animals mentioned
above have 1 compartment in the intestinal tract
with high fermentation intensity (Figure 1). Bacterial density in the cecum, colon, and rumen
exceeds 1011/g of digesta, the habitats are anoxic,
and their redox potential is in the range of 200
to 400 mV [13, 14, 15, 16]. Typically, such
bacterial communities are methanogenic or sulfidogenic, and the major end products of fermentation are highly reduced volatile fatty acids,
such as acetic, propionic, and butyric acids and
gases, such as CO2 and CH4. Not all animals
have such bacterial concentrates in their intestine. The GIT of Atlantic salmon has a stomach,

APAJALAHTI: INFORMAL NUTRITION SYMPOSIUM

proximal intestine with a number of pyloric ceca,
and distal intestine. All these compartments are
characterized by flat bacterial densities lower
than those in the ileum of the homeothermic
animals and modest bacterial fermentation, producing lactic and acetic acids [17] (Figure 1).
Such low bacterial density and the lack of bacterial gradients are likely to reflect the low body
temperature and highly digestible, carbohydratepoor diet of these carnivorous species.

PROTEIN FERMENTATION IN THE

GUT PRODUCES HARMFUL
METABOLITES
In the GIT, we can name obvious pathogenic
bacteria, which invade host tissues and produce
potent toxins. Well-known examples are some
species of the genera Salmonella, Campylobacter, and Clostridium, which lead to acute
diarrhea, intestinal inflammation, and sometimes
lethal necrosis of the intestinal tissue [18, 19,
20]. In addition to such indisputably recognized
pathogens, there are many functional bacterial
groups, which under certain conditions release
metabolic products considered harmful for the
well-being of the host. Such functional bacterial
groups include putrefactive, protein-fermenting
bacteria, which produce toxic end products in
their amino acid metabolism. The metabolism
of all amino acids produces, in addition to shortchain fatty acids, ammonia, and amines, which
tend to increase the pH of the intestinal contents
(Figure 2). Saccharolytic fermentation (fermentation of carbohydrates) produces mainly organic acids, which tend to reduce luminal pH.
The general assumption is that a low intestinal
pH is good for intestinal health, because the
growth of many known pathogens is inhibited
under such conditions [21, 22, 23, 24, 25]. Indeed, the relative intensity of the fermentation
types determines the effect of the bacteriological
activity on the acidity of the intestinal contents
(Figure 2).
Products of putrefaction have several undesired effects on the host health, which is why
high-protein diets are often considered unhealthy for humans. Firstly, amino acid assimilation always produces ammonia, which may lead
to neoplastic growth of colon epithelium and
harmful accumulation of ammonia in body fluids

447

[26]. For risk groups, this can be especially hazardous. Aromatic amino acids, such as tyrosine,
phenylalanine, and tryptophan are converted to
phenols and indoles, which have been shown
to express hypertensiveness, schizophrenia, and
migraine, and phenols are, in addition, cocarcinogens [27, 28, 29].
Generally, bacteria favor carbohydrates, if
those are available. In practice, this means that
saccharolytic fermentation is characteristic of
proximal colon, which is rich in carbohydrates.
Carbohydrates in the distal colon become depleted; thus, putrefaction becomes the dominating type of fermentation. This largely accounts for the health effects of prebiotics and
dietary fibers; as slowly digestible structures,
they provide carbohydrates also to distal colon,
thus suppressing putrefaction [30]. An obvious
alternative for the inclusion of nonstarch polysaccharides in the diet is to limit the intake of
poorly digestible protein. If dietary protein became readily digested in the upper GIT by the
digestive system, there would be less substrate
available for the colonic putrefaction. Predatory
animals have their meals raw, which keeps the
meat-derived proteins native and far more susceptible to digestive enzymes. Little is known
about the extent to which the end products of
putrefaction affect the performance of production animals. It seems likely, however, that any
protein escaping digestion in the small intestine
of chickens and pigs is a potential risk for the
health of the animal.

MICROBIAL GROWTH IN THE

SMALL INTESTINE IS COSTLY
FOR THE PRODUCTION ANIMALS
Taking into account that small intestinal digesta is very rich in nutrients, it is striking how
efficiently animals can keep bacterial numbers
low in this critical part of the digestive tract.
Monogastric animals have several mechanisms
that restrict bacterial growth in the proximal
GIT, including chemical inhibition (e.g., acid
and bile), highly competitive rate of nutrient
absorption (large absorptive surface and active
transport), high passage rate of digesta (washout
of free bacteria), continuous sloughing of the
epithelial cells and mucus (washout of adhered
bacteria), and the immunological defense mech-

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JAPR: Symposium

FIGURE 2. Schematic presentation of characteristic pathways and potential health effects of saccharolytic and
putrefactive fermentations by intestinal bacteria.

anisms (IgA production). Failure to prevent bacterial growth in the proximal small intestine
would, no doubt, lead to starvation of the host.
Physiological functions, such as those mentioned above, prevent the wild competition for
substrates, and bacteria are restricted to densely
populated sites, such as rumen, cecum, or colon.
In these compartments, bacteria benefit the host
by converting unavailable dietary compounds to
end products of fermentation, which can then
be utilized by the host. However, in spite of all
the measures to limit bacterial growth to described compartments, bacteria do grow
throughout the GIT. Even though the density of
actively metabolizing bacteria in the low-density
regions rarely exceeds 1% of those in the most
active sites, bacteria can still capture a significant proportion of simple substrates that could
be readily absorbed and utilized by the host. In
broiler chickens, as much as 20% of the total
absorptive surface in the small intestine can be
bacterial. If we assume that the respected absorp-

tive surfaces would be evenly distributed in the

small intestine and the rate of uptake through
bacterial membranes would be comparable to
that of the epithelial membranes of the host,
bacteria could capture up to 20% of the nutrients.
The true figure is most likely somewhat lower
because of an increasing gradient of bacterial
density and a decreasing concentration of simple
substrates towards the distal end of the small intestine.
Animal production has been commonly using prophylactic antibiotics to support the animals inherited measures to fight bacteria in the
intestinal regions that are essential for the nutrient extraction and absorption. The total bacterial
numbers in the ileum of broiler chickens are
reduced by 90% with Avoparcin [13]. Possibly
based on such bacterial growth reduction,
growth-promoting antibiotics improve weight
gain and feed conversion efficiency in pigs and
broiler chickens [31, 32]. Recently, the use of
some prophylactic antibiotics has been banned

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449

FIGURE 3. Effect of antibacterial agents on the growth of broiler chickens. Birds were grown on a wheat-based
diet amended with monensin, bacitracin methylene disalicylate (BMD), and phenoxy methyl penicillin as indicated
in the figure. Bars in columns indicate SE between replicate animals [33].

in many countries in an attempt to limit the

spread of antibiotic-resistant organisms. The
trend will likely expand to limitation of the remaining antimicrobial growth promoters and
perhaps even the coccidiostats. The consequence
of the present development varies and will continue to vary from country to country. In countries in which the feed production and farm management practices follow high standards, the situation is relatively well under control. However,
in many countries, abandoning the prophylactic
antibiotics has led to serious problems in animal
health and performance. Figure 3 summarizes
the effect of a coccidiostat (monensin) alone
and in combinations with a growth promoter
bacitracin methylene disalicylate (BMD) or a
therapeutic antibiotic (penicillin) on the growth
of broiler chickens [33]. At the age of 11 d, none
of the antimicrobials had an effect on the
growth of the animals, whereas 1 wk later all
the treatment groups had gained significantly
more weight than the control group. It was not
until after 4 wk from hatching that the antibiotics
combined with monensin started to affect the
growth of the chicken (Figure 3). Even though
some antimicrobial compounds are called and
registered as coccidiostats instead of growth pro-

moters, their effect on the growth of total microflora may not differ significantly.
The presence of antibiotics in feed has been
one of the major selective factors affecting the
composition of microflora in the GIT of animals.
For decades, microbe selection has favored those
tolerating antibiotics in comparison to those sensitive to antibiotics. This antibiotic-tolerant microflora has become the normal intestinal microflora as we know it, and indeed, most of our
knowledge and experience on animal management leans against this background. Veterinarians have been able to recognize the microbemediated disorders developing from this normal
microflora base. Likewise, most feed companies,
animal breeders, and so on have developed their
products for conditions using growth-promoting
antibiotics. Removing antibiotics from feeds has
changed the rules of competition (selection pressure) in the intestinal microbial community from
favoring antibiotic-resistant microbes to the advantage of microbes growing efficiently on feed
residues in the absence of antibiotics. Since most
microbes in the GIT of animals have been and
are still unknown, the new selection criteria may
lead to the outgrowth and establishment of unforeseeable bacterial species.

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450

FIGURE 4. Flow chart illustrating the role of rumen fermentation in ruminant nutrition.

Following the recent ban of growth promoters, feed and feed ingredient companies are actively seeking for alternative strategies to control
unwanted growth of small-intestinal microflora.
As the tools for microbial community analysis
have become more accurate and independent of
bacterial culturability we have learned that bacteria in the GIT can be modulated by numerous
dietary vehicles, such as grain, feed types, and
feed enzymes [13, 34, 35].

RUMEN BACTERIA BUILD

PROTEIN AND ENERGY
FOR THE HOST
Gastrointestinal bacteria have a central role
in the life of ruminants, not only on the health
of individual animals but also on the evolution
of a whole animal species. In a nutrient-poor
environment, animals with emerging characteristics of ruminants would benefit from diversi-

fied selection of utilizable feed and retrieval of

energy from cellulose and hemicellulose. Rumen-like sites with intense bacterial fermentation are found in most animal species, but no
other group of animals depends on bacterial fermentation to the same extent as the ruminants,
which have been estimated to derive 70% of their
energy from the products of rumen fermentation,
mainly acetic, propionic, and butyric acids [36].
Not all acids have the same value for the host.
Different fatty acids are preferred energy sources
for different tissues; therefore, their relative
value for the host varies, depending on the physiological status of the animal. Monogastric animals, such as swine and human, may significantly gain from bacterial fermentation; they
have been estimated to achieve 10 to 20% of
their energy requirements by absorbing shortchain fatty acids produced by bacteria in the
hindgut [36, 37].

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451

FIGURE 5. Yield of volatile fatty acids and microbial biomass in rumen fermentation simulation. In vitro fermentation
was carried out for 12 h, and then fermentation products were quantified. Volatile fatty acids were analyzed by
gas chromatography and bacteria by flow cytometry as described previously [17, 38]. Bars in columns indicate
SE between the replicate fermentation vessels [33].

The bulk of dietary nitrogen is assimilated

by the actively growing rumen microflora. Subsequently, the majority of the protein assimilated
by the animal comes from the digestion of bacterial biomass in abomasum, the true stomach of
the ruminants.
Optimization of the diet for ruminants is
challenging due to the involvement of the complicated rumen parameters. How to optimize dietary protein and energy when they cannot be
readily analyzed from the components of the
ration? True protein and energy available for the
host are not in the diet but are being produced
by rumen bacteria from the components of the
diet (Figure 4). Feed materials used for ruminant
rations are many, and their quality may vary.
Quality of forages depends on plant varieties,
fertilization, and seasonal conditions, such as
light, temperature, and humidity. Quality of fermented silages depends, in addition to the factors
listed above, on the bacteria present and the
fermentation conditions. Also, farmers use many
types of byproducts, the specifications of which
are difficult to control. One way to evaluate the
response of rumen bacteria to feed components
is to use an in vitro rumen simulation and to

measure relevant fermentation parameters, such

as the yield of bacterial biomass (protein) and
volatile fatty acids (energy). As an example, we
tested fermentation characteristics of different
fractions of corn silage, using such an approach.
Significant differences were observed in the
yield of bacterial biomass, volatile fatty acids,
and gas production of different fractions (Figure
5). The highest yields of both microbial protein
and volatile fatty acids were found with the grain
fraction; rumen fermentation produced 800 g of
acids and bacteria from 1 kg (DM) of ensiled
grain [33]. The other fractions and the mixed
silage produced about 500 g of the fermentation
products. It is noteworthy that not only the total
yield, but also the energy-to-protein ratio of different fractions of corn silage varied significantly (Figure 5). The approximate ratio of microbial biomass to volatile fatty acids was 1:4
in mixed corn silage but close to 1:2 in the leaf
fraction of the corn silage.
How accurately can protein and energy
yields of different feed raw materials be estimated from their chemical composition? Is it
possible that the production efficiency of dairy
and beef cattle could be significantly improved if

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452
we knew how to optimize the relative production
kinetics of the true protein and energy?

FUTURE POTENTIAL
OF KNOWLEDGE-BASED
MICROBIAL MANAGEMENT
The structure and function of gastrointestinal
bacteria affect our daily life and society. Gut
bacteria may become recognized because of the
intestinal disorders caused by enteric pathogens
or the high price of farm products affected by
the efficiency of rumen fermentation. Obviously,
we should regulate the growth of beneficial and
harmful bacteria and thus improve the quality
of life. However, it is only within the last decade
that scientists have shown that there is an undiscovered microbial world in the GIT of animals.
This discovery has emerged with the new tools
for microbial community analysis. Earlier methods were based on the cultivation of bacteria
under artificial laboratory conditions, whereas
the new analytical tools are based on direct extraction of bacterial DNA from intestinal samples and subsequent sequencing of the taxonomically relevant genes. Such approaches have re-

vealed that we used to know only a fraction of

the total bacteria. Now we acknowledge the true
diversity of bacteria, but for most newly discovered species, we only know a partial DNA sequence.
In the present situation, microbial management based on true understanding is challenging.
Modulation of the intestinal bacterial community to a beneficial direction by feeding of live
bacteria (probiotics) or specialty carbohydrates
for the beneficial bacteria (prebiotics) are currently under active research and development.
Since the growth requirements of bacteria differ,
it should be possible to shift the microbial community from harmful to nonharmful direction
by changing the gut dynamics through dietary
modulations. Probiotic bacteria are meant to improve the health of the GIT, but these are likely
to be effective only if the requirements for their
growth are fulfilled or if their physiological effect is independent of their viability. If viability
is a requirement, a synbiotic product that contains both a probiotic strain and a prebiotic substance, favoring the growth of that particular
probiotic, might be a good solution.

CONCLUSIONS AND APPLICATIONS

1. Modern tools to detect each member of the microbial communities have brought up new questions
about the role of microflora. What are all the bacterial species doing? Are they good or bad
for the health of the host?
2. Thorough epidemiological studies together with well-designed animal trials, measuring parameters that reflect animal performance and health should be carried out to improve our understanding
of gastrointestinal interactions and the role of individual bacteria in the gastrointestinal microbial community.
3. When relevant indicator bacteria and their metabolites are known, it is possible to design animal
trials and laboratory simulations, which can be effectively used for product evaluation and
screening of novel microbial modulators.

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