ANAEROBIC FERMENTATION OF LYSINE1
PATRICIA M. DOHNER AND B. P. CARDON
Departments of Animal Husbandry and Animal Pathology, University of Arizona, Tucson, Arizona
Received for publication November 18, 1953
The importance of microbial synthesis in the
bovine rumen is well recognized (Baker et al.,
1947). Catabolic processes involving decomposi-
tion of the complex carbohydrates have also been
studied extensively. However, catabolic changes
in the protein fraction brought about by rumen
microorganisms are much less understood. Rumen
microorganism destruction of protein or amino
acids may be extensive in high protein rations.
Considerable amounts of ammonia may accumu-
late in the rumen fluid with rations containing
only average amounts of protein (McDonald,
1952). This ammonia could only come from the
breakdown of amino acids.
Some proteins, particularly those of plant
origin, may have a low content of one or more
essential amino acids (Maynard, 1951). There-
fore, rations composed of these proteins would be
low or even deficient in these essential amino
acids. Extensive rumen bacterial destruction
would further aggravate this condition.
Lysine is an essential amino acid and occurs in
very low concentrations in plant proteins such
as zein and edestin. In rations composed of these
or similar proteins extensive destruction could
influence the nutritive value.
Very little data are available on the fermenta-
tion or destruction of lysine by bacteria. The
present paper deals with the isolation and study
of two strains of Escherichia coli from the bovine
rumen which together in growing cultures
ferment lysine.
MATERIALS AND METEODS
Fermentation of lysine in an aerobic enrich-
ment culture medium inoculated with rumen
fluid and incubated at 37 C was obtained in 48
hours. The enrichment medium consisted of the
following: L-lysine, 0.5 g; yeast extract (Difco),
0.3 g; sodium thioglycolate, 0.01 g; FeSOc47HOH,
0.001 g; MgSO4-7HOH, 0.005 g; M/1 phosphate
1 Arizona Agricultural Experiment Station
Technical Paper No. 331.
608
buffer pH 7.2, 2.0 ml; saturated CaSO4, 2.0 ml;
tap water, 100 ml.
Decomposition of lysine was indicated by the
formation of ammonia and of volatile fatty acids
in equimolar amounts. The predominant organ-
isms which developed were small, nonmotile,
gram negative rods. Ten successive transfers were
made. In each transfer the same types of organ-
isms developed, and ammonia and volatile fatty
acids were produced in equimolar amounts.
The organisms were isolated from the tenth
transfer using the anaerobic shake culture
method. The medium was the same as that used
for enrichment, except the yeast extract was
increased to 0.6 per cent and two per cent agar
was added. Two types of colonies developed in
these shake cultures after two days' incubation.
One colony was cream colored and disc shaped,
and the other was white and fimbriate. Mi-
croscopic examination showed that the organisms
from both types of colonies were similar to those
appearing in the enrichment cultures. Isolations
were made from both colonies. The white
fimbriate colony was designated as Type E, and
the other, as Type F.
Five enrichment cultures were started from
rumen fluid. The same two organisms were
isolated from each enrichment culture series.
The organism designated as Type E is a
slender, nonmotile, gram negative red, about 1.1
by 0.3 ,u in size. It is a facultative anaerobe.
Vigorons growth appears in aerobic sugar media
and numerous two- or three-rod chains appear;
but in anaerobic stab cultures the cells usually
occur singly. Colonies are white to light cream
colored and are fimbriate. No spores or capsules
have been observed.
The organism referred to as Type F is a non-
motile, nearly ovoid, gram negative bipolar rod,
about 0.9 by 0.6 Iu in size. The celLs usually occur
singly. It also is a facultative anaerobe and
readily ferments sugars. Individual colonies
forming in anaerobic shake tubes are cream
colored and disc shaped.
1954] ANAEROBIC FERMENTATION OF LYSINE 609
Examination of the cultural characteristics of rate and amount of lysine fermented. Lysine was
these organisms in diagnostic media indicates that not fermented at concentrations of yeast extract
they both fit into the classification of Escherichia below 0.005 per cent or above 1.50 per cent. The
coli as defined in Bergey's Manual (Breed et al., optimum amount was in the range from 0.06 to
1948). Since they both belong to the same 0.10 per cent.
species, they will be differentiated in this paper Lysine is fermented incompletely in mixed
as Types E and F. cultures. During enrichment and later with pure
mixed cultures it was observed that only 60
EXPERIMENTAL RESULTS to 65 per cent of L-lysine was decomposed. In-
Physiological characteritis. Inoculation made vestigation of the influence of the amount of
from pure cultures of either Type E or F grown lysine on the fermentation showed that for con-
in nutrient broth into lysine medium failed to centrations up to 0.4 per cent the rate and per
produce growth or a fermentation of lysine. cent of decomposition were fairly constant (62
Varying the percentages of each ingredient in the per cent), but that at higher concentration
medium still failed to bring about a fermentation fermentation was inhibited. At the 1.5 per cent
of lysine by pure cultures of either organism. level less than 1.0 per cent was fermented.
Fermentation of lysine did occur, however, when The dehydrogenase activity of an organism
both organisms were inoculated into the same toward a specific substrate can be determined by
culture flask. use of the methylene blue reduction method
In order to determine whether a metabolite of (Umbreit et al., 1945). The following tests were
one organism was required for the fermentation of made to determine the ability of Types E, F, and
lysine by the other, cultures of each organism mixed cultures to activate lysine and glucose as
were autoclaved and varying amounts added to H-donors to methylene blue.
lysine medium. Inoculations of each organism into Cell suspensions of pure cultures of the two
lysine medium containing sterile metabolites of types were prepared (Cardon and Barker, 1947).
the other organism failed to produce a fermenta- One ml of this cell suspension, 1 ml 0.5 per cent
tion of lysine. Only when living cells from each solution of glucose or lysine, and 1.0 ml m/15
type were inoculated into the same flask was phosphate buffer, pH 7.0, were placed in a
lysine fermented. Thunberg tube. One-half ml 0.025 per cent
The specific interrelationship of Types E and F methylene blue was placed in the side arm. The
in this fermentation is unknown. It seems un- contents of the side arm and tube were mixed
likely that the fermentation of the lysine molecule after the tube was evacuated and brought to
requires the direct action of both organisms. constant temperature of 37 C. Time required for
Further work must be done to clarify this point. decolorization was used as a measure of the de-
Since both Types E and F are essential to the hydrogenase activity.
fermentation of lysine, mixed cultures were used Glucose was activated as a hydrogen-donor by
in all the following studies. The two types were both Types E and F. Lysine was completely in-
kept in anaerobic stab cultures, and the medium active with Type E or F or with mixed cell
to be fermented was inoculated with a loop from suspensions.
each type. Cell suspensions of each type as well as mix-
The influence of various constituents on the tures of the two were incubated under anaerobic
growth of mixed cultures of Types E and F was conditions in a Thunberg tube with I-lysine.
determined using the same medium that was After 12 hours' incubation analysis for ammonia
used for the enrichment. and volatile fatty acids also indicated that lysine
Using this medium it was shown that fermenta- was not fermented by cell suspensions of these
tion of lysine would occur over the pH range 6.6 organisms.
to 7.5, the optimum being between 7.0 to 7.1. The Lysine fermentation. The foregoing discussion
optimum concentration of thioglycolate was has indicated that lysine fermentation was ob-
shown to be 0.01 per cent. The rate of fermenta- tained in enrichment cultures, mixed growing
tion was considerably slower at higher or lower cultures, but not in cell suspensions of Types E
concentrations. and F. Thus, it was necessary to use growing
The amount of yeast extract also influenced the cultures in the studies of lysine fermentation.
610 PATRICIA M. DOHNER AND B. P. CARDON
TABLE 1
Fermentation of z-1ysine by growing mixed culture.
of Types E and F
(pM of product per 100 pm lysine fermented)
PODVCTS
PK
Carbon dioxide ................. 0.4
Ammonia ...................... 200.0
Butyric acid .................... 94.6
Acetic acid ..................... 94.6
Carbon recovery (per cent) ...... 94.7
Hydrogen recovery (per cent). 94.7
Redox value .................... 0.93
The fermentation medium used in these experi-
ments was the basic enrichment medium varied
in accordance with the optimum component con-
centrations previously determined. These were as
follows: xlysine, 0.4 g; yeast extract (Difco),
0.08 g; FeSO4-7HOH, 0.001 g; MgO04-7HOH,
0.001 g; sodium thioglycolate,0.01 g; m/1 phos-
phate buffer pH 7.0, 2.0 ml; saturated CaSO4,
0.6 ml; tap water, 100 ml.
Sterile medium was inoculated from anaerobic
stab cultures of Types E and F and incubated
anaerobically for 3 days at 37 C. A suitable
blank prepared as above, except for the absence
of L-lysine, was also fermented and analyzed at
the same time, and suitable corrections were
made.
Ammonia production was mesured by micro-
Kjeldahl distillation. The amount produced was
used as a measure of lysine fermented since pre-
liminary experiments had shown that from 96 to
100 per cent of the decrease in amino nitrogen
could be accounted for as ammonia (Van Slyke,
1929). No primary amines other than ammonia
could be detected by paper chromatographic
analysis (Bremner and Kenten, 1951).
Volatile fatty acids were collected by steam
distillation, and the kind as well as the amount of
each was determined by Duclaux distillation or
separation on a silica gel column (Elsden, 1946).
Neutral volatile compounds or nonvolatile acids
were not detected.
The results of a typical analysis are given in
table 1.
These data show that this fermentation of
irlysine conforms to the following equation:
CH2NH2CHsCH2CH2CHNH2COOH + 2HOH
- CH,COOH + CHzCH2CHsCOOH + 2NH,
[VOL. 67
The carbon and hydrogen recovenes, as well as
the redox value, are satisfactory for cultures of
this type since some of the substrate will be
incorporated in the bacterial cells.
In several experiments the ratio of acetic to
butyric varied only two per cent from 1:1. There
was more variation in the amount of carbon
dioxide produced, but in no cae did it amount to
more than 1.5 umM per 100 pM of L-lysine fer-
mented. In all probability this carbon dioxide
came from the yeast extract (Difoo) added to the
medium.
DISCUSSION
Stickland (1935) was the first to show that
several anaerobic bacteria could satisfy their
energy requirements by catalyzing oxidation-
reduction reactions between pairs of different
amino acids. Later it was shown (Clifton, 1942)
that certain of the substituted amino acids, such
as aspartic and glutamic acids, serine, etc., also
could be fermented. Cardon and Barker (1947)
demonstrated that unsubstituted amino acids
such as glycine and alanine also could be fer-
mented readily. The mechanism of fermentation,
however, still appeared to be reactions between
pairs of the same amino acid.
The formation of acetic and butyric acids in a
1:1 ratio indicates that the decomposition of
lysine reported here may be a true fermentation
involving the direct plit of a six-carbon chain
into a two-and-four-carbon fragent. However,
the posibility that these acids are formed by a
condensation reaction cannot be ruled out. Other
than decarboxylation, the dimet splitting of a
carbon chain has not been demonstrated hereto-
fore in anaerobic decomposition of amino acids.
The importance of this fermentation reaction
in rumen catabolic changes cannot be evaluated
at the present time. It can be stated that these
two organisms are common inhabitants of the
rumen since they were isolated from each of the
five enrichment cultures started. Further work
is in progres to study this point.
Two strains of EwAerichia coli, which together
ferment lysine, were isolated from the bovine
rumen by use of enhment culture technique.
Their moxphological, cultural, and physiological
characteristics have been described.
19541 ANAEROBIC FERMENTATION OF LYSINE 611
Analysis of the end products of fermentation CLIFTON, C. E. 1942 The utilization of amino
in growing cultures indicates that L-lysine is acids and related compounds by Clostridium
fermented according to the following equation: tetani. J. Bact., 4, 179-183.
ELSDEN, S. R. 1946 Silica gel method for vola-
CH2NHsCH2CH,CH2CHNH2COOH + 2HOH tile fatty acids. Biochem. J. (London), 40,
CHJCOOH + CH.CH2CH,COOH + 2NHa 252-256.
MAYNARD, L. A. 1951 Animal nutrition. 3rd
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