Vol. 44, No. 2
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1982, p. 346-350
1,4-Naphthoquinone and Other Nutrient Requirements of
ROGELIO A. GOMEZ-ALARCON,1t COLLEEN O'DOWD,1t JANE A. Z. LEEDLE,1§ AND MARVIN P.
Department ofDairy Science' and Department ofMicrobiology,2 University ofIllinois, Urbana, Illinois 61801
Received 7 December 1981/Accepted 27 April 1982
Three strains of Succinivibrio dextrinosolvens isolated from the rumen of cattle
or sheep under diverse conditions grew well in a minimal medium containing
glucose, minerals, cysteine, methionine, leucine, serine, ammonia, 1,4-naphtho-
quinone, p-aminobenzoic acid, and bicarbonate-carbonic acid buffer, pH 6.7.
When menadione or vitamin K5 was substituted for 1,4-naphthoquinone, the
growth rate was somewhat depressed. Growth was poor with vitamin K1 and
phthiocol, and other related compounds were inactive. In the absence of
ammonia, further addition of the amino acids aspartic acid, arginine, histidine,
and tryptophan was necessary for good growth oftype strain 24, but the other two
strains grew well only in media containing ammonia. Strains C18 and 22B
produced urease and grew well when ammonia replaced urea. When urea replaced
ammonia, strain 24 grew poorly and urease activity could not be detected. Strain
24 required no B-vitamins, but the other two strains were stimulated by p-
aminobenzoic acid. The methionine requirement was not replaced by vitamin B12,
betaine, or homocysteine. Cysteine was replaced by sulfide in strain 24 but less
well in the other two strains. Very poor growth was obtained when sulfate
replaced cysteine. The half-saturation constant for ammonia during growth of S.
dextrinosolvens is more than 500 p,M, a much higher value than that of many
Although much is known about the nutrition
of many important species of rumen bacteria (4,
6, 7, 10), Succinivibrio dextrinosolvens (2, 8) has
received little attention. It is usually isolated in
large numbers only from the rumen of cattle or
sheep fed relatively high starch diets or other
diets containing large amounts of rapidly fer-
mented carbohydrate (2, 8, 13) and, under some
conditions, may be one of the major rumen
bacteria fermenting dextrins and grass levan. Its
fermentation products include mainly acetate
and succinate, with a relatively large uptake of
CO2 (8, 13). It was the most numerous bacterium
producing urease in the rumen of cattle fed a
relatively high grain and corn silage diet (15).
In early studies with glucose media, S. dex-
trinosolvens was shown to require a high level of
HCO3 -CO2 for good growth (5). In a basal
medium containing glucose, minerals, enzymat-
ic casein hydrolysate, B-vitamins, cysteine, and
HCO3 -CO2, growth of a relatively large inocu-
lum of one strain was highly stimulated by
rumen fluid and ammonia, and factors in casein
t Present address: CIPES, Carbo, Sonora, Mexico.
t Present address: Department of Microbiology, University
of Texas Medical Center, San Antonio, TX 78284.
§ Present address: Department of Microbiology, University
of Illinois, Urbana, IL 61801.
hydrolysate were essential (6). Acetate or a
mixture of isobutyrate and valerate isomers had
no effect on growth, but one or more B-vitamins
was stimulatory (6). When NH4', peptides, or a
mixture of free amino acids was added as nitro-
gen source, little NH4+ was produced or used,
and 14C from free amino acids was very effec-
tively incorporated into cellular material (7).
Scardovi (13), working with another strain,
found that a factor present in rumen fluid was
necessary for good growth.
In the present study we describe minimal
chemically defined culture media for growth of
three strains of S. dextrinosolvens and show that
the rumen fluid requirement is replaced by 1,4-
MATERIALS AND METHODS
The strains used were from the culture collection of
the Microbiology Division, Department of Dairy Sci-
ence, University of Illinois. Strain 24 (ATCC 19716) is
the type strain ofthe species and was isolated from the
rumen ofa cow on a high-grain diet in Maryland (5, 8).
Strain 22B was isolated from the rumen of a sheep fed
dried grass cubes and hay in England (S. N. Wilson, J.
Gen. Microbiol. 9:i-ii, 1953) and was provided by
S. R. Elsden. Strain C18 was a more recent isolate
from an Illinois cow fed mainly grain and corn silage
NUTRITION OF SUCCINIVIBRIO DEXTRINOSOLVENS
TABLE 1. Composition of the basal medium for
nutritional studies of S. dextrinosolvens
Mineral solution ...................... 5.0 (vol/vol)
Trace mineral solution"................ 0.5 (vol/vol)
FeSO4 (10 mM) ...................... 0.2 (vol/vol)
Resazurin (0.1% [wt/vol]).............. 0.1 (vol/vol)
Na2CO3 solution (8% [wt/vol])d ........ 5.0 (vol/vol)
CO2 gas phase (pH 6.7)
MgCI2-6H2O, 0.4; MnCI2-4H2O, 0.2;andCoC12'6H20,
0.02 in distilled water.
b Trace mineral solution contained 10 mg each of
ZnSO4 7H2O, H3BO3, Na2SeO3, Na2MoO4'2H20, and
NiCl2-6H2O, 5 mg of CuSO4 5H2O, and 2 mg of
Al(SO45)2412H20 made to 100 ml with distilled water.
c The cysteine solution was autoclaved separately,
tubed, and stored with an N2 gas phase (3).
d The Na2CO3 solution was autoclaved separately,
aseptically equilibrated with CO2, and stored with a
CO2 gas phase (3).
........ 0.3 (wt/vol)
... 2.0 (vol/vol)
and was one of many urease-forming S. dextrinosol-
vens strains isolated (15).
Anaerobic methods and media. The anaerobic tech-
niques were those of Hungate as modified by Bryant
The composition of the basal medium is shown in
Table 1. The medium, with additions, was autoclaved
at 121°C for 15 min. The separately sterilized cysteine
and Na2CO3 solutions were then added, and the medi-
um was tubed in 5-ml amounts into rubber-stoppered
culture tubes (13 by 100 mm) as previously described
Some solutions added to the basal medium were as
follows: 0.3% (vol/vol) volatile fatty acid (VFA) solu-
tion was added, containing 2.0 ml each ofDL-2-methyl-
n-butyric, n-valeric, isobutyric, and isovaleric acids
adjusted to pH 7.0 with NaOH and made to 50 ml with
distilled water. The B-vitamin solution contained 20
mg each of thiamin-hydrochloride, calcium-D-panto-
thenate, nicotinamide, riboflavin, and pyridoxine-hy-
drochloride, 1 mg of p-aminobenzoic acid, 0.25 mg
each of biotin and folic acid, and 0.2 mg of cobalamin
in 100 ml of distilled water, and 0.5% (vol/vol) was
added to the medium. Five percent (vol/vol) of 120
mM (NH4)2SO4 was added as a source of ammonia
nitrogen. These and other additions to the basal medi-
um are indicated in the respective experiments.
Strains were maintained in carbohydrate medium
maintenance slants (6). For nutrition experiments, one
loop (about 0.01 ml) from the inoculum medium into 5
ml of experimental media (triplicate tubes) was used.
The inoculum medium was the basal medium with
Casitone (0.2% [wt/vol]), VFA mixture, (NH4)2SO4
solution, B-vitamins, and 1% ofrumen fluid (6) added.
In later experiments, the inoculum was from the
simplest defined medium, relevant to the experiment,
which allowed good growth.
Growth was measured by absorbance at 600 nm
(A6w),using a Bausch and Lomb Spectronic 70 with
triplicate culture tubes (13 by 100 mm). Cultures were
usually serially transferred on a given medium three
times before the final growth measurements were
made. An absorbance value of 1.0 was equal to about
0.67 mg of cellular dry weight per ml of medium.
Naphthoquinone requirement. The basal medi-
um with B-vitamins, Casitone (0.2% [wt/vol]),
and 6 mM (NH4)2S04 added did not support
growth through three transfers of any of the
three strains. However, with the addition of 1%
(vol/vol) of rumen fluid or certain vitamin K-like
compounds growth was good. Results with these
additions to the growth medium, using strain 24
(Fig. 1), indicated that 1,4-naphthoquinone sup-
ported the best growth. Somewhat slower
growth rates were obtained with rumen fluid or
menadione, and vitamin K5 gave a long lag in
growth followed by good growth. Phthiocol and
vitamin K1 supported poor growth after long
lags, and other compounds tested did not sup-
port growth. Similar results were obtained with
strains 22B and C18 except that vitamin K5
supported somewhat faster growth than mena-
Strain 24 grew best with 2.6 ,uM or more 1,4-
naphthoquinone added; below this level growth
rates were lower. Below 0.26 ,uM, both growth
rates and extent of growth were drastically re-
Incubation Time (h)
FIG. 1. The effect ofcompounds related to vitamin
K in replacing the rumen fluid requirement for growth
of S. dextrinosolvens strain 24 in the basal medium
(Table 1) supplemented with Casitone (0.2% [wt/vol]),
6 mM (NH4)2SO4, VFA mixture, and B-vitamins.
Symbols: 0, rumen fluid (1% [vol/vol]); 0, 52 ,uM
menadione; [, 52 ,uM 1,4-naphthoquinone; U, 52 ,uM
phthiocol; A, 52 FLM vitamin K1; A, 52 ,M vitamin
K5; and x, no additions or 52 ,uM naphthaline, hydro-
quinone, toluoquinone, or coenzyme Q. Points are the
means of three tubes after three transfers on a given
VOL. 44, 1982
GOMEZ-ALARCON ET AL.
TABLE 2. The effect of certain amino acids on the
growth of S. dextrinosolvens strain 24 in a mediuma
with and without ammonia
0.01 (l00)C 0.02 (48)
1.60 (20)d 0.07 (40)
(20)d 0.09 (32)
ser, leu, arg, asp, his, trp
met, ser, leu, arg, asp, his, trp 1.90
met, ser, leu, arg, asp
met, ser, leu
aThe medium was that in Table 1 supplemented
with the VFA mixture and 52 ,M 1,4-naphthoquinone.
(NH4)2SO4, 1.34 mM DL-methionine (met), 2.85 mM
L-serine (ser), 3.04 mM L-leucine (leu), 0.95 mM L-
arginine (arg), 2.25 mM L-aspartate (asp), 0.52 mM L-
histidine (his), and 0.49 mM L-tryptophan (trp).
c Values in parentheses indicate hours to reach
dCultures with abnormal growth as indicated in the
strain 24 was unable to grow in the basal medium
supplemented with 52 ,uM 1,4-naphthoquinone,
B-vitamins, and VFA mixture or in the same
medium supplemented with 6 mM (NH4)2SO4.
However, growth in the latter medium was good
if Casitone (0.2% [wt/vol]), Casamino Acids
(0.2% [wt/vol]), or a complex mixture of amino
acids was added. If (NH4)2SO4 was deleted,
growth in the medium with Casitone was some-
what depressed, but not in the medium with
Casamino Acids or in the medium with a com-
plex mixture of amino acids.
These results indicated that ammonia and
cysteine alone would not satisfy the nitrogen
requirements of strain 24 but that other amino
acids were required. The results for media with-
out ammonia added also suggested that free
amino acids were better sources of nitrogen than
peptides (Casitone) were.
The effect of various amino acid combinations
on the growth of S. dextrinosolvens strain 24 in
the presence and absence of ammonia is shown
in Table 2. Methionine was necessary for good
growth regardless of the other amino acids pre-
sent; however, methionine, cysteine, and am-
monia supported only feeble growth. If serine
and leucine were also added, growth was excel-
lent. If either serine or leucine was deleted from
this medium, growth was good, but abnormal,
and was characterized by large swollen cells,
longer chains ofcells, and by clumps ofcells that
were hard to disrupt. The methionine require-
ment could not be replaced by betaine, homo-
cysteine, or vitamin B12, and the VFA mixture
had no effect on growth regardless ofthe number
of amino acids present in the medium.
In the absence of ammonia further amino acid
supplementation was necessary to obtain good
growth of strain 24 as shown in Table 2 and in
other experiments not shown. The medium with
just those amino acids necessary for good
growth when ammonia was present, i.e., methi-
onine, serine, leucine, and cysteine, supported
very poor growth in the absence of ammonia.
Further addition of aspartate and arginine al-
lowed much better growth, but the cultures were
abnormal as indicated above. The further addi-
tion of histidine and tryptophan resulted in the
best growth observed without ammonia, and the
addition of other amino acids had little effect. In
contrast to strain 24, strain 22B grew more
slowly, and strain C18 grew very poorly without
ammonia even when a complete mixture of
amino acids was added to the medium; yet all
three strains grew very well with only ammonia,
methionine, cysteine, serine, and leucine as ni-
The minimal amount of ammonia required to
give near optimal growth of strain 24, with only
methionine, cysteine, serine, and leucine as add-
ed amino acids, was about 2 mM (Table 3). With
ammonia levels of 1 mM or less, the extent of
growth was drastically lower, and the growth
rate was lower.
Urea added to the medium in place of ammo-
nia supported excellent growth of strains 22B
and C18 but only poor growth of strain 24 (Table
4). Strong urease activity, indicated by ammonia
production in the spot test of Wozny et al. (15),
was obtained with strains 22B and C18 in the
culture containing 25 mM urea, but no ammonia
was detected with strain 24. Tests on the uninoc-
ulated medium and on the stock urea solution by
TABLE 3. The effect of levels of ammonia on the
growth of S. dextrinosolvens strain 24 in a medium
containing the amino acids necessary to give good
growth when ammonia was in excessa
aThe medium was that in Table 1, with 52 F.M 1,4-
naphthoquinone, 1.34 mM DL-methionine, 3.04 mM L-
leucine and 2.85 mM L-serine added.
bGrowth rate during exponential growth.
Hours to reach maximum absorbance.
APPL. ENVIRON. MICROBIOL.
NUTRITION OF SUCCINIVIBRIO DEXTRINOSOLVENS
TABLE 4. Effect of the addition of ammonia or urea
on the growth of S. dextrinosolvens strains in a
medium containing only those amino acids necessary
to allow good growth when ammonia was in excess'
(NH4)SO4 (5 mM)
Urea (5 mM)C
Urea (25 mM)Y
aThe medium was that described in Table 1 supple-
mented with 52 ,uM 1,4-naphthoquinone, 50 ng of p-
aminobenzoic acid per ml, 1.34 mM DL-methionine,
3.04 mM L-leucine, and 2.85 mM L-serine.
bHours to reach maximum absorbance.
c Urea was filter sterilized.
the spot test or by the indophenol test (9)
indicated no ammonia contamination.
B-vitamin requirements. Strain 24 grew very
well when none of the B-vitamins was added to
the basal medium supplemented with
naphthoquinone and various utilizable nitrogen
sources (Tables 2 and 3), and none of the B-
vitamins stimulated the growth of strain 24.
However, although none of the B-vitamins was
essential to the growth of the other two strains,
p-aminobenzoic acid was stimulatory to their
growth. In the absence ofp-aminobenzoic acid,
both strains 22B and C18 had reduced growth
rates, and the extent of growth was greatly
reduced in strain C18.
Sulfur requirements. Little or no growth was
obtained in the basal medium supplemented with
leucine, with NH4Cl and FeCl2 replacing their
sulfate salts and with 1 mM dithiothreitol replac-
ing cysteine. The addition of cysteine to this
medium allowed excellent growth of all three
strains. The replacement of cysteine with 1 mM
sulfide allowed excellent growth of strain 24,
slower growth of strain 22B, and much less
growth of strain C18. When cysteine was re-
placed with 1 mM Na2SO4, strain 24 grew with a
greatly reduced growth rate, and strains 22B and
C18 did not grow.
This is the first documentation of a naphtho-
quinone, menadione, or vitamin K requirement
by a bacterium known to be important in the
rumen. Bacteroides levii, previously thought to
be a strain ofBacteroides melaninogenicus, has
long been known to require menadione or 1,4-
naphthoquinone (11) but was found in only small
numbers in the rumen.
The amino acids required for good growth of
S. dextrinosolvens are somewhat greater in num-
ber than those required by most known species
of rumen bacteria. A few other species also
require methionine and cysteine but not serine
and leucine (4). When ammonia was deleted
from the medium, strain 24 then required addi-
tional amino acids for good growth. These in-
cluded arginine and aspartic acid and, to a lesser
extent, histidine and tryptophan. The other two
strains were stimulated by ammonia even in a
medium containing many amino acids. That
strain 24 grew well with mixtures of free amino
acids fits well with previous results showing that
it very effectively incorporated 14C from a mix-
ture of 14C-amino acids as compared with many
other rumen bacteria when grown on a medium
containing free amino acids, peptides, and am-
monia as possible nitrogen sources (7). The fact
that ammonia was more stimulatory to growth in
media containing mainly oligopeptides (Casi-
tone) as a nitrogen source than in media contain-
ing many free amino acids suggests that strain 24
is not very effective in the utilization of oligo-
peptides as compared with species such as Bac-
teroides ruminicola (12). Previous results indi-
cated that S. dextrinosolvens did not produce
ammonia in a medium containing a large amount
of amino acid and peptide nitrogen (1).
The present results show that strain 24 does
not have a very good affinity for ammonia as the
maximum growth rate with 0.5 mM ammonia in
the medium was less than halfofthat in the same
medium with 2.0 mM or more (Table 3). Thus,
the half-saturation constant for ammonia for
growth in media in which ammonia is required is
in the order of 500 ,uM or more. Many species of
rumen bacteria have half-saturation constants
for ammonia of less than 50 ,uM (14).
The present results show that S. dextrinosol-
vens strain C18, shown earlier to contain urease
(15), retained it, and strain 22B, not previously
shown to have urease, also has the enzyme.
This, along with the study of Wozny et al. (15),
suggests that most strains of the species contain
urease. However, although the type strain 24 of
the species grew very slowly when urea replaced
ammonia (Table 4), no ammonia was detected
and no 14CO2 was detected in a urease assay
involving [14C]urea (J. A. Patterson and R. B.
Hespell, personal communication). Thus, the
type strain either contains no urease or a greatly
The valuable discussions with R. B. Hespell are acknowl-
edged. C. J. Smith suggested the possibility of a menadione
This research was supported by Consejo Nacional de Cien-
cia y Tecnologia of Mexico, grant 35-331 from the U.S.
Department of Agriculture, and the Agricultural Experiment
Station of the University of Illinois.
VOL. 44, 1982
350GOMEZ-ALARCON ET AL.
1. Bladen, H. A., M. P. Bryant, and R. N. Doetsch. 1961. A
study of bacterial species from the rumen which produce
ammonia from protein hydrolysate. Appl. Microbiol.
2. Bryant, M. P. 1959. Bacterial species ofthe rumen. Bacte-
riol. Rev. 23:125-153.
3. Bryant, M. P. 1972. Commentary on the Hungate tech-
nique for culture of anaerobic bacteria. Am. J. Clin. Nutr.
4. Bryant, M. P. 1974. Nutritional features and ecology of
predominant anaerobic bacteria of the intestinal tract.
Am. J. Clin. Nutr. 27:1313-1319.
5. Bryant, M. P., and L. A. Burkey. 1953. Cultural methods
and some of the more numerous groups of bacteria in the
bovine rumen. J. Dairy Sci. 36:205-217.
6. Bryant, M. P., and I. M. Robinson. 1962. Some nutritional
characteristics of predominant culturable ruminal bacte-
ria. J. Bacteriol. 84:605-614.
7. Bryant, M. P., and I. M. Robinson. 1963. Apparent incor-
poration of ammonia and amino acid carbon during
growth of selected species of ruminal bacteria. J. Dairy
8. Bryant, M. P., and N. Small. 1956. Characteristics of two
APPL. ENVIRON. MICROBIOL.
new genera of anaerobic curbed rods isolated from the
rumen of cattle. J. Bacteriol. 72:22-26.
9. Chaney, A. L., and E. P. Marbach. 1962. Modified re-
agents for determination of urea and ammonia. Clin.
10. Hungate, R. 1966. The rumen and its microbes. Academic
Press, Inc., New York.
11. Lev, M. 1959. The growth-promoting activity of com-
pounds of the vitamin K group and analogues for a rumen
strain of Fusiformis nigrescens.
12. Pittman, K. A., S. Lakshmanan, and M. P. Bryant. 1967.
Oligopeptide uptake by Bacteroides ruminicola. J. Bacte-
13. Scardovi, V. 1963. Studies in rumen microbiology. I. A
succinic acid producing vibrio: main physiological charac-
ters and enzymology of its succinic acid forming system.
Ann. Microbiol. Enzymol. 14:171-187.
14. Schaefer, D. M., C. L. Davis, and M. P. Bryant. 1980.
Ammonia saturation constants for predominant species of
rumen bacteria. J. Dairy Sci. 62:1248-1263.
15. Wozny, M. A., M. P. Bryant, L. V. Holdeman, and W.
Moore. 1977. Urease assay and urease-producing species
of anaerobes in the bovine rumen and human feces. Appl.
Environ. Microbiol. 33:1097-1104.