Archives of
Arch. Microbiol. 120, 61-66 (1979)
Competition for L-Glutamate
between Specialised and Versatile Clostridium Species
H. J. L a a n b r o e k , A. J. Smit, G. Klein N u t e n d , a n d H. V e l d k a m p
Laboratory of Microbiology, Biological Centre, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
Abstract. Clostridium cochlearium c o u l d be r e p r o -
d u c i b l y e n r i c h e d in an L - a s p a r t a t e - a n d L - g l u t a m a t e -
limited, a n a e r o b i c c h e m o s t a t i n o c u l a t e d with a n a e -
r o b i c sludge. L-glutamate, L-glutamine a n d L-histidine
were the o n l y f e r m e n t a b l e substrates. Less specialised
c l o s t r i d i a o f the C. tetanomorphum t y p e c o u l d only be
i s o l a t e d f r o m b a t c h e n r i c h m e n t s with L - g l u t a m a t e a n d
L - a s p a r t a t e as energy sources. C o m p e t i t i o n experi-
m e n t s with C. cochlearium a n d C. tetanomorphum in a
L - g l u t a m a t e - l i m i t e d c h e m o s t a t resulted in the selective
e l i m i n a t i o n o f the l a t t e r species. A d d i t i o n o f glucose to
the m e d i u m resulted in coexistence o f b o t h species. T h e
m o l a r g r o w t h yields for L - g l u t a m a t e a t different di-
l u t i o n rates at 30 ~ C were d e t e r m i n e d f o r b o t h species.
T h e m a x i m u m specific g r o w t h rates o n L - g l u t a m a t e
were 0 . 5 5 h -~ for C. cochlearium a n d 0 . 3 5 h - 1 for
C. tetanomorphum.
Key words: Clostridium cochlearium - Clostridium
tetanomorphum - Glutamate fermentation -
A n a e r o b i c m i x e d cultures.
A s r e p o r t e d p r e v i o u s l y ( L a a n b r o e k et al., 1977) the
result o f a n e n r i c h m e n t e x p e r i m e n t with an a n a e r o b i c
e n e r g y - l i m i t e d c h e m o s t a t in which L - a s p a r t a t e a n d L-
g l u t a m a t e were a p p l i e d as energy sources was the
d o m i n a n c e o f two different b a c t e r i a l species. One o f
these, a Campytobacter spec., f e r m e n t e d L - a s p a r t a t e
a n d its characteristics have been d e s c r i b e d ( L a a n b r o e k
et al., 1977). T h e o t h e r b a c t e r i u m was a Clostridium
species a n d its i d e n t i f i c a t i o n ~411 be p r e s e n t e d in this
p a p e r . Its f e r m e n t a t i v e abilities a p p e a r e d to be re-
stricted to L-glutamate, L-glutamine a n d L-histidine.
The e n r i c h m e n t o f this species in an a n a e r o b i c L-
g l u t a m a t e - l i m i t e d c h e m o s t a t , i n o c u l a t e d with anae-
r o b i c sludge, was r e p r o d u c i b l e . H o w e v e r , b a t c h en-
r i c h m e n t s with L-glutamate, i n o c u l a t e d with sludge
II ialw
9 by Springer-Ver|ag 1979
f r o m the s a m e source, s h o w e d the occurrence o f L-
g l u t a m a t e - f e r m e n t i n g c l o s t r i d i a w h i c h were m o r e ver-
satile with respect to o t h e r f e r m e n t a b l e substrates.
H e n c e c o m p e t i t i o n e x p e r i m e n t s were m a d e to de-
t e r m i n e the ecological niche o f b o t h types o f o r g a n i s m s .
T h e results o f these e x p e r i m e n t s will be d e s c r i b e d b e l o w
a n d c o m p a r a t i v e d a t a o f the energetics o f L - g l u t a m a t e
f e r m e n t a t i o n o f b o t h types o f b a c t e r i a will be presented.
Materials and Methods
Cultures Used
Ctostridium cochlearium and Clostridium tetanomorphum (ATCC
15920) were used in the experiments described. C. cochlearium was
isolated from an anaerobic, L-aspartate and L-glutamate-limited
chemostat inoculated with sludge from an anaerobic digester, which
was fed with amino acid-rich waste water (Laanbroek et al., 1977).
C. tetanomorphum was obtained from Professor G. Gottschalk,
University of G6ttingen, Federal Republic of Germany.
Growth Conditions
The anaerobic growth medium contained the following compounds
per litre of demineralised water: MgSO,- 7H20, 0.5 g; CaC12,0.02 g;
K2HPO4, 0.75 g; NaH2POa*HzO, 0.25 g; cysteine. HCI, 0.5 g;
resazurin, 1 rag; 2 ml trace elements solution of Vishniac and Santer
(1957) in a 5 fold dilution, together with a carbon- and energy source.
The pH was adjusted to 7.1 before autoclaving. The type and
concentration of the carbon- and energy source was dependent upon
the experiment made. Substrate utilization tests were made at 30~ C in
anaerobic agar shake tubes, which contained the mineral medium
plus 0.5 % (w/v) carbon- und energy source, 0.1% (w/v) yeast extract
and 1.0 % (w/v) agar. The carbon- and energy source was omitted in
the blank.
The maximum specific growth rate on L-glutamate was de-
termined in 3 litre conical flasks, which contained 100 ml cell
suspension under a nitrogen atmosphere. Turbidity was measured in
a tube, which had been connected to the conical flask and which also
fitted into a Vitatron colorimeter.
Differential Tests
All differential tests were made according to Holdeman et al. (1977) at
30~ C in 20 ml anaerobic Hungate tubes, which contained 10 ml of
test medium under a gas atmosphere of oxygen-free nitrogen.
0302-8933/79/0120/0061 / $ 01.20
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Enrichment Techniques
Enrichments of L-glutamate-fermenting clostridia were made at
30~ C in both batch and continuous culture; the latter at 4 different
dilution rates, which varied from 0.02-0.20 h -~. Batch culture
enrichments were made in screw-cap bottles completely filled wich
mineral medium supplemented with 0.35 % (w/v) L-aspartate, 0.35
(w/v) L-glutamate and 0.1% (w/v) yeast extract. Continuous culture
enrichments were made in a chemostat described previously
(Laanbroek et al., 1977) and fed with the same medium. Anaerobic
sludge from a digester fed with waste water rich in L-aspartate, L-
glutamate and their amides, was used as inoculum in every case. After
3 or 4 days incubation in batch culture, or after 5 volume changes in
continuous culture, samples from the cultures were pasteurized and
agar shakes were made. These cultures contained the mineral salts
medium supplemented with 0.5 % (w/v) L-aspartate, 0.5 ~s (w/v) L-
glutamate, 0A ~ (w/v) yeast extract and 1.0 % (w/v) agar. After
4 days incubation at 30~ single colonies were tested for growth
on L-aspartate, L-glutamate and glucose.
Cell Yield Studies
Cell yieldstudies of C. cochleariumand C. tetanomorphumwere made
in L-glutamate-limited chemostats. Steady-state cell carbon was
determined as follows: the culture was centrifuged for 20 rain at
10.000 x g in a Sorvall refrigerated superspeed RC2-B centrifuge at
4~ C. The pellet was washed first with 3.2 mM and then with 1.6 mM
phosphate buffer (pH 7.0). Finally the sediment was resuspended in
CO2-free water and the content of organic carbon determined with a
Beckman 915A carbon analyzer, connected to a Beckman 865
infrared analyzer. The molar growth yield was calculated from the
following equation:
Y =x/(SR-s)
where x is the celt carbon determined and Sa-s is the amount of L-
glutamate used,
Competition Experiments
Competition experiments were made in an anaerobic energy-limited
chemostat. The mineral medium was supplemented with 0.35 % (w/v)
L-glutamate and 0.1% (w/v) yeast extract. In one case 0.35 % (w/v)
glucose was also added.
Total cell number was determined from a dilution series of
anaerobic agar shakes in Hungate tubes under oxygen-free nitrogen
at 300 C. The number of C. teta~omorphu~z cells were similarly
determined at 45~ C, since C. eochIeariumwas unable to grow at this
temperature. The agar shake tubes contained the mineral medium
supplemented with 1.0% (w/v) L-glutamate and 0.1% (w/v) yeast
extract. The organic cell carbon was determined as described above.
The percentage number of spores in the culture was estimated by
direct counting.
Chemical Analyses
L-glutamate was determined according to the method of Bernt and
Bergmeyer (1970). Volatile fatty acids, carbon dioxide and ammonia
were estimated as described previously (Laanbroek et al., 1977).
Hydrogen was determined by the method of Laanbroek et al. (1978).
Results
Isolation of Clostridium cochlearium
and Its Differentiation from Clostridium tetanomorphum
I n the first e n r i c h m e n t m a d e ( L a a n b r o e k et al., 1977) a n
a n a e r o b i c c h e m o s t a t was fed with L - a s p a r t a t e a n d L-
Arch. Microbiol., Vol. 120 (1979)
g l u t a m a t e as g r o w t h - l i m i t i n g substrates. T h e L-
g l u t a m a t e - f e r m e n t i n g Clostridium spec. t h a t b e c a m e
d o m i n a n t was i s o l a t e d in p u r e culture b y m a k i n g
successive d i l u t i o n series in a n a e r o b i c a g a r shake
cultures, a p p l y i n g a m i n e r a l m e d i u m with yeast extract,
L - a s p a r t a t e a n d L-glutamate.
T h e characteristics o f this b a c t e r i u m were as fol-
lows. Cells were r o d - s h a p e d (0.8 g m wide a n d 4 . 0 -
10.0 p m long) a n d were m o t i l e b y m e a n s o f p e r i t r i c h o u s
flagella. T h e y were a b l e to f o r m t e r m i n a l o v o i d en-
d o s p o r e s . T h e swollen end o f the s p o r e - b e a r i n g cells
gave t h e m a s p o o n - l i k e a p p e a r a n c e . C o l o n i e s in a g a r
were c r e a m - c o l o u r e d a n d lenticular. T h e b a c t e r i u m was
a strict a n a e r o b e a n d the only f e r m e n t a b l e substrates
were L-glutamate, L-glutamine a n d L-histidine. O t h e r
s u b s t r a t e s tested with a negative result were: glucose,
fructose, p y r u v a t e , L-lactate, citrate, a - o x o g l u t a r a t e ,
succinate, f u m a r a t e , DL-malate, o x a t o a c e t a t e , L-ala-
nine, L-arginine, L-aspartate, L-asparagine, L-cysteine,
glycine, L-isoleucine, DL-leucine, L-lysine, L-methio-
nine, L-phenytalanine, L-proline, L - t r y p t o p h a n , L-tyro-
sine, L-threonine, L-serine, L-valine a n d xanthine.
Little g r o w t h was o b t a i n e d unless y e a s t e x t r a c t was
i n c l u d e d in the m e d i u m . N o further analysis was m a d e
o f the specific g r o w t h f a c t o r requirements. The stoi-
c h i o m e t r y o f L-glutamate f e r m e n t a t i o n was as fol-
lows:
1.00 g l u t a m a t e --, 1.30 acetate + 0.35 butyrate
+ 0.95 CO2 + 0.25 H z + 0.96 N H 3
T h e m a x i m u m specific g r o w t h rate at 3 0 ~ in a
m e d i u m c o n t a i n i n g L-glutamate plus 0.1 ~o y e a s t ex-
t r a c t was 0.55 h -1. T h e characteristics as described
a b o v e are similar to those given for C. cochlearium
(Smith a n d H o b b s , 1974) in Bergey's M a n u a l .
A s will be d e s c r i b e d below, the sludge f r o m which
C. cochtearium was i s o l a t e d also c o n t a i n e d c l o s t r i d i a
which c o u l d in a d d i t i o n to L - g l u t a m a t e ferment glu-
cose. These o r g a n i s m s were p r e s u m a b l y similar o r
identical to C. tetanomotThum, a species which for
u n k n o w n r e a s o n s was n o t i n c l u d e d in the latest edition
o f Bergey's M a n u a l (Smith a n d H o b b s , 1974).
A n t i c i p a t i n g a m o r e detailed s t u d y o f the ecological
niches o f b o t h types o f o r g a n i s m s , a c o m p a r i s o n was
m a d e o f several characteristics o f the newly i s o l a t e d
C. cochlearium a n d C. tetanomorphum ( A T C C 15920)
which is s u m m a r i s e d in T a b l e 1. A l s o i n c l u d e d are the
d a t a o f S p r a y et al. (1957) for b o t h species d e s c r i b e d in
Bergey's M a n u a l (1957). T h e a b i l i t y o f C. tetanomor-
phum to g r o w at 45 ~ C was used to differentiate between
b o t h species in the c o m p e t i t i o n studies to be described
below. C. tetanomorphum h a d a m a x i m u m specific
g r o w t h rate at 30 ~ C in a m e d i u m which c o n t a i n e d L-
g l u t a m a t e plus 0 . 1 % yeast extract o f 0.35 h - 1 .