FEMS MicrobiologyEcology62 (1989) 275-284
Published by Elsevier
275
FEC 00214
Growth of mixed cultures of Actinomyces viscosus and
Streptococcus mutans under dual limitation of glucose and oxygen
H a n s S. v a n der H o e v e n 1 a n d J a n C. G o t t s c h a l 2
I Laboratoryfor Oral Microbiology, Medical Faculty, University ofNijmegen, Nijmegen,
and 2 Laboratoryfor Microbiology, State University Groningen, Haren, The Netherlands
Received 10 October 1988
Revision received 27 November 1988
Accepted 29 November 1988
Key words: Mixed culture; Coexistence; Actinomyces viscosus," Streptococcus mutans,"
Oxygen consumption; Mathematical model
1. S U M M A R Y
Streptococcus mutans and Actinomyces viscosus
are among the dominant species in human dental
plaque. In their natural environment, carbohydrate- and oxygen-limited conditions are likely to
occur frequently. Therefore, mixed cultures of the
2 species were studied under dual limitation of
glucose and oxygen. Over a wide range of
oxygen-supply rates, coexistence of A. viscosus
and S. mutans was observed, within this range A.
viscosus increased almost linearly with oxygen
supply. A mathematical model based on Monodtype kinetics and accounting for uncompetitive
inhibition of growth by oxygen was developed to
simulate these mixed cultures. The model predicted coexistence over a fairly large range of aeration rates. This finding, in combination with the
results of the chemostat experiments, led to the
conclusion that coexistence of the two species
Correspondence to: J.C. Gottschal, Laboratory for Microbiology, State University Groningen, Kerklaan 30, 9751 NN
Haren, The Netherlands.
depends upon a certain degree of growth inhibition by oxygen.
Growth was also studied under more natural
conditions in gnotobiotic rats. In accordance with
the above findings, the relative number of A.
viscosus was found to increase at sites of the
dentition more easily accessible to oxygen, than in
narrow fissures.
2. I N T R O D U C T I O N
The bacterial accumulations on human teeth
are characterized by a predominance of Grampositive aerotolerant cocci and rods. The microflora includes high numbers of different Streptococcus and Actinomyces species. This ecosystem of
the mouth is continuously exposed to saliva and
oxygen. In saliva, carbohydrate is the growth
limiting nutrient implying that competition for the
available sugars is likely to be a major interaction
between the micro-organisms, most of which depend upon carbohydrate for their growth [1]. In
addition oxygen is likely to be an important environmental factor, but its role in the mouth has
0168-6496/89/$03.50 © 1989 Federation of European Microbiological Societies
276
received little attention so far. The oral streptococci may consume some oxygen by partially
oxidizing sugars to acetic acid, carbon dioxide and
water [2,3]. The slight increase in the yield of the
streptocci grown in the presence of oxygen could
be accounted for by the increased production of
acetic acid [4]. Recently, it was found that oxygen
utilization in A. viscosus leads to a shift to fully
aerobic metabolism, including citric-acid cycle activity coupled to electron transport phosphorylation [5]. The high yield of A. viscosus, resulting
from aerobic metabolism [5,6], could be of considerable competitive advantage in their natural
environment [7].
In m a n y regions of the mouth ecosystem conditions are likely to prevail in which both glucose
and oxygen are'present in limiting amounts [1,4].
Therefore, mixed cultures of these two species
were studied under dual limitation of oxygen and
glucose. Over a wide range of rates of aeration,
coexistence of two species may be anticipated if
two or more nutrients are growth limiting [7-10].
However, based on the/~max and K s values of the
two organisms it was to be expected that S. mutans
would outcompete A. viscosus at all combinations
of oxygen and glucose supply. To investigate
whether inhibition by oxygen as an additional
factor could explain this coexistence, a mathematical model was developed to simulate growth in
such mixed cultures.
Simultaneous growth of A. viscosus and S.
mutans was also studied in gnotobiotic rats. These
animals provide the possibility to monitor growth
of these organisms at aerobic and anaerobic sites
of the dentition, in the absence of other microorganisms.
tained 21 amino acids, in total 2.6 g / l , vitamins
nucleotides, inorganic salts, trace elements, and 10
m M of glucose. The medium supports growth of a
variety of oral bacteria including Actinomyces and
Streptococcus species.
3.3. Continuous cultivation
The bacteria were grown at a dilution rate of
D = 0.2 h-1 at 370C in a chemostat (500 ml) as
described before [12]. The p H was kept constant
at 7.0 by the automatic titration of 2 M K O H . The
appropriate gas phase was obtained by mixing
(95% N 2 + 5% C02) with (75% N 2 + 5% CO 2 +
20% 02). The filter-sterilized gas was sparged into
the culture at a rate of 5 1. h 1, with the gas inlet
positioned below the stirrer (constant stirring rate
= 200 revs. min 1).
Mixed cultures of A. viscosus and S. mutans
were obtained by mixing equal volumes of pure
cultures. Runs were performed, at least in duplicate, with step-wise increase of the oxygen tension
in the gas phase. Dissolved oxygen in the culture
liquid was measured continuously with a polarographic sterilizable oxygen electrode (Ingold AG,
Urdorf, Switzerland). Cultures were generally allowed to stabilize for 15 volume changes. Duplicate samples (0.5 ml) were taken daily to estimate
the proportions of the organisms in the culture
and for the determination of organic acids and
ethanol. The purity of the cultures was routinely
checked on aerobically and anaerobically incubated blood agar plates. The identity of isolates
was confirmed using the API 20A and the API 20
Strep (API, Montallieu, France) identification systems.
3. 4. Bacteriological composition o f m i x e d cultures
3. M A T E R I A L S A N D M E T H O D S
3.1. Micro-organisms
A. viscosus Ut2 was originally isolated from
h u m a n dental plaque [11]. S. mutans N C T C 10449
also is a h u m a n isolate. The strains were kept in
skim milk (Difco Labs., Detroit, MI) at - 8 0 o C.
3.2. Media
In all experiments a filter-sterilized chemically
defined medium [12] was used. The medium con-
Samples from the cultures were sonicated for
20 s at 0 ° C using a Kontes K-881440 sonifier
provided with a microtip at maximal output. This
procedure disrupts the streptococcal chains without damaging the cells, to give optimal viable
counts. Suitable dilutions were plated onto blood
agar plates. The plates were incubated for 48 h at
3 7 ° C in an atmosphere of 95% N 2 + 5 % CO 2.
Colonies of A. viscosus and S. mutans can easily
be distinguished and counted separately on blood
agar.
277
3.5. Analytical procedures
For routine purposes, glucose was assayed by
the glucose oxidase test (Boehringer, Mannheim).
To estimate K~ values residual glucose in cultures
growing at half the maximal rate was determined
by a sensitive fluorimetric method [13]. Formic,
acetic, lactic, and succinic acids were assayed using
isotachophoresis [14]. Ethanol was determined by
gas chromatography. Oxygen consumption was
measured polarographically with a biological
oxygen monitor (BOM) using suspensions of
aerobically grown cells [15]. The suspensions containing 1 mg dry weight of cells per ml were made
up in chemically defined medium.
D r y weights were determined in 40-ml samples
taken directly from the culture by syringe. The
samples were centrifuged and washed 3 times in
demineralized water. The cells were dried in crucibles at 105 ° C until constant weight.
The inocula consisted of 18-h cultures of the
organisms in chemically defined medium with 10
m M glucose. Each rat received 0.1 ml of the 1 : 1
mixed cultures, by using a syringe. The rats received a gamma-irradiated powdered diet containing 16% of glucose [16]. Diet and autoclaved tap
water were available ad libitum. 30 days after
inoculation the rats were killed using CO 2. Plaque
was removed from smooth surfaces of the first and
second molars in the lower jaw to give one pooled
sample per rat. Another plaque sample was collected from the fissures in the first and second
molars in the lower jaw. Plaque samples were
dispersed in 0.5 ml of saline (0.85%) by ultrasonication, and plated onto blood agar plates as described above. Colonies of A. uiscosus and S.
mutans were counted separately. The identity of
random isolates was checked with API 20A and
API 20 Strep.
3. 6. A n i m a l experiment
16 germ-free Sprague-Dawley rats, approx. 35
days old, were used to estimate the proportions of
A. viscosus and S. mutans on various sites of the
dentition. The rats were kept in a plastic isolator
in cages with wire mesh bottom. The animals were
inoculated with a combination of A. viscosus Ut 2
and S. mutans N C T C 10449 on 2 successive days.
3. 7. A mathematical model of competition
A model was constructed to describe the growth
of mixed chemostat cultures of A. viscosus and S.
mutans with glucose a n d / o r oxygen as the only
growth limiting nutrients. For the description of
the specific growth rates modified Monod-type
growth kinetics were assumed including a simple
expression to account for uncompetitive inhibition
0.7 -A
Streptococcus
0.7 "B
0.6
Act inomyces
08
i
~" 0.5
~ O.E
.
0.4
~ 0z
o 03
p o.:
g 02 i ,
~, 02
0.1
o
\
~
0.1
i
~
~
~
: 8
Oxygen concentrotion (pl'A)
~
totol
~ i i i
~ilii
erobtc
IX\.
0'
o
e,
2
4
&
8
Oxygen concentration (pM)
q,
1o
Fig. ]. Relationship between specific growth rates ( h - ] ) and concentration of dissolved oxygen (@M) assuming the @m~, K~ and K i
values presented in Table 1. A S. mutans, O, anaerobic growth rate. B. A. viscose, +, total growth rate; 0, anaerobic growth rate; II,
aerobic growth rate.
278
Table 1
List of symbols used in the model with their standard preset values
Symbol
Units
Description
Value
D
/z
Sr
s
[Oz]
[O2]sa t
x
R
(h - ] )
(h - ] )
(mM)
(mM)
(mM)
(mM)
(mg-1-1)
Dilution rate
Specific growth rate
Substrate concentration in the reservoir medium
Substrate concentration in the culture
Oxygen concentration in the culture liquid
Idem at air saturation (37 ° C)
Biomass concentration in the culture
Rate of aeration (including oxygen transfer efficiency
impeller speed etc.; arbitrary units)
0.2
variable
10
variable
variable
0.215
variable
variable
p a¢,
p,. . . . .
/z~ x
/~Z r
(h-l)
(h - 1)
(h - l )
(h 1)
Ks(gluc )
Ks(O2)
Ki.. (O2)
K i..... (02)
Ya~r(gluc)
Y a . . . . (gluc)
[02 ]/ OXmax
(,aM)
(/~M)
(,uM)
(/LM)
(g-mol 1)
(g-mol 1)
Specific aerobic growth rate (oxygen dependent)
Specific anaerobic growth rate (oxygen independent)
Maximum specific aerobic growth rate
Maximum specific anaerobic growth rate
Substrate saturation constant for glucose
Substrate saturation constant for oxygen
Inhibition constant for oxygen (aerobic growth)
Inhibition constant for oxygen (anaerobic growth)
Growth yield for aerobic growth with glucose
Growth yield for anaerobic growth with glucose
Stoichiometry of glucose oxidation as a function
of oxygen concentration
by oxygen. Since the observed maximum specific
growth rates and the growth-yields of both species
were quite different under anaerobic and aerobic
conditions the resulting actual rate of growth and
substrate consumption in the model were assumed
to be composed of an aerobic and an anaerobic
component. The aerobic part was described by
including both oxygen dependence and oxygen
inhibition. Appropriate choice of the value of the
oxygen inhibition constants allowed good description of very oxygen sensitive anaerobic growth
( = fermentation) and oxygen dependent, fairly
oxygen-insensitive aerobic growth (Fig. 1).
Description of the population densities and
substrate concentrations was based on the standard differential equations describing growth in
continuous culture. For an explanation of the
symbols see Table 1.
I. d x /
II.
ds/
dt = ~x-
Dx
dt = DS r - Ds - I~x/Y
The anaerobic and the aerobic component of the
specific growth rate were formulated according to
Actinomyces
variable
variable
0.64
0.51
15
1
1000
0.2
150
62.5
0-6
Streptococcus
variable
variable
0.0
0.64
5
6
1 000
0.2
45
-
modified Monod-kinetics:
Ill. /~.....
=
t~mna~r" s / ( K s + S"
(1 + [O2] Ki(O2)))
I V . /xaer = ~ T r x " s / ( K s + S" (1 + [ O 2 1 / K i ( O 2 )
•[02]/([02]
)
+ Ks(O2)))
The resulting total specific growth rate was described as:
V . ~total = ~anaer _{_ ~taer
The change in dissolved oxygen concentration in
the culture liqui d was described as follows:
VI. d[O2J dt = (Uptake by the culture)
-
-
(Outflow by dilution)
(Consumption by biomass)
with:
(Uptake
by t h e culture) = ( ( [ 0 2 ]sat -- [ 0 2 ] ) " R )
(Outflow by dilution) = ( D . [02] )
(Consumption by biomass)
= (~aer X/Yaer) . ([O2]/OXmax)
279
For the numerical solution of the differential
equations the straight-forward method of Euler
was used. To overcome computational problems
due to overshooting the zero values of the substrate and oxygen concentrations (resulting in
'negative concentrations') automatic adjustment
of the step size of the time intervals was built in
the program. The criterium for lowering the step
size involved checking the fraction of the residual
nutrient concentration consumed by the biomass
and its subsequent comparison with an adjustable
preset maximum value. This procedure in combination with automatically repeated attempts to
double the step size to an adjustable preset maximum value allowed automatic computational
speed optimization.
The program was written in Turbo Pascal 3.02
supporting the 8087 mathematical coprocessor for
IBM (compatible) personal computers. The program supported both numerical and graphical representation of the output.
oxygen saturation constants thus obtained were
assumed to represent the true Ks(O2) values for
growth [17]. Using glucose as substrate, Ks(O2) of
A. viscosus was 1 /~M, while the high affinity
oxygen uptake system of S. mutans had a K s
value of 6 /LM. This value was in accordance with
earlier published data [3].
Yields (g per mol of glucose) were obtained
from earlier work [4,5].
K i values for oxygen were chosen in accordance with the sensitivities of the organisms for
oxygen. For S. mutans, strong inhibition has been
observed in glucose-limited chemostat cultures
when the concentration of dissolved oxygen rose
above 0.4/~M. A K i value of 0.2/~M was used for
S. mutans. The same standard preset value was
used for inhibition of anaerobic metabolism of A.
viscosus. Aerobic metabolism of A. viscosus was
supposed to be uninhibited by oxygen (K~ = 1.0
mM).
3.8. Account of parameter values
4. RESULTS
Anaerobic /~m~x values were measured under
anaerobic conditions in the chemostat using washout kinetics. The values for S. mutans and A.
viscosus were 0.64 h -a and 0.51 h -1, respectively.
Aerobic ~max of A• viscosus (0.64 h - 1 ) was
estimated in an aerated batch culture. S. mutans is
obligately fermentative, and therefore its aerobic
growth rate was assumed to be zero. As a consequence, the calculated oxygen consumption by
this organism becomes zero, because in the model
oxygen consumption is related linearly to the
aerobic growth rate (see equation IV). This is an
acceptable simplification as the maximal rate of
oxygen consumption by S. mutans was found to
be small compared to that by A. viscosus (21
n m o l . m i n - a. m g . dr.wt. - x and 180 nmol- m i n - 1
• mg- dr.wt.- 1, respectively).
K s values for glucose were approached by measuring steady state glucose concentrations at half
maximal growth rate under anaerobic conditions.
K s values of 5 # M and 15/~M were obtained for
S. mutans and A. viscosus, respectively.
K s values for oxygen were estimated from progress curves obtained by measuring oxygen uptake
by cell suspensions in the BOM (see above)• The
4.1. Growth in m i x e d cultures
Anaerobic chemostat-grown pure cultures of A.
viscosus UT2 and S. mutans N C T C 10449 were
mixed in a ratio of 1 : 1 and grown anaerobically
under glucose limitation at a dilution rate of 0.2
h a. Under these conditions S. mutans became
dominant and reached a density of 0.45 mg dry
weight per ml, equivalent to 4 × 109 cells per ml
with A. viscosus persisting at a low level of approximately 10 6 cells per ml. In subsequent experiments various amounts of oxygen were included in
the gas mixture used to flush the culture. In Fig. 2
the densities of A. viscosus and S. mutans in
mixed cultures are given as a function of the
percentage of oxygen in the gas. The densities
were calculated from the viable counts (CFU) of
the 2 organisms in the mixed culture. To this end,
the relationship between C F U and dry weights
were estimated using pure chemostat cultures. It
can be seen that the density of A. viscosus rose
with the increasing supply of oxygen. Total dry
weight of the mixed culture also increased with the
oxygen concentration in the gas. Glucose was
always below the detection level (50 /xM) of the
280
g l u c o s e - o x i d a s e test. T h e c o n c e n t r a t i o n of dissolved o x y g e n in the culture was b e l o w 0.22 /~M
(20 Pa). E l e v a t i o n of either the glucose c o n c e n t r a tion in the m e d i u m ( d a t a n o t shown) or the o x y g e n
c o n c e n t r a t i o n in the g a s - p h a s e resulted in an increase of the cell-density, i n d i c a t i n g that the m i x e d
c u l t u r e grew u n d e r d u a l l i m i t a t i o n of glucose a n d
oxygen. All o t h e r n u t r i e n t s were p r e s e n t in excess
a n d no i n h i b i t o r y s u b s t a n c e s were p r o d u c e d . This
was c o n c l u d e d f r o m the o b s e r v a t i o n that supern a t a n t fluids of p u r e cultures of each of the strains,
s u p p l e m e n t e d with glucose to the original level,
s u p p o r t e d g r o w t h of the c o m p e t i n g o r g a n i s m at
an equal rate, a n d to the s a m e d e n s i t y as o b t a i n e d
in fresh m e d i u m . It was also s h o w n in p u r e cultures of A. oiscosus that g r o w t h at the e x p e n s e of
f e r m e n t a t i o n p r o d u c t s f r o m S. m u t a n s (formic
acid, acetic acid a n d ethanol, see [4]) d i d n o t
occur. This e x c l u d e d the p o s s i b i l i t y of coexistence
as a result o f cross-feeding. M o r e o v e r , in cell
s u s p e n s i o n s of A. viscosus these p r o d u c t s d i d not
s u p p o r t m e a s u r a b l e r e s p i r a t o r y activity.
S u p p l y o f gas c o n t a i n i n g m o r e than 16% ( v / v )
of o x y g e n failed to give stable m i x e d cultures of
the 2 organisms. B e y o n d this level of oxygen, S.
m u t a n s always w a s h e d o u t from the culture, suggesting s t r o n g i n h i b i t i o n of the o r g a n i s m b y
oxygen.
total
$00
7OO
6QQ
Table 2
Degradation products (mM) from glucose in mixed cultures of
S. mutans NTCC 10449 and ,4. viscosus UT2 at different
oxygen concentrations in the gas-phase
% 02
in gas
formic
acid
acetic
acid
lactic
acid
succinic
acid
ethanol
0
0.3
0.7
1.5
3.0
6.0
9.0
11.1
15.6
16.6
16.3
15.5
16.4
14.6
14.9
12.4
9.1
9.1
10.0
12.9
14.4
15.6
17.1
18.0
18.2
11.7
9.0
0.5
0.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0"
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
7.5
5.7
4.4
4.3
1.7
0.0
0.0
0.0
0.0
* detection limit is 0.05 mM.
4.2. Degradation products f r o m glucose
T h e c o n c e n t r a t i o n s of formic, acetic, lactic, succinic acid a n d e t h a n o l in m i x e d cultures g r o w n in
the presence of various o x y g e n c o n c e n t r a t i o n s in
the gas p h a s e are given in T a b l e 2. T h e f e r m e n t a tion p r o d u c t s in the a n a e r o b i c culture are representative of a p u r e culture of S. m u t a n s [4]. A t
increasing levels of o x y g e n in the gas, acetate
i n c r e a s e d at the e x p e n s e of ethanol. B e y o n d 6%
o x y g e n in the gas mixture, S. m u t a n s s t o p p e d
p r o d u c i n g ethanol. T h e i n c r e a s i n g d e n s i t y of A.
viscosus in the m i x e d culture w h e n m o r e o x y g e n
was supplied, d i d n o t result in the p r o d u c t i o n of
a n y s u b s t a n t i a l a m o u n t of succinic acid, a typical
e n d - p r o d u c t of a n a e r o b i c glucose d e g r a d a t i o n b y
this organism.
o~ 5 O C
E
~, 4 0 (
4.3. E s t a b l i s h m e n t o f A. viscosus a n d S. m u t a n s in
gnotobiotic rats
T h e recovery of A. viscosus U T 2 a n d S. m u t a n s
~; 3 0 0
200
0
10C
O
2
4
6
8
10
°/o Oxygen in gas phase
12
14
lb6
Fig. 2. Total cell dry weight, and dry weights of S. m u t a n s
NCTC 10449 and A. viscosus UT2 (calculated from cel num-
bers, see MATERIALSAND METHODS) in mixed chemostat cultures at steady state growing at a dilution rate of 0.2 h 1 under
varying supply of oxygen in the gas phase. II, total dry weight;
Q, S. mutans; A, A. oiscosus.
N C T C 10449 from s a m p l e s of d e n t a l p l a q u e is
p r e s e n t e d in T a b l e 3. T h e relative n u m b e r s of A.
viscosus a p p e a r e d to be higher in p l a q u e from
s m o o t h surfaces of the d e n t i t i o n t h a n f r o m fissures. It is n o t e d that fissure p l a q u e s c o n t a i n e d a
higher a b s o l u t e n u m b e r of b a c t e r i a t h a n s m o o t h
surface plaques. This c o r r o b o r a t e d the o b s e r v a t i o n
that m u c h less p l a q u e was p r e s e n t on s m o o t h
surfaces t h a n in fissures.
281
Table 3
Occurrence of A. viscosus UT2 and S. m u t a n s NCTC 10449 on
various sites of the dentition in gnotobiotic rats
.4. viscosus
S. m u t a n s
dentinal
fissures
smooth
surfaces
C F U x 107 * *
%*
CFU X 107 * *
%*
13.5 (1.2)
82.8
1.9 (0.3)
17.2
0.7 (0.2)
49.9
0.7 (0.2)
50.1
* Percentage of total CFU.
* * Mean of 16 samples with standard error within brackets.
4. 4. Mathematical model o f competition
A crucial part of the model is the appropriate
description of the specific growth rate of the two
species A. viscosus and S. mutans. This was
accomplished by separating the actual if-description into an aerobic and anaerobic component
and by including a term for inhibition by oxygen.
In Fig. 1 the values for ~ta~r, ilJ,anaer and ~tt°tal h a v e
been plotted versus an increasing oxygen concentration for the choice of parameter values
shown in Table 1. These results clearly show that
A. viscosus maintains a relatively high specific
growth rate at all oxygen concentrations tested
SOOF !/o~
L P
,~1o7
&
"°°t~'""/, ......... ......//..
[ \/"'..
/
3ooL \/
",, /
2 L/\ ,
U
/
1OO
0~"
O
,
1
~2o) .
...................-'""~
",,
...-""
~
s ...........
whereas S. mutans exhibits a progressively declining specific growth rate for the same range of
oxygen values.
Using the parameter values as indicated in
Table 1, but assuming that no inhibition by oxygen
occurs (all K i values ~ l m M ) , the model predicts
that coexistence of A. viscosus and S. mutans is
not possible. Both under strictly anaerobic conditions and at various rates of aeration (R = 0-5),
S. mutans outcompetes A. viscosus completely.
Different values for the dilution rate ( D = 0.01-0.4
h - 1 ) did not affect this result. However, including
an inhibition term in the description of ~anaer of S.
mutans and of A. viscosus, resulted in coexistence
of the two species over a wide range of rates of
aeration. The span of this range depended primarily on the ratio of the inhibition constants of
S. mutans and A. oiscosus ( g i ( S . m u t a n s ) / K i
( A . viscosus); Fig. 3). This is true not only for K i
values used in the present calculations (Fig. 3), but
also for higher values. However, when simulating
stronger inhibition (lower K i values) by oxygen,
the absolute value of K i increasingly affected the
composition of the mixed culture. For example, at
a 10-times lower g i value of 0.02 /zM, coexistence
could no longer be obtained.
Of course, a change of dilution rate also affects
the range of aeration-rates leading to coexistence
since such a change affects the availability of
oxygen to the culture through the change in the
availability of oxidizable substrate per unit of
time.
.......
..'"
..........
5. D I S C U S S I O N
",,
This study describes the influence of oxygen on
mixed cultures of S. mutans N C T C 10449 and A.
viscosus UT2. The cultures were glucose limited
and it appeared that in the presence of low concentrations of oxygen the 2 strains could coexist.
Since the dry weight increased with the oxygen
supply, it can be concluded that, in addition to
glucose, oxygen was also limiting, Recently, it was
demonstrated that oxygen consumption by A.
viscosus [5] strongly stimulated the production of
biomass, whereas biomass formation by S. mutans
was only slightly affected by oxygen [4]. The ques-
",,,
~
2
Rate o~ aeration
"]'"",
3
,
4
Fig. 3. Cell dry weights of S. mutans NCTC 10449 and A.
viscosus UT2 in mixed chemostat cultures as predicted by the
mathematical model as a function of the extent of aeration, R
(see also Table 1). Different values for g i of .4. viscosus were
used leaving the standard preset value for K i of S. m u t a n s
unchanged to give ratios of K i (S. r a u t a n s ) / K i (A. viscosus) =
0.5, 1.0, and 2.0. Lines designated A. represent the cell dry
weight of A. viscosus, and those designated S. represent cell dry
weight of S. mutans.
282
tion is raised whether coexistence of the strains
could be explained, according to current theories
[7-10], by either the presence of two growth-limiting substrates or by the occurrence of cross-feeding through fermentation products. This latter
possibility can be excluded, as in pure cultures of
A. viscosus no growth at the expense of fermentation products from S. mutans could be observed.
Under anaerobic conditions S. mutans outcompeted A. viscosus due to its high affinity for
glucose i.e. its low K s and high ~max relative to A.
viscosus. It is tempting to assume that in the
presence of oxygen as a second limiting nutrient
the good aerobic growth of A. viscosus would
make coexistence possible. Yet, preliminary analysis of the growth parameters of the competing
species casts some doubt on the validity of the
above hypothesis. Assuming M o n o d - t y p e of
growth kinetics, /~-S curves constructed from /Lmax
and K S values revealed that at a growth rate of
0.2 h -1, S. mutans would become dominant
anaerobically as well as aerobically (Fig. 4). This
would mean that for coexistence to occur other
mechanisms must operate in addition to mixed
substrate utilization. It is logical to assume that
inhibition by oxygen could represent such a mechanism as cultures of S. mutans in particular were
found to be sensitive to dissolved oxygen [4].
To find support for this assumption a mathematical model was developed, based on simple
07
1
06
£
o5
3
La 0.4
~ 02
0.1
0
0
~0 40 do gO 16o ~bo 1~,0 1~9 1gO 200
Glucose (pM)
Fig. 4. Theoretical /~-S curves of S. m u t a n s N C T C 10449 a n d
A. viscosus U T 2 w i t h glucose as g r o w t h - l i m i t i n g substrate. 1. S.
m u t a n s (anaerobically), 2. A. oiscosus (aerobically), 3. A.
viscosus (anaerobically).
Monod-type kinetics, to describe the growth of S.
mutans and A. viscosus in mixed chemostat cul-
ture. In order to account for oxygen dependent
growth (ffaer) at very low oxygen concentration the
specific growth rate was assumed to follow saturation kinetics for both oxygen and the carbon
substrate (eq. IV) in much the same way as described earlier [18,19]. The additional feature of
inhibition by oxygen was obtained by including an
inhibition constant (Ki; eqs. III and IV). These
features together with the separation of the specific
growth rate in an aerobic and an anaerobic component make the model extremely versatile. It can
thus be used for modelling mixed cultures of
m a n y different types of bacteria such as strict
anaerobes, facultative anaerobes, aerotolerant and
microaerophilic species and strict (oxygen sensitive?) aerobes.
When using the model to predict the outcome
of the competition between A. viscosus and S.
mutans for growth limiting substrate(s) all but one
set of organism-specific growth-parameters were
known. Only the values of the inhibition constants
were chosen freely in combination with various
rates of aeration to find conditions allowing
coexistence of both species. The thus chosen combinations are by no means exhaustive, but only
serve to stress the important finding that coexistence is predicted over a fairly large range of rates
of aeration. This finding, in combination with the
chemostat experiments, also indicating coexistence
over a range of aeration-rates, lends support to the
conclusion that coexistence of these two species is
possible due to a certain degree of inhibition of
the anaerobic growth of both species.
The competition between S. mutans and A.
viscosus was also analyzed under more natural
conditions in gnotobiotic rats. Plaque analyses
revealed a preference of .4. viscosus for smooth
surfaces of the dentition, while S. mutans was
dominant in fissures. Smooth surface areas are
considered to be far more accessible to oxygen
than the narrow fissures of the teeth. A similar
distribution of these species is found in the human
mouth [20] and in rats harbouring a complex
microflora [21], suggesting that the gnotobiotic rat
is a relevant model to study the interaction between S. rnutans and A. oiscosus. In view of the
283
p r e s e n t e x p e r i m e n t s , it is t e m p t i n g t o a s s u m e t h a t
o x y g e n plays a m a j o r role in g o v e r n i n g the p o p u l a t i o n s o f A . viscosus o n v a r i o u s sites o f t h e d e n t i tion.
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