FEMS Microbiology Ecology 31 (1985) 249-254
Published by Elsevier
249
FEC 00031
The use of compound continuous flow diffusion chemostats to
study the interaction between nitrifying and nitratereducing bacteria
(Nitrate respiration; nitrification; interactions; continuous culture)
G.T. Macfarlane * and R.A. H e r b e r t
Department of Btologtcal Sciences. The Unwerst(v. Dundee DDI 4HN. U K.
Received 7 May 1985
Revision received 8 July 1985
Accepted 11 July 1985
1. S U M M A R Y
The interactions occuring between populations
of a nitrate-respiring Vibrio sp. and autotrophic
nitrifying bacteria belonging to the genera Nitrosomonas and Nitrobacter have been investigated in
a compound bi-directional flow diffusion chemostat at a dilution rate of 0.025 h - 1 and a temperature of 25°C. When grown under N O 3 limitation,
the Vibrlo sp. produced NH~- as the principal
end-product of nitrate respiration, and there was a
corresponding significant increase in cell numbers
of the Nitrosomonas sp. population, which derived
energy by the oxidation of N H J to NO_,-. Nitrite
in turn was used by the Nitrobacter sp. population
as an energy source with the concomitant regeneration of N O ~ . Under NO~ excess growth conditions the Vibrto sp. produced N O Z rather than
NH~- as the major product of N O 7 dissimilation,
and growth of the Nitrobacter population was
stimulated as increased quantities of NO~ became
available. In contrast, the Nitrosomonas sp. popul* Present address: Dunn Clinical Nutrition Centre, Addenbrookes Hospital, Trumpington Street, Cambridge CB2
tQG, U.K.
ation declined sharply as the energy source NH~became limiting. These data demonstrate that defined mixed populations of obligately aerobic
nitrifying bacteria and facultatively anaerobic
nitrate respiring bacteria can co-exist for extended
time periods and operate an internal nitrogen cycle
which is energetically beneficial to both populations.
2. I N T R O D U C T I O N
Most natural habitats are heterogeneous and
contain a diverse range of metabolically distinct
groups of microorganisms, often spatially organised in response to a variety of physical and
chemical gradients. The results is a heterogeneous
microbial ecosystem in which diffusion is the
primary mechanism of solute transfer [1,2].
Estuarine sediments, particularly the surface layers
(0-50 m m depth) are regions of intense microbial
activity [3-5]. The vertical distribution of bacteria
within these sediments is to a large degree governed
by the presence and absence of oxygen and the
distribution of individual physiological types follows a profile similar to the physico-chemical
0168-6496/85/$03.30 © 1985 Federation of European Microbiological Societies
25O
t~ves of th~s study were to mvesugate the relatum~hlps betv, een a mtrate-respmng l~9brto sp. and
mtnfying bacteria belonging to the genera NttrosotHotl~ts and Nttrobacter m relatton to the generation of inorganic nitrogen intermediates.
gradJents which develop [6.7]. The minerahsatum
of organic detritus ill estuarine and coastal marine
sedunents is a complex process me&ated by a
.succession of respiratory processes of which those
involving O,, NO~ . SO4 and CO, are quantttat~vely the most important.
Autotrophic nitrifying bacteria, which generate
energy b~ the oxidation of NH 4 and NO~ are
obligate aerobes and the surface layers of estuarine
sediments, where O, and NH 4 are snnultaneously
present, are ideal envtronments for mtrification to
occur. In contrast, mtrate respiration performed
by facultatlvely anaerobic heterotrophic bacteria is
favoured bv the absence of (A and the presence of
NO~ and oxi&sable organic matter. The series of
morgan,c nitrogen intermediates revolved in the
aerobic oxidation of NH~ to NO~ by mtnfylng
bacteria is the reverse of those generated during
the anaerobic &ssimilat]on of NO~ to NH 4 by
nitrate resp,ring bacteria (Fig. 1). A number of
recent field studies ha,; demonstrated unequivocally that mtrification and nitrate respiration occur simultaneously in manne and estuanne sediments, but the nature of the interactions involved
between these physiologically distinct groups of
bacteria is poorly understood [8-10]. The objec-
NO3(
NO20xldlslng
bacteria
_
3. M A T E R I A L S A N D M E T H O D S
3.1. Or,gants,ls
Nttrosomonas N3, Nttrobacter Nit 1 and Vthrto
V48 were tsolated from Kingoodie Bay sediments
m the Tay estuary as described by Macfarlane and
Herbert [11,12].
3.2. Enumeration o/ mu'roblal populanons
('ell population densitms were determined b~y
direct counting methods using a Neubauer counting chamber [13].
3.3. Analysts of spent rnedta
Spent media were analysed for NO:~ , N O 2 and
NH 4 after filtration through 0.22 /*m Millipore
filter, as described by MacFarlane and Herbert
[11].
SO2~
+
NH 4 Oxidlslng
bacterl~
4
Aerobic Zone
Anaerobic Zone
,NH 4
I
Nitrate reduclng
bacteria e.g. Vibrio spp.
Denitrifying bacterla e.g. Pseudomona8 spp.
Fig. 1 R e l a t i o n s h i p b e t w e e n n i t r a t e r e s p i r a t i o n a n d n i t r i f i c a t i o n in e M u a r l n e ~ e d u n e n t s
N2
I
251
3.4. Growth medium for defined mixed population
studies
The basal mineral salts growth medium for each
chamber had the following composition (g. 1 1):
N a H C O 3, 0.5; MgCI 2 • 6H20, 0.1; K 2 H P O 4, 8.0;
K H z P O 4, 2.4; Na2SO 4, 0.5; FeSO4-7H20, 0.03;
COC12.6H20, 0.02; pH 7.6. In addition, the
growth medium in chamber 1 was supplemented
with either 50 mM glycerol and 5 mM KNO3
(N-limitation); or 25 mM glycerol and 10 mM
KNO3 (C-limitation).
3.5. Effect of nitrogen availability on the growth of
defmed mixed populations
A 3-chamber continuous flow diffusion chemostat, as described by Keith and Herbert [14] was
used to study the effect of nitrogen availability on
the populations of Nttrosomonas N3, Nitrobacter
Nit 1 and Vibrio V48 (Fig. 2). To avoid possible
problems due to the leaching out of inhibitory
compounds, the butyl rubber membrane sealing
gaskets were replaced with ones fabricated from
silicone rubber. Vibno V48 was inoculated into
chamber 1, the only vessel to receive an input of
organic carbon and inorganic nitrogen (KNOB).
Vibrio V48 was allowed to grow for 24 h prior to
inoculating chamber 2 with Nttrosomonas N3.
After a further 24 h, chamber 3 was inoculated
with Nttrobacter Nit 1. Growth chamber 1 was
sparged continuously with oxygen-free nitrogen (1
1 • m i n - 1) to maintain anaerobic conditions for the
growth of Vibrio V48. Chambers 2 and 3 were
sparged with sterile air (1 1-min 1) to ensure
aerobic conditions for the growth of nitrifying
Fig. 2. Bi-directional flow chemostat in operation. Chamber 1 (left) contains VtbrzoV48, chamber 2 (centre) contains Nttrosomonas
N3 and chamber 3 (right) NttrobacterNit 1
(a)
3"5"
-2 "5
N-lzmit
C-limit
3.0.
.2"0
0 / o
o0~
2- 5-
-1-5
o/
Z
o/
E
20-
__o/
/'-
'~.~_~./'~"
1"5t/
I------~ I ~
2
0
(b)
0 .....~
0
M
..:
°~o~
/
7
4
e
•
~o
~:j..
./~
-~.___
,---
/'/
I ~
•
8
1o
,'2
,4
Daya
,~
2'2
2~,
2~
~$
N-]imit
C-limit
•2 "0
30-
/
-=
c-"
_
0-5
2b
i~
1-0
2-5
/
'
0
I
o
\
i"
-1-5
',
Z
I
,
,
k
-1"0
C~
2.0-
/ o
.
1-5"1
0
/
o
O-
o/
o....
,
~
,
/ o
2
4
6
El
8
10
12
14
Days
_ /
~
16
~
0 ~ 0
i
mm~U~gll"~
I
"
18
.05
20
22
24
26
28
--
253
(c)
3-0.
-2.5
C-limit
N-I i m i t
-2-0
2"5'
~"o
2"0
.~
-1.5
/
N
Z
•1.0
1.5
o------o~
o /
o~
I
I
1.01 ~
•
~
I
=---- • ~
',
..~. II
_
=~
• /
•
• ~
~
,i
~
~
lb
I
1'2
14
1'6
l'e
2b
2'2
{
LO.5
iu ...-.--. Ii.......__
o
=
,
24
, ~
2e
20
0
Days
Fig. 3. C h a n g e s in cell n u m b e r s of (a) Yihr,~ V48. (b) Nitrosomonas N3 and {c) Nttrohacter Nit 1 when gt-own m c o n t m u o u s culture
on KNO~ as N-source under either C or N - l i m i t a t i o n at 25°C. D = 0.025 h - ~. (3, cell numbers: •
• . nitrate concentration:
•
• , NO_, c o n c e n t r a t i o n : and •
0, N H ~ c o n c e n t r a t i o n
bacteria. The cultures were grown at a dilution
rate of 0.025 h -1 and temperature control was
achieved by placing the experimental system in a
constant-temperature room at 25°C. To avoid
photo-inhibition of the nitrifying bacteria chambers 2 and 3 were covered with aluminium foil.
Samples (5 ml) weri~ aseptically removed from
each chamber at 24-h intervals for determination
of cell numbers and spent media analysis.
4. RESULTS AND DISCUSSION
4.1. Growth of defined mixed populations
The data in Fig. 3 show the effect of N-availability on the population dynamics of 3 microorganisms. Under N-limiting conditions, Vtbrio
V48 produced NH~- as the principal end-product
of nitrate respiration, which in turn provided an
inorganic energy source for the NH~'-oxidising
bacterium, Nitrosomonas N3. As a consequence,
there was a marked stimulation of the Nitrosomonas N3 population under these growth conditions (Fig. 3b). Nitrite, the end-product of NH~oxidation, was in turn utilised as an energy source
by the NO2--oxidising bacterium Nitrobacter Nit
1. However, NH~ oxidation by Nitrosomonas N3
was the rate-limiting process, and insufficient NO~was generated under N-limiting conditions to
stimulate the growth of the Nitrobacter Nit 1
population (Fig. 3c).
Upon changing to C-limiting (NO~-excess)
growth conditions, marked changes in the 3 microbial populations occurred. Under these growth
conditions, l/ibrio V48 produced NO2- rather than
NH~ as the principal end-product of NO 3 respi-
254
ration, and this resulted in a sharp decrease in the
Nitrosomonas N3 population as the NH~- concentration declined (Fig. 3b). As a consequence of
these elevated N O £ concentrations, the Nttrobacter Nit 1 population was stimulated due to the
increased availability of an oxidisable energy
source. These data demonstrate unequivocally that
the 3 microbial populations are able to co-exist for
extended time periods in the experimental system
without washing out. The end-products of nitrate
respiration produced by Vibrio V48 are dependent
upon the availabihty of N O r . Under N O 3 limitation the Vibrto V48 population is essentially e - acceptor limited, and N O r is reduced through to
N H 4 mediated by dissimilatory nitrate and nitrite
reductases, whereas under N O ~ excess growth
conditions excess e -acceptor capacity is available
and reduction only proceeds as far as NO2- [15].
The net effect is that under N O ~ limitation the
N H 4 oxidiser population is stimulated, whilst the
converse occurs under N O f excess conditions with
stimulation of the N O 2- oxidiser population. These
data validate the results of field investigation which
has demonstrated the simultaneous occurrence of
nitrification and nitrate respiration [8-10] and
provide experimental evidence to substantiate the
hypothesis that an internal nitrogen cycle may
operate, in which bacteria respiring N O 3 to N O ~
and NH~- generate a source of inorganic energy
for autotrophic nitrifying bacteria. In turn, the
nitrifying bacteria regenerate the e - acceptor for
the nitrate-respiring bacteria. The operation of
such an internal cycle is clearly energetically advantageous to both groups of bacteria.
ACKNOWLEDGEMENT
This work was financed by Grant G T / 3 / 4 2 0 8
from the Natural Environment Research Council.
REFERENCES
[1] Bazm, M.T., Saunders, P T . and Prosser, J 1 (1976) C R C
Crlt. Rev. MJcroblol 4, 463-469
[2] Wimpenny, J.W.T., Lovttt, R.W. and Coombs, J.P. (19831
m Microbes in their Natural Environments (Slater, J H ,
Whlttenbury, R and Wlmpenny, J W T., Eds ) pp. 66 117
Cambridge Umverslty Press, Cambridge
[3] Wood, E.J.F (19651 Marine Microbaal Ecology, Chapman
and Hall, London.
[4] Jorgensen, B.B (1980) m Contemporary Microbial Ecology (Ellwood, D.C., Hedger, J . N , Latham, M J., Lynch.
J M and Slater, J H., Eds ). Academic Press, London.
[5] Nedwell, D.B. (1982) m Sediment Microbiology (Nedwell,
D.B. and Brown, C M., Eds ), pp 73-106, Academic
Press, London.
[6] Mechalas, B.J. (1974) in Natural Gases m Marine Sediments (Kaplan, I.R., Ed.) pp. 12-25. Plenum, New York.
[7] Blllen, G. (19821 in Sediment Microbiology (Nedwell, D.B
and Brown, C.M., Eds.), pp 15-52 AcademLc Press,
London.
[8] KoJke, l. and Hatton, A (19781Appl Envlron. Microblol
35, 853-857.
[9] Sorensen, J (19841 m Current Perspectives in MJcrohal
Ecology (Klug. M.J. and Reddy. A C , Eds.I, pp. 447-453
American Sooety for Microbiology Pubhshmg. WashmgIon
110] Macfarlane, G.T and Herbert, R A (19841J Gen Microbiol. 130, 2301-2308
[11] Macfarlane. G T and Herbert. R A (19841 FEMS Microbiol. Lett, 23, 107-111
[12] Macfarlane. G.T. and Herbert, R A. (19821J Gen Microblol 128, 2463-2468
[13] Crulckshank. R (19651 Medical Microbiology, pp
886-889. Livingstone, Edinburgh.
[14] Koth, S.M. and Herbert, R A (1985) FEMS MJcroblol.
Ecol. 31, 239-247.
[15] Herbert, R A. (19821 m Sediment Microbiology (Nedwell,
D.B. and Brown, P.M., Eds.) pp. 53-72 Academic Press,
London