University of Groningen
Growth of bacteria at low oxygen concentrations
Gerritse, Jan
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Chapter 2
Mixed chemostat cultures of obligately aerobic and fermentative
or methanogenic bacteria under oxygen-limiting conditions
Jan Gerritse, Frits Schut and Jan C. Gottschal
In: FEMS Microbiology Letters (1990) 66:87-94
Cbapter 2
Mixed chemostat cultures of obligately aerobic and fermentative
or methanogenic bacteria grown under oxygen-limiting conditions
Jan Gerritse, Frits Sc hut a nd Jan C. G o ttscha l
Deparlmenl of Microbiology. University of Groningen, Haren, The Netherlands
Received and accepted 8 Augus t 1989
Key words: Pseudomollas leslosleroni; VeilIonelIa a/ca/escens; Methanogens: Oxygen limitati on;
Mjxed cultures
1. SUMMARY
Defined mixed cultures of an obligatel y aerobic
Pseudomonas leSIOSleroni and an anaerobic Vei//onella a/ca/escens strain were grown under oxygen
and lactate limitation in chemostats with different
oxygen supply rates. The aerobic and the anaerobic
bacteria were shown to coexist and to compete for
common substrates over a wide range of oxygen
supply rates. Under similar conditions but with
formate as the major substrate chemostat enrichments gave ri se to undefined mixed cultures of
aerobi c, fermentati ve and methanogenic bacteri a.
The relevance of these observati ons to natural
mineralization processes is discussed.
2. INTRODUCTION
Strict aerobes depend upon molecular oxygen
for their metaboli sm, whereas strict anaerobes can
only grow in an oxygen-free environment [1]. Considering these definitions one may concJude that
Correspondellce 10: J. Gerritse, Depa rlmen t of M icrobiology,
U ni versity of Groni ngen, Kerkl aan 30, 975 1 NN Hare n, The
Netherlands.
16
strictly aerobic a nd anaerobic bacteria li ve separated in time and/ or in space. The occurrence of
large numbers and acti vities of strict anaerobes
like sulph ate reducers a nd meth anogens in apparently aerobic environments in therefore usuall y
explained by the ability o f many anaerobes to
survive periods of aerobi osis [2- 5] or by the presence of anaerobic microniches [6,7.9,10]. However,
some in vestigation indica te, th at under conditi ons
with very low dissolved oxygen concentrati ons
aerobes and anaerobes may grow simultaneously
in one and the sa me habitat [11 ,12]. Microaerobic
zones occur near the borders of aerobic and
anaerobic habita ts in many natural ecosystems
like soil particJes [13,14], sediments [1 5,16], periodontal pockets [17,18], bacteri al colonies (1 9] and
stratified lakes and seas [20,2 1]. Often high (micro)bi ologica l acti vities coincide with th ese aerobic/ anaerobi c interfaces wru ch illustrates the significance of these sites in mineraliza tion processes
(1 5,20- 23]. In tru s paper we show, that the
metaboli sm o f aerobic and anaerobic bacteri a is
not necessa rily link ed only by diffusion o f
anaerobic end-products into the aerobic zone or
by f1u ctuating oxygen gradients. but th at these
two physiological types of orga nisms can grow
simultaneously under mi croaerobic conditions
created in labora tory cultures.
Mixed cultures of aerobic and anaerobic bacteria
3. MATERlALS AND METHODS
3.1. Organisms
VeilloneJla alcalescens NS.L49 was isolated from
the intertidal sediments of lhe Ems Dollard
estuary, and has been described by Laanbroek et
al. [24]. It fermen ts L-Iactate 10 propionate, acetale,
H , and CO, . This organ ism is not able to grow at
the expense of aerobi c metabolism on ly and its
growth is inhibiled completely at oxygen concenlralions above lhe detection limit of convenlional oxygen eleclrodes ( > 0.1 !.lM; unpubli shed
results).
The aerobic organi sm used in lh is sludy was
isolated from a L-Iaclale Iimited microaerobic
chemostal enrichment (dissolved oxygen concen lration < 1 !.lM), that had been inoculated
with a sample from a fresh-waler sedimen t and
was lentatively identified as a Pseudomollas
testosteron i strain. The metabolism of thi s organism is strictly respiratory with oxygen as electron
acceptor. Lactate, propionate and acetate are used
as sole ca rbon and energy sources.
A sample for the methanogenic enrichment culture was taken from a fresh-water sediment. After
pre-enrichment of methanogens on formate in a
batch cu lture, and filtration over a membrane
filter (1.2 !.lM) :0 remove protozoa, 20- 30 mI were
used as an inoculum for th e continuous culture.
Strictly anaerobic techniques were used throughout
these procedures.
3.2. Media
The medium used for the chemostat cultivation
of P. testosteron i and V alcalescens (PV-medium)
contained in deionized water (gi l): KH , PO. ,
(1.0) ; Na , SO. , (0.2); MgCI, · 6H,o, (0.2) ; NaCI,
(1.0) ; NH.CI, (1.0); CaCl , · 2H , O, (0.015); resazurin, (0.001) ; and yeast extract, (0 .1). A trace
elements and a selenium solution were added (1
mijl). Af ter autoclaving a filter- sterilized vitamin
solution (1 mijl) and an autoclaved potassium-Llactate solution to a final concentration of 19 mM
we re added. For batch media and media solidified
with 2% w/ v agar, KH , PO. was omitted a nd 30
mM of a sodium j potassium phosphate buffer
(pH 7.0) was added. For pure cultures of V.
alcalescens the medium was additionaUy supplied
with 1 mi of 1 N HCIjI; 3 mI of 1 N NaHCOJ/I;
and 0.2 mI of a 10% w/ v Na 2 S· 9Hp soluti on/ l.
M-medium used fo r the methanogenic enrichment was composed of (gi l): MgCl 2 · 6H , O, (0.2):
NaCI (1.0); NH .CI, (0.5) ; CaCl 2 · 2H 20, (0.015):
Na , S20 ) · 5H,o, (0 .5); L-cysteine· HCI, (0.16) ; resazurin, (0.001); yeasl ex tract, (2.0); and gelysate
peptone, (2.0). This solution was autoclaved af ter
add iti on o f the lrace elements and selenium solutions (1 ml/ l) and adjustmen t of the pH to 7.5.
Af ter cooli ng down , sterilized vitamin soluti on (1
mijl) and 7.5 mM phosphate buffer (pH 7.5) we re
added. Formic acid was pumped separately from a
2.58 M solution into the culture vessel resulting in
an S, of 277 - 284 mMo Batch culture medi a with a
pH of 7.5 contain ed 30 mM phosphate buffer and
50 mM potassium formate.
The trace elements, selenium and vitamin soluti ons were as described previously [12].
3.3. Growth conditions
Bacteria were grown at 30 0 C in chemostats
with a working volume of 450 mI. The pH was
maintained at a constant value by automatic additi ons of 1 N KOH or 1 N HCI . Aeration was
controlled by the combination of a constant stirring rate, a constant flow and a variabIe composition of the innowi ng (airi N,) gas-mixture. The
gas-f1owrate was approximately 1700 mljh and
contained 0.0 to 5.2% (v I v) oxygen. Nitrogen gas
was stripped of traces of oxygen by leading it over
hot copper filin gs. Butyl and PVC tubing was used
for gasses and media. Marprene tubing was used
for the peristaltic medium pumps. Oxygen concen trat ions we re measured continuously with a
polarographic Ingold oxygen electrode. The redox
potential was measured with a platinum eleclrode
with an exposed surface of 0.16 cm2 The reference
cell (Agl AgCI) of the pH-electrode was used as
the reference electrode for the redox readings. The
redox electrode was calibrated in buffer solutions
containing a saturating concentration of chinhydron (approximately 5 gi l) and 200 mM phosphate buffer (pH 6, 7 and 8).
Mixed cultures of P. testosteron i and V. alcalescens were grown at a dilution rate of 0.10
H - 1 and pH 7.0. The stirring rate was 400 r.p.m.
Purity was checked each day be microscopic ob-
17
Chapter 2
servations and every three days by streaking on
agar-plates containing PV-medium with 0.2% yeast
extract, incubated both aerobically and in anaerobic jars. The methanogenic population was grown
at pH 7.5 at a dilution rate of 0.06 h - I. The
stirring rate was 500 r.p.m.
3. 4. Chemical analysis
Cells were washed and suspended in PVmedium without yeast extract, vitarnins, trace elements and L-lactate (= PV -buffer), or in Mmedium without yeast extract, gelysate peptone,
L-cysteine· HCl, vitamins, trace elements and formate (= M-buffer). The supernatant and ceU suspensions were stored at - 20 0 C for further analysis.
Organic acids were determined on a Packard
437 gaschromatograph as described by Nanninga
et al. [25J. Formate was measured by the method
of Lang and Lang [26J. CeU carbon was analyzed
with a Shimadzu TC-500 carbon analyzer with
biphthalate as a standard for dissolved organic
carbon. Protein was measured with the method of
Lowry et al. with bovine serum albumin as the
standard [27J.
3.5. Gas analysis
Gas-flowrates through the chemostats were
measured accurately with film-flow pipettes and
were corrected for changes in gas volume caused
by the consumption or production of gasses.
Wash-out of H 2' CH. and O 2 with the culture
fluid was neglected in the calculations. Oxygen
concentrations of input and output gasses were
continuously monitored with a Servomex 1100
oxygen analyzer connected to a personal computer
for process con trol and data processing. Methane,
hydrogen and carbon dioxide concentrations were
determined in samples (0.5 ml) wi thdrawn from
the output gas on a Pye Unicarn 104 gaschromatograph equipped with a katharometer and a Poropack Q (Waters Associates Inc., Milford, Mass. ,
U.S.A.) mesh 100- 120 column (4 mrn X 1.2 m).
The flowrate of the nitrogen carrier gas was 20
mI/min. The temperature of the column was kept
at 20 0 C and that of the detector at 50 0 C. The
bridge current was 125 mA. Dissolved CO 2-con-
18
centrations were measured with the carbon
analyzer.
Maximum oxygen consumption rates of cell
suspensions were measured polarographically in a
YSI-Biological Oxygen Monitor, in washed suspensions of cells in air saturated M-buffer.
3.6. Cell quantification
The ratio between P. testosteron i (rods) and V.
alcalescens (cocci) was determined by combining
cell-counts and carbon measurements. Cells were
counted microscopically af ter appropriate dilution
of culture samples in PV-buffer containing 0.4%
formaldehyde. The amount of P. testosteroni and
V. alcalescens, in terms of cell-carbon, in the mixed
cultures was based on a calibration of cell-carbon
and cell-numbers in pure cultures. For V. alcalescens 1 mg of eell-carbon in batch culture eorresponded to 4.44 X 10 7 eells, whereas in oxygen
lirnited batch cultures of P. testos/eroni 1 mg of
ceU-carbon was equivalent to 2.09 X 10 7 cells.
3.7. Microscopy
A Carl Zeiss G42-110-e Axioseope equipped
with a Vertieal Illuminator IV FL and an Osram
high-pressure mercury lamp was used for phasecontrast and fluoreseenee microscopy. A Zei ss BP
400- 440, FT 460, LP 470 filter-eombination was
used for excitation at 420 nm and detection of
fluoreseence of methanogenic bacteria.
4. RESULTS AND DISCUSSION
4.1. Cocultivation of P. testosteroni and V. alcalescens
Under strictly anaerobic conditions lactatelimited growth at a dilution ra te of 0.1 h - 1 resulted in a pure culture of V. alcalescens. However, supplying the culture with various arnounts
of oxygen yielded stabIe mixed cultures of P.
testosteroni and V. alcalescens over a range of
oxygen eonsumption rates from 0.41 to 2.45 mmol
. 1- 1 • h- ' (Fig. 1). In sueh mixed cu ltures the
oxygen eoneentration always remained bel ow 0.1
~M (the detection limit of the oxygen probe used).
Under these condi tions both lactate and oxygen
are growth limiting. The ratio between the two
Mixed cultures of aerobic and anaerobic bacteria
11.0 , - - - - - - - - - - - ,
T
T~
12 .0
T/
ëi
E
!
' .0
c:
o
~
•
"
.!.
'ij
"
&0
'0
0 .0
11
/l-~~
1
/
T
T/ i-!--i
.,...._-+-_--+__>-_-<-_..J
0 .0
0.5
1.0
1.5
2 .0
2.1
O.-conaumption (mmol.l· 1.h· 1 )
Fig. 1. Changes in the numbers or the individual organisms, in
tenns or ceU·carbon, calculated rrom steady states or mixed
cultures or V. alcaleseens and P. testosteroni with increasing
oxygen supply. P. testosteroni , ( ... ); V. alcaleseens, (e ). Bars
represent standard devialÎons with 9.:s;; n .$ 28 (n = numbers or
microscope.rields counted).
species appeared strictly dependent on the input
of O2, Thus a gradually increasing supply of O2
resulted in an increase of P. testosreroni , yet V.
alcaleseens decreased only slightly. In the strictly
anaerobic steady state the fermentation pattem of
this V. alcaleseens strain is similar to that found in
pure cultures [24]. With increasing oxygen availability an increasing amount of the available carbon
and energy sources are oxidized by P. testosreron;
to CO2 and H 20 as indicated by the decrease in
acetate and propionate concentration, the increased CO 2-production, and the elevated 02-consumption of the mixed culture under these conditions (TabIe 1). H r Production in the mixed cul-
ture showed little variation as did the numbers of
V. alcaleseens. This indicates the L-lactate fermentation by V. alcaleseens was not influenced strongly
by the increased 0 2-flux. Since H 2 cannot be
oxidized by P. testosteron; (unpublished results)
the H 2 produced in the mixed culture provides a
measure of the amount of lactate fermented by V.
alcaleseens. Thus the reaction stoichiometry for
the conversion of L-lactate by the individual
organisms can be calculated for the different
steady states. For example, in the steady state
with the largest supply of oxygen (TabIe 1; Steady
State Nr: 6) the reactions are:
- V. alcaleseens: 1.20 Lactate ---+ 0.25 CO, + 0.38
H 2 + 0.67 Acetate + 0.64 Propionate + 0.29 Cell
carbon
- Po testosteron;: 0.60 Lactate + 0.20 Acetate +
0.53 Propionate + 2.22 O2 ---+ 1.39 CO 2 + 1.33 Cell
carbon
Thus it can be seen that the disappearance of
acetate and propionate (TabIe 1) is due mainly to
the consumption by P. testosteron; rather than a
decreased formation by V. alcaleseens. These results indicate a mutualistic relationship between
the aerobic and the anaerobic species. The oxidation by P. testosteron; of propionate and acetate
produced by V. alcaleseens creates an environment
sufficiently devoid of oxygen for V. alcalescens to
continue L-lactate fermentation. Apparently P.
testosteron; does not outcompete V. alcaleseens for
L-lactate as long as growth proceeds under
oxygen-limited conditions and the concentrations
of propionate and acetate are non-limiting. In
order to further clarify the carbon flow in these
Table 1
Analysis or the mixed chemostat cultures or P. testosteron; and V. alcaleseens grown at several oxygen supplies at a dilulÎon rale or
0.1 h - 1 under lactate and oxygen !imitation
Sleady
state
Eh
(mV)
-97
-1 4
-12
-19
-10
-23
Consumption (mmol · l '·h ')
Production (mmol · 1 '·h ')
0,
Lact.
CO,
H,
Ace.
Pro.
CeIiC
0.00
0.39
1.06
1.31
1.86
2.22
1.74
1.78
1.78
1.79
1.78
1.80
0.37
0.92
1.08
1.49
1.39
1.64
0.55
0.53
0.32
0.36
0.34
0.38
1.00
0.90
0.71
0.67
0.52
0.47
0.93
0.66
0.52
0.25
0.20
0.11
0.42
0.82
1.03
1.13
1.51
1.62
Recovery
(%)
107
103
95
88
85
84
Abbreviations: Lact. = l-lactate; Ace. = Acetate; Pro. - Propionate; Cell C = Cell·carbon.
19
Chapter 2
mixed cultures a mathematical model has been
constructed based upon the growth characteristics
of both organisms, which predicts the outcome of
these mixed cultures quite accurately (in preparation).
4.2. Methanogenesis in oxygen-Iimited chemostats
A chemostat enrichment culture was set up
under strictly anaerobic conditions with fo rmate
as a major ca rbon and energy source. FoUowing
an initial period of batch cultivation the dilution
rate was gradual!y increased to 0.06 h - I, resulting
in a formate-limited methanogenic mixed culture.
The methanogens, as observed by Ouorescent mi crocopy, were mai nly short rods, growing singly or
in pairs, and long Oexible rods forming aggregates
of about 10- 30 cel!s. In addition large numbers of
non-methanogenic spiril!um-shaped organisms and
spore-forming rods were present. Methane and
carbon dioxide were the maj or end products of the
mixed culture (Tabie 2; Steady State N r: 1).
A gradual!y increasing supp ly of oxygen did
not significantly interfere with methanogenesis. In
fa ct a stimulation of methane production of about
20% was found at low oxygen Ouxes (T abie 2).
Similar results have been reported in anaerobic
digestors and in batch cultures in which O2 was
introduced in the head space [10,28]. These authors
suggested this to be a consequence of an increased
supply of ' growth factors' to the methanogens by
facultative organisms. In our experiments the
supply of oxygen could be increased without a ffectin g methane formation until only very low
concentrations of ferm entation products were de-
tected in the culture. Further increase of the oxygen
supply resulted in washout of the methanogens
and accumul ati on of fo rma te. T he presence of
acetate, propionate. iso-butyrate, butyrate, methyl-bulyrate and iso-valerale (Tab ie 2) in dica les the
activi ty of fermenta live organisms in lhe mixed
culture growing at the expense of the relati vely
high amoun t of d issolved organic carbon sources
originating from the yeast extract and pep lone
present in the med iu m. Direct plating on aerob ic
agar medi a and subsequent testing for anaerobic
growth indicated the presence o f large numbers of
ob ligately aerobic bacteria. Especially the nu mber
of spiril!um-shaped organisms increased with increasing oxygen supply. G rowth of aerobic
organi sms in the mi xed cultu re is furth er indica ted
by increasi ng 0 2-consumption. CO 2-prod ucti on
and cel! yield at higher 0 2-0uxes. The cell yield
(ass uming a cel! composition of C H 1.70 0.3No.2 ) at
an 02-consumpti on rate of 6.82 mmol · I- I . h - I
(Tab Ie 2) was 18.1 g dry-weight per mol methane
p roduced, whi ch is much hi gher than reported
yields for methanogens alone wh.ich is l.6- 6.9
g. mol- I [29]. Oxygen uptake rates of washed cel!
suspensions increased with lhe increasi ng oxygen
uptake ra tes in the chemostat (Ta bie 3). Hi gh
0 2-uptake rates wi th yeast extract ind ica te that a
substantial am ount of the 0 2-uptake in the
chemostat was due to the oxidation of yeast extract (and peptone). The considerabl e potentia l for
forma te oxidation strongly suggests competition
for formate between aerobes and meth anogens.
Steady states in the presence o f high oxygen
Ouxes and methane production coul d only be ob-
Tab'e 2
Analysis of the methanogenic mixed culture grown al a dil utio n rale of 0.06 h - 1 wit h an increasing supply of oxyge n
Slcady
state
Eh
(mV)
-
332
232
237
191
Consumption (mmo)-] '· h ')
0,
0.09
1.38
4.37
6.82
Farm .
16.43
16.35
16.35
16.07
D.O.C.
10.92
13.49
13.49
13.49
Production (mmol -'
CH ,
3.92
4.95
4.56
3.87
CO,
14.42
15.31
17.56
23.89
,h
Ace.
1.06
1.27
0.7 1
0.02
Re-
')
Pro.
0.33
0.23
0.10
0.00
150-8.
0.04
0.05
0.05
0.00
Bu t.
0. 16
0.02
0.01
0.00
Met.
0.07
0.07
0.07
0.03
lso-
v.
Cell
C
0.04
0.03
0.02
0.01
1.91
2.87
3.09
3.39
cov-
ery
(%l
90
91
93
106
Abbrevia tions: From. = Formate; D.O.c. = Dissolved Organi c Carbo n subst rates origi nating (rom the yeast ex tract and pepto ne
present in the medium ; 150-B. = Iso-butyrate; Met. = Methyl-bu tyrate. Iso-V. = Iso-valerate. Qther abb reviatio ns are as in Tab le 1.
20
Mixed cultures of aerobic and anaerobic bacteria
Table 3
Maximum oxygen consumption rales in samples taken fro m th e steady-state methanogc nic mixed chemostat cultures. Oxygen
co nsumpLions by supernatant of culture sa mpl es were measured immediatcly following ce ntrifuga tion . Steady state numbers are lhl.!
same as those give n in Table 2
Steady
Supernatant
state
0.06
0041
4
0.72
2.51
Maximum Orconsumplion ra le (mmol I I· h I)
Washed-cclls
End.
Form.
Y.E.
Tatal
0.27
1.07
3.03
7.51
0.08
0.59
2.75
2.97
0.30
1.84
2.83
9.82
0.7 1
3.9 1
9.33
22.8 1
0 2-consump ti on
in (h e c hemostat
(mmol·I - I . h - I )
0.09
1.38
4.37
6.82
Abbrevia lions: End. = Endogenous; Form . = +2.5 mM Formatc; Y.E. = +0.01 % yeas t ex tract.
tained if the oxygen input was increased gradually. A sudden switch of the metanogenic culture,
grown under strictly anaerobic conditions for 7
volume changes to an input of oxygen even less
than that used in the steady state Nr: 2 (Tabie 2)
resulted in an immediate, though reversible, inhibition (> 25 %) of the methanogenesis, an increase of the redox potential from - 327 to - 103
mV and an accumulation of formate within
minutes.
ACKNOWLEDGEMENT
This investigation was supported by the
Netherlands Integrated Soil Resea rch Programme.
The authors thank R. Palmen for the identification of the Pseudomonas leSIOSleroni strain.
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Although strict anaerobes and aerobes are generally considered to grow spatially separated in
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Chapter 2
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