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Appl Microbiol Biotechnol (1995) 43:1061-1066
© Springer-Verlag 1995
S. Salgueiro Machado • M. A. H. Luttik
J. P. van Dijken • J. A. Jongejan • J. T. Pronk
Regulation of alcohol-oxidizing capacity
in chemostat cultures of Acetobacterpasteurianu$
Received: 20 October 1994/Received revision: 27 January 1995/Accepted: 2 February 1995
Abstract Acetobacter pasteurianus LMG 1635 was
studied for its potential application in the enantioselective oxidation of alcohols. Batch cultivation led to
accumulation of acetic acid and loss of viability. These
problems did not occur in carbon-limited chemostat
cultures (dilution rate--0.05 h -~) grown on mineral
medium supplemented with ethanol, L-lactate or acetate. Nevertheless, biomass yields were extremely low in
comparison to values reported for other bacteria. Cells
exhibited high oxidation rates with ethanol and racemic glycidol (2,3-epoxy-l-propanol). Ethanol- and
glycidol-dependent oxygen-uptake capacities of
ethanol-limited cultures were higher than those of cultures grown on lactate or acetate. On all three carbon
sources, A. pasteurianus expressed NAD-dependent
and dye-linked ethanol dehydrogenase activity.
Glycidol oxidation was strictly dye-linked. In contrast
to the NAD-dependent ethanol dehydrogenase, the activity of dye-linked alcohol dehydrogenase depended
on the carbon source and was highest in ethanol-grown
cells. Cell suspensions from chemostat cultures could
be stored at 4°C for over 30 days without significant
loss of ethanol- and glycidol-oxidizing activity. It is
concluded that ethanol-limited cultivation provides an
attractive system for production of A. pasteurianus biomass with a high and stable alcohol-oxidizing activity.
Introduction
Bacteria of the genus Acetobacter are well known for
their application in the production of acetic acid from
S. Salgueiro Machado - M. A. H. Luttik • J. P. van Dijken
J. A. Jongejan • J. T. Pronk ([])
Department of Microbiology and Enzymology, Kluyver
Laboratory of Biotechnology, Delft University of Technology,
Julianalaan 67, 2628 BC Delft, The Netherlands.
Fax: (31) 15 782355
ethanol (Ebner and Follmann 1983). These bacteria
also catalyse the oxidation of other aliphatic and aromatic alcohols to the corresponding aldehydes and
organic acids (De Ley and Kersters 1964; Asai 1968).
Because of the broad substrate specificity of the dehydrogenase systems involved in ethanol oxidation,
Acetobacter species hold great promise for the production of a variety of aldehydes and organic acids.
An attractive property of microbial dehydrogenases
with regard to the production of fine chemicals is that
they often exhibit enantioselectivity (Hummel and
Kula 1989). This also holds for the oxidation reactions
catalysed by Acetobacter species. For example, when A.
pasteurianus cell suspensions are incubated with racemic (R,S)glycidol (2,3-epoxy-l-propanol), the Sisomer is preferentially oxidized (Geerlof et al. 1994).
This enantioselective bioconversion can in principle be
used for the industrial production of optically pure
(R)-glycidol, which is a building block for the synthesis
of a number of pharmaceutical products (Klunder et al.
1986; Kloosterman et al. 1988; Avignon-Tropis et al.
1991).
In biotechnological processes, optimum conditions
for biocatalyst production (growth) and the bioconversion itself may differ strongly. Therefore, industrial
production of fine chemicals by whole-cell biotransformations generally involves separate process steps for
production of biomass and bioconversion processes.
Optimization of the first phase of the process, i.e. development of efficient cultivation techniques yielding
highly active, stable biomass, is a prerequisite for industrial-scale application.
The broad substrate specificity of dehydrogenase
systems from Acetobacter species is in sharp contrast
with the narrow range of carbon substrates for growth.
For example, the only known carbon substrates for
growth of A. pasteurianus are ethanol, acetate and
lactate (De Ley 1958). Ethanol and lactate are rapidly
oxidized to acetic acid, which accumulates in batch
cultures and is only slowly oxidized to carbon dioxide
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(De Ley 1958; De Ley and Schell 1959). Dissipation of
the trans-membrane pH gradient by acetic acid (Alexander et al. 1987) is likely to contribute to the very low
biomass yields of Acetobacter species in batch cultures
and the sensitivity of such cultures to storage, in particular in the absence of vigorous aeration (Ebner and
Follmann 1983).
In theory, it should be feasible to prevent acetic acid
formation by growth under carbon-limited conditions
in fed-batch or chemostat cultures. However, although
the intermediary carbon metabolism and the molecular
biology of electron transport in Acetobacter species
have received much attention in the literature (for reviews see Asai 1968 and Matsushita et al. 1994), data on
the growth of these organisms in carbon-limited
chemostat cultures have not been reported.
The aim of the present study was to investigate the
suitability of carbon-limited chemostat cultivation for
the production of alcohol-oxidizing biomass of A. pasteurianus. Research focused on factors relevant for the
application of this organism in bioconversions such as
the nature, regulation and stability of the alcoholoxidizing activity.
Control of culture purity
The purity of chemostat cultures was routinely examined by phasecontrast microscopy at 1000 x magnification. Furthermore, absence
of catalase activity, a striking characteristic of this aerobic organism
(Visser't Hooft 1925) was checked daily by adding 10 gl hydrogen
peroxide to a 1-ml culture sample. Contaminations were detected by
visible gas formation.
Determination of culture dry weight
Dry weights of culture samples were determined using a microwave
oven and 0.45-gin membrane filters as described by Postma et al.
(1989). The dry weight of parallel samples varied by less than 1%.
Analysis of substrates and metabolites
Concentrations of ethanol, acetate and lactate in reservoir media
and culture supernatants were determined by H P L C on a Rezex
organic-acid column (Phenomenex, 300 x 7.8 ram) at 60°C. Detection of acetate and lactate was by means of a Waters 441 UV
detector at 214 nm, coupled to a Waters data module. Detection of
ethanol was by means of an Erma 7515 A refractive-index detector.
Peak areas were linearly proportional to concentrations. The detection limits for acetate, lactate and ethanol were approximately
0.05 raM, 0.05 mM, and 0.5 m M respectively.
Materials and methods
Gas analysis
Microorganism and maintenance
Acetobacter pasteurianus L M G 1635 (formerly called A. peroxydans)
was obtained from the culture collection of the Laboratory of
Microbiology, University of Gent, Belgium, and stored at - 70°C
with 10% (v/v) dimethylsulphoxide in 1-ml aliquots. Samples from
these frozen stock cultures were grown on slants of a solidified
medium containing, per litre of demineralized water: Difco peptone
10 g, Difco yeast extract t0 g, Difco agar 20 g, ethanol 10 g, and
CaCO3 20 g. Inoculum cultures for chemostat cultivation were
grown in 500-ml shake flasks containing 100 ml same medium but
without agar. These cultures were incubated for 48 h in a rotatory
shaker (200 rpm) at 30°C.
Chemostat- cultivation
Aerobic chemostat cultivation was performed at 30°C in 2-1 Applikon laboratory fermenters at a stirring speed of 1000 rpm and at
a dilution rate of 0.05 h-1. The cultures were flushed with air
(11 rain-1/. The dissolved-oxygen concentration in the cultures was
measured with an Ingold polarographic oxygen electrode and remained above 25 % of air saturation. The culture pH was controlled
at 6.0 by automatic addition of 2 M K O H . The condenser was
connected to a cryostat and cooled at 2°C. The working volume of
the culture was kept at 1.0 1 by a peristaltic pump coupled to an
Applikon level controller. Biomass concentrations in samples taken
directly from the culture differed by less than 1% from biomass
concentrations in the culture effluent. The mineral medium contained, per litre of demineralized water: (NH4)2SO,~ 5 g, KH2PO,~
3g, M g S O 4 . 7 H 2 0 0.5g, EDTA 15mg, Z n S O 4 ' 7 H 2 0 4.5rag,
MnC1 z • 2H20 1.0 mg, CoC12 • 6H20 0.3 mg, CuSO 4 • 5H20 0.3 rag,
N a z M o O 4 • 2H20 4 mg, CaC12 . 2H20 4.5 mg, FeSO 4 • 7H20 3 rag,
KI, 0.1 mg, BDH silicone antifoam 50 bd. The mineral medium was
autoclaved at 120°C. Pure ethanol, acetic acid or L-lactic acid was
added aseptically to the sterile media without prior sterilization.
On-line measurement of oxygen-uptake rates of chemostat cultures
was performed as described by Van Urk et al. (1988).
Preparation of cell-free extracts
Samples of steady-state chemostat cultures containing 25-50 mg dry
weight were harvested by centrifugation, washed twice and resuspended in 4 ml ice-cold 0.1 M potassium phosphate buffer (pH 7.5),
containing 1 m M dithiothreitol and 2 mM MgC12. Extracts were
prepared by sonication with 0.7-mm-diameter glass beads at 0°C for
2 min at 0.5-min intervals with an MSE sonicator (150 W output,
8 I.tm peak-to-peak amplitude). Unbroken cells and debris were
removed by centrifugation at 4°C (20 min at 75 000 9). The clear
supernatant, typically containing 1-3 mg protein ml-1, was used as
the cell-free extract.
Protein determination
Protein concentrations in cell-free extracts were estimated by the
Lowry method. The protein content of culture samples was estimated by a modified biuret method (Verduyn et al. 1992). In both
assays, bovine serum albumin (fatty-acid-free, Sigma) was used as
a standard.
Substrate-dependent oxygen' uptake
Substrate-dependent oxygen uptake by culture samples was assayed
polarographically with a Clark-type oxygen electrode (Yellow
Springs Instruments Inc., Yellow Springs, Ohio, USA) at 30°C after
appropriate dilution in air-saturated buffer (100mM potassium
phosphate, 10 m M MgSO 4, pH 6.0). Calculations of specific rates
1063
were based on an oxygen concentration in air-saturated buffer of
236 pM at 30°C. Endogenous respiration rates by chemostat-grown
cells were negligible.
Enzyme assays
Enzyme assays were performed immediately after preparation of the
cell-free extracts. All enzyme assays were carried out at 30°C. In all
activity measurements, the reaction rate was linearly proportional to
the amount of cell-free extract added.
Alcohol dehydrogenase (pyridine-nucleotide dependent)
The reaction mixture (1 ml) contained: 50 gmol glycine/KOH buffer
(pH 9.0), 1 Ixmol N A D or NADP, 0.05% (v/v) Triton X-100 and
cell-free extract. The reaction was started by the addition of either
10 gmol ethanol or 50 gmol racemic glycidol. Formation of
NAD(P)H was monitored at 340 nm (e = 6.22 mM -1 cm -~) in
a Hitachi spectrophotometer model 100-60.
Acetaldehyde dehydrogenase (pyridine-nucleotide dependent)
The reaction mixture (1 ml) contained 100 gmol potassium phosphate buffer (pH 8.0), 15 gmol pyrazole, 0.4 gmol dithiothreitol, 0.4
gmol N A D or NADP, 10 gmol KC1, 0.05% (v/v) Triton X-100 and
cell-free extract. The reaction was started by the addition of 5 p-mol
acetaldehyde. Formation of NAD(P)H was monitored at 340 nm in
a Hitachi spectrophotometer model 100--60.
Dye-linked alcohol and acetaldehyde dehydrogenases
Activities of dye-linked [probably pyrroloquinoline-quinone (PQQ)
dependent; Matsushita et al. 1994] alcohol and aldehyde dehydrogenases were assayed polarographically with a Clark-type oxygen
electrode (Yellow Springs Instruments Inc., Yellow Springs, Ohio,
USA) at 30°C, using the artificial electron acceptor phenazine
methosulphate (PMS) as a mediator. The values presented have
been calculated based on an oxygen concentration in air-saturated
buffer of 236 gM at 30°C. The reaction mixture (4 ml) contained
400 gmol potassium phosphate buffer (pH 6.0), 8 gmol MgC12,
1.2 gmol (PMS), 26 000 U catalase (from bovine liver, Boehringer)
and cell-free extract. The reaction was started by the addition of
either 40 gmol ethanol, 200 gmol racemic glycidol or 20 gmol acetaldehyde.
Stability of alcohol-oxidizing capacity
To determine the stability of the alcohol-oxidizing activity of A.
pasteurianus upon storage; 100-ml samples from steady-state
chemostat cultures were transferred tO 100-ml serum flasks. The
closed flasks were incubated statically at 4°C and 30°C. At appropriate intervals, the contents of the flasks were mixed by inverting them,
and samples were withdrawn for determination of substrate-dependent oxygen uptake as described above.
Chemicals
Racemic (R, S)-glycidol was obtained from Janssen Chimica and
distilled before use to remove polymerized material.
bsulN
Growth of A. pasteurianus in chemostat cultures
Preliminary experiments with shake-flask cultures
demonstrated that A. pasteurianus LMG 1635 is capable of growth on defined mineral salts media without
vitamins. However, growth in shake-flask cultures led
to rapid loss of viability and alcohol-oxidizing activity.
This was probably due to the observed accumulation of
acetic acid and concomitant acidification of the growth
medium (results not shown). Since the maximum specific growth rate in shake-flask cultures grown in mineral
medium with ethanol was about 0.1 h - 1, it was decided
to study growth in chemostat cultures at a dilution rate
of 0.05 h - 1.
Problems related to the accumulation of acetate were
also encountered during the start-up phase of A. pasteurianus chemostat cultures on ethanol. These problems could be avoided by keeping the initial concentration of ethanol in the growth medium at 5 g 1-1. Once
all substrate had been consumed, it was then possible
to feed the cultures with higher concentrations of
ethanol. Using this strategy, steady-state chemostat
cultures could be obtained at reservoir concentrations
of ethanol up to 20 g 1-1. Steady-state conditions (i.e.
no significant changes in biomass yield and oxygen
consumption over a period of 3 days) were generally
established after about 10-15 volume exchanges.
In ethanol-grown chemostat cultures, the biomass
yield on ethanol was identical at reservoir concentrations of 10 g1-1 and 20 g1-1, and no residual ethanol
or acetate was detectable in culture supernatants. These
observations confirmed that ethanol was the growthlimiting nutrient. Biomass yields on ethanol were very
low and growth was accompanied by high oxygenuptake rates (Table 1). In practice, these high oxygen-uptake rates imposed a limit to the biomass
concentrations that could be achieved in the chemostat
cultures with normal aeration. Nevertheless, biomass
Table I Biomass yield on substrate (Y~x), protein content and specific oxygen-uptake rates (qO2) in chemostat cultures of A. pasteurianus L M G 1635 with ethanol, acetate or L-Iactate as the
growth-limiting nutrient. Growth conditions: dilution, rate =
0.05h -1, T = 30°C, pH 6, PO2 > 25% air saturation. Data are
presented as average ± standard deviation of measurements from at
least two independent chem0stat cultures grown at different reservoir concentrations of the growth-limiting nutrient (10g1-1 or
20 gl - 1)
Carbon
source
Y~x
(g dry weight
mol - 2)
Protein
content (%)
qO2
(mmol g- 1 h - 1)
Ethanol
Acetic acid
L-Lactic acid
6.1 _+ 0.1
4.1 _+ 0.1
11.4 _+ 0.6
68 _+ 1
63 _+ 1
64 _+ 1
23 ± 2
23 + 1
12 + 1
1064
concentrations exceeding 2 g dry weight 1-1 could be
attained in cultures grown on ethanol.
Also with acetic acid or lactic acid as the sole carbon
source, steady-state chemostat cultures were obtained
at a dilution rate of 0.05 h - 1. Again, no residual substrate was detected in these cultures and biomass concentrations were proportional to the concentration of
acetic acid or lactic acid in the reservoir medium (Table
1). As observed with the ethanol-limited cultures, biomass yields were very low. The carbon source for
growth did not appear to have a substantial effect on
the protein content of the biomass, which was about
65% (Table 1).
Regulation of alcohol-oxidizing capacity of intact cells
To investigate regulation of ethanol- and glycidoloxidizing activity in A. pasteur±anus, substrate-dependent oxygen uptake rates were measured in cell suspensions harvested from the chemostat cultures.
Although the ability to oxidize ethanol and glycidol
appeared to be expressed constitutively, activities were
three- to sixfold higher in ethanol-grown cells than in
samples from acetate- or lactate-limited chemostat cultures (Tables 1, 2). The ethanol-dependent oxygen-uptake capacity of ethanol-grown cells was fourfold higher than the in situ oxygen uptake rate in ethanol-limited chemostat cultures. The ratio between ethanoland glycidol-oxidizing activity was not constant in the
different cultures (Table 2), suggesting the involvement
of at least two enzymes in the oxidation of these compounds. To investigate further which enzymes are involved in the oxidation of ethanol and glycidol, activities of alcohol-oxidizing enzymes were measured in
cell-free extracts.
Regulation of alcohol and acetaldehyde
dehydrogenases
Cell-free extracts of A. pasteur±anus exhibited both
NAD-dependent and dye-linked (probably PQQ-dependent; Matsushita et al. 1994) ethanol dehydrogenase activities, but no activity was detected with N A D P
as the electron acceptor (Table 3). Oxidation of glycidol
was observed with the artificial electron acceptor PMS,
but not with NAD or N A D P (Table 3). Acetaldehyde
oxidation was observed with NAD, N A D P and the
artificial electron acceptor PMS (Table 3).
NAD-dependent ethanol dehydrogenase was expressed at a constant level in ethanol-, lactate- and
acetate-grown cultures. In contrast, the activity of dyelinked ethanol dehydrogenase depended on the carbon
source on which the cells were grown, the highest
activity being measured in ethanol-grown cells (Table
3). A similar pattern was observed for the dye-linked
glycidol-oxidizing activity (Table 3).
Table 2 Ethanol- and glycidol-dependent oxygen-uptake rates of
Acetobacter pasteur±anus L M G 1635 grown in carbon-limited
chemostat cultures (growth conditions as in legend to Table 1). Data
are presented as average ± standard deviation of data from at least
two independent chemostat culture experiments
Growth-limiting
substrate
Ethanol
Acetic acid
L-Lactic acid
Substrate-dependent oxygen-uptake capacity
(mmol 02 g - 1 h - 1)
10 mM ethanol
50 m M (R, S)-glycidol
97 + 4
39 _+ 2
29 ± 2
56 ± 3
10 + 1
11 ± 1
Table 3 Activities of ethanol-, glycidol- and acetaldehyde-oxidizing
enzymes in cell-free extracts of A. pasteur±anus L M G 1635 grown in
aerobic, substrate-limited chemostat cultures. Activities are given as
average ± standard deviation of data from at least two independent
chemostat culture experiments. (ND not determined, PMS
phenazine methosulphate)
Dehydrogenase
reaction
Enzyme activity (U mg protein-1)
with growth-limiting substrate:
Ethanol
Lactate
Acetate
Ethanol: N A D
0.21 ± 0.02
0.21 ± 0.02
0.23 ± 0.04
Ethanol: N A D P
< 0.01
n.d.
< 0.01
Ethanol: PMS
0.74 _+ 0.27
0.24 ± 0.03
0.11 ± 0.01
Glycidol: NAD
< 0.01
< 0.01
< 0.01
Glycidol: N A D P
< 0.01
ND
< 0.01
Glycidol: PMS
0.45 ± 0.17
0.15 ff 0.06
0.04 ± 0.01
Acetaldehyde: N A D
0.11 ± 0.01
0.26 ± 0.08
0.10 ± 0.01
Acetaldehyde: N A D P
0.34 + 0.06
0.89 ± 0.02
0.40 ± 0.01
Acetaldehyde: PMS
0.14 ± 0.01
0.15 ± 0.10
0.11 ± 0.01
Dye-linked acetaldehyde dehydrogenase was expressed constitutively at a constant level. Both NADand NADP-dependent acetaldehyde dehydrogenase
activities were two- to threefold higher in lactate-grown
cells than in extracts from ethanol- or acetate-grown
cultures (Table 3).
Stability of alcohol-oxidizing activity
of cell suspensions
Stability of the biocatalyst upon storage is a prerequisite for whole-cell biotransformations in which growth
of the biomass and the conversion itself are separated
in time. To investigate the stability of the ethanol- and
glycidol-oxidizing activity of A. pasteur±anus, cell suspensions from ethanol-limited chemostat cultures were
stored at 4°C and 30°C without further treatment.
At 4°C, no significant loss of ethanol- or glycidoloxidizing activity was observed after storage for 30
days (Fig. t). At 30°C, the activity declined with a halflife of approximately 10-15 days (Fig. 1). Since the
1065
100
T
" " • O-"
¢
B~II •
i
0
E
E
so
0
c
®
X
0
0
0
10
20
30
40
Time (days)
Fig. 1 Stability of ethanol- and glycidol-oxidizing activities of cell
suspensions of A. pasteurianus pregrown in ethanol-limited chemostat cultures. Culture samples were incubated at 4°C (O I ) or 30°C
(O []). Ethanol (0 O)- and glycidol ( I [])- dependent oxygen-uptake rates were measured at 30°C with a Clark-type oxygen electrode
samples were not stored under aseptic conditions, fungal contaminations occurred after prolonged storage at
30°C. This problem did not occur after 30 days of
storage at 4°C.
extracts of ethanol-grown cells are consistent with the
recent report of Takemura et al. (1993) who showed
that the alcohol dehydrogenase gene cluster in a different A. pasteurianus strain is induced by ethanol.
The biomass yields of A. pasteurianus in carbonlimited chemostat cultures (Table 1) are three- to fivefold lower than those reported for other heterotrophic,
aerobic bacteria (Heijnen and Roels 1981). The fact that
these low yields were found despite the absence of
acetate accumulation indicates that the low growth
efficiency in batch cultures is not solely due to uncoupling by this metabolite. Instead, the low biomass yields
must be due to intrinsic metabolic properties of the
organism, which remain to be identified. The biomass
yields reported in Table 1 have been measured at
a single, low growth rate. It is therefore not clear if the
low yields are due to a low maximum ('true') yield,
a high maintenance-energy requirement, or both.
The high ethanol- and glycidol-oxidizing activities of
A. pasteurianus are consistent with the relation between
growth efficiency and maximum rate of production of
catabolic metabolites proposed by Linton (1990). A.
pasteurianus clearly represents an extreme case with
respect to its apparent low energetic efficiency. Therefore, in addition to the potential benefits for the production of glycidol-oxidizing biomass, further research
into the bioenergetics of this and other acetic acid
bacteria is also of fundamental interest.
Discussion
Carbon-limited chemostat cultivation of A. pasteurianus biomass was applied and resulted in high
alcohol-oxidizing activities (Tables 2, 3) and an excellent storage stability (Fig. 1). Indeed, carbon-limited
cultivation appeared to be a prerequisite for the production of stable alcohol-oxidizing biomass, since attempts to grow A. pasteurianus in batch cultures invariably resulted in loss of viability as well as alcoholoxidizing capacity (data not shown). On the basis of the
alcohol-oxidizing capacities of cell suspensions (Table
2) and cell-free extracts (Table 3), ethanol was identified
as the best carbon source for production of A. pasteurianus biomass.
Enzyme activity assays in cell-free extracts indicated
that a PQQ-dependent (dye-linked) alcohol dehydrogenase (Matsushita et al. 1994) was responsible for the
oxidation of glycidol. The periplasmic localization of
this enzyme (Matsushita et al. 1994) may be advantageous for bioconversions that involve toxic substrates
and/or products. The observed ratio of dye-linked and
NAD-dependent ethanol dehydrogenase activities, and
the similar regulation of ethanol-oxidizing activity of
cell suspensions and dye-linked ethanol dehydrogenase
activity in cell-free extracts, indicate that PQQ-dependent ethanol dehydrogenase is probably predominantly responsible for ethanol oxidation in A. pasteurianus under conditions of substrate excess. The high
activities of PQQ-dependent alcohol dehydrogenase in
Acknowledgements Sonia Salgueiro Machado acknowledges the
financial support of the Conselho Nacional de Desenvolvimento
Cientifico e Technologico (Brazil). We thank our colleague Dr. Arie
Geerlof for many stimulating discussions.
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