CURRENT MICROBIOLOGYVol. 25 (1992), pp.

149-155
Current
Microbiology
9 Springer-Verlag New York Inc. 1992

Glycerol Inhibition of Growth and Dihydroxyacetone Production by

Gluconobacter oxydans
C. Claret, 1 A. Bories, 1 and P. Soucaille 2
1Laboratory of Biotechnology of the Environment of the IAA National Institute of Agronomic Research, Narbonne; and
2Department of Biochemistry and Nutrition, National Institute of Applied Sciences, Toulouse, France

Abstract. The evidence, kinetic aspects, and modelization of the inhibitory effect of glycerol
on dihydroxyacetone (DHA) production by Gluconobacter oxydans have been studied. The
comparison of the maximal productivities and specific rates evaluated for initial concentrations
of 31,51, 76, 95, and 129 g L - ~of substrate showed that glycerol exerts an inhibitory effect both
on growth and DHA production: decrease of the growth-specific rate and of the specific rate of
DHA production with increase of the initial glycerol content. The inhibition phenomenon was
attributed to an immediate effect of glycerol on the biological activity. It was also established
that the presence of glycerol at high concentration induces an increase in the time necessary for
the cells to reach their maximal level of specific rates. This result tends to show that glycerol
brings into play on the biological system the capacity to reach its optimal range of activity. The
main models found in the literature dealing with substrate inhibition phenomena were then tested
on experimental data. The exponential model describes at best the glycerol inhibition on growth
(/x = 0.53 e(-S/93'6)) 1 and on DHA production (qp = 7 e(-S/767)). The kinetic study and modelization
of the inhibition effect of glycerol on DHA production allows one, therefore, to fill the gap in
the fundamental knowledge of this industrial fermentation, to show the maladjustment of the
classical fermentation process used (batch), and to reconsider the conception for the optimization
of the production (proposition of more adapted process like fed-batch and/or biphasic systems).

Dihydroxyacetone (DHA) production from glycerol
by Gluconobacter oxydans is an industrial fermenta-
tion that was studied and developed during the 1950s
and 1960s. Studies principally looked at the meta-
bolic pathways of glycerol assimilation by acetic
bacteria, especially the characterization of enzymes
involved in these transformations and determination
of their regulation modes [8, 11, 12, 15, 18, 30].
Concerning the implementation of cultures, the
nutritive requirements of cells growing on glycerol
were defined so as to specify the optimal composi-
tion of culture medium [2, 24, 26, 29]. The process
of DHA production was described as a batch fermen-
1 Terms used herein: X(S, P), biomass (substrate, product) con-
centration; rx, rs, rv, growth (consumption, production) rate;
bt, qs, qv, specific rate of growth (glycerol consumption, DHA
production); Yx/s, Yv/s, yields of conversion of substrate into
biomass and product; Ks, affinity constant for the substrate; Kis,
constant of inhibition by the product; Sin, threshold concentration
in substrate.
Address reprint requests to: Dr. C. Claret, Laboratoire de Biotechnologie de l'Environnement des IAA, Institut National de la
Recherche Agronomique, 11100 Narbonne, France.
ration, realized at pH 6, with good oxygenation, in
a medium containing initially a high glycerol concen-
tration (about 150-200 g L-~), and it was character-
ized by a high yield of conversion of glycerol into
DHA (90%) [6, 7, 25, 28]. Since then, the only pub-
lished data have dealt with the setting up of bioreac-
tors with immobilized cells [1, 10, 13, 16, 19, 23, 31],
and a continuous two-staged system of DHA produc-
tion [14] in order to improve the performance of this
bioconversion (productivities, yields). It seems inter-
esting to update the knowledge of this fermentation by
consideration of the misunderstandings or the kinetic
aspects, which have been passed over.
One of the gaps in the bibliographic data on the
microbial conversion of glycerol into DHA is the
lack of kinetic and physiological description of this
fermentation process. In fact, in view of the high
initial glycerol concentration used, problems of inhi-
bition of the fermentation kinetics could exist. Such
a phenomenon was pointed out in some works, but
it was never characterized or detailed. These obser-
150 CURRENTMICROBIOLOGYVol. 25 (1992)

vations led us to study in detail the kinetic aspects of Table 1. Comparison of characteristic parameters obtained
during Gluconobacter oxydans cultures on glycerol at different
the glycerol fermentation by G. oxydans and their
concentrations
modelization. A previous study showed, for the first
time, that this bioconversion was subject to a double Glycerol (g L -1) 31 51 76 95 129
inhibitory effect: by the substrate and by the product.
This work concerns the kinetic and physiologi- Fermentation 12 20 30 48 78
time (h)
cal analysis and the modelization of the inhibition
Producted 1.62 1.98 1.95 1.90 1.85
by glycerol. In order to specify and to quantify the biomass (g L -s)
inhibition mode, we used cultures with increased Yx/s (%) 5.2 3.9 2.6 2.0 1.4
initial substrate concentrations. Relating to the mod- Producted 28.5 47 66 86 106
elization, the main bibliographic models that express DHA (g L -s)
YP/s (%) 92 92 87 90 82
inhibition by the substrate were applied to the exper-
rXmax (g L -1 h -s) 0.25 0.22 0.15 0.10 0.07
imental phenomenon and compared. rsmax (g L -1 h -l) 5.76 5.14 4.56 3.79 3.54
rpmax (g L -1 h I) 5.19 4.96 4.15 3.67 3.46
Materials and Methods /~max (h-l) 0.38 0.30 0.24 0.21 0.13
qSmax (g . g - I h-~) 8.73 7.49 6.75 3.07 2.76
Organism. Gluconobacter oxydans ATCC 621 was used. The qPmax (g " g-I h-l) 7.79 6.32 5.04 3.01 2.17
strain was maintained on agar slants containing glycerol, 20 g
L - 1; yeast extract, 10 g L - ~; agar, 20 g L - ~, and it was transferred Inoculation, 5.9% v/v by a 1-day-old culture; agitation, 800 rpm;
to new slants every month. aeration, 1 vvm; pH, 6; temperature, 28~

Cultures. Two hundred milliliters of inocula (medium: glycerol,

50 g L-I; yeast extract, 10 g L -1) were prepared in 500-ml flasks
and placed on an orbital shaker (130 rpm) in a thermostated room
(28~ for 1 day. The growth medium contained glycerol at various
concentrations between 31 and 129 g L i and Difco yeast extract
at 10 g L -I. The cultures were in an LSL Biolafitte bioreactor
with a total capacity of 2 L and initially contained 1.5 L of growth
medium. Inoculation was done by the sterile addition of 100
ml of precultures. The pH and the temperature were adjusted
respectively to 6 (addition to NaOH, 2 N) and 28~ The agitation
and aeration (800 rpm, 1 vvm [supplied air volume/reactor vol-
ume/mini) caused the oxygen partial pressure to be higher than
10% during all experiments. Addition of 10-fold diluted Sigma
silicone emulsion avoided the formation of foam.
Analytical methods. Estimation of biomass concentration. Opti-
cal density (OD) was measured at 620 nm with a Varian spectro-
photometer. Centrifuged samples (20,000 rpm, 5~ 10 min) were
used as blanks and for dilutions. It was found that 1 0 D unit
corresponded to 450 mg L-1 dry weight. For dry-weight determi-
nation of organisms, 40-ml samples were removed from culture,
centrifuged, washed twice with distilled water, and dried to a
constant weight at 105~
Determination ofmetabolites. Glycerol and DHA were de-
termined by HPLC after filtration and dilution of samples. A
Brownlee Polypore Calcium column thermostated at 86~ and
eluted by continuous flow (0.4 ml rain -I with a Shimadzu L6A
pump) of double-distilled, filtered, and deaerated water was used.
Metabolites were detected with a Waters refractometer, and their
concentrations were evaluated with a Shimadzu Integrator CR
3A.
Results
Analysis of glycerol effect. The influence of glycerol
content on Gluconobacter oxydans growth and its
conversion into DHA was determined from cultures
initially containing various concentrations of glyc-
erol: 31, 51, 76, 95, and 129 g L -1, under identical
experimental conditions (inoculation level 5.9% v/v,
pH 6, temperature 28~ pO2 > 10%). The evolution
of integral (X, S, P) and specific (/x, qs, qe) parame-
ters with time was determined, as characteristic pa-
rameters of each culture: DHA and biomass final
content, yields maximal kinetic parameters (Table
1).
Glycerol effect on growth. Whatever the initial
substrate content was (from 31-129 g L-1), the bio-
mass concentration at the end of the cultures was
more or less the same. As a result, the yield of
conversion of glycerol into biomass Yx/s varied in
an inversely proportional way to the initial substrate
content (Table 1). This phenomenon was imputed to
an inhibition of growth by increase of the glycerol
concentration. The hypothesis of a deficiency in nu-
triments cannot explain the growth limitation for
the following reasons: (a) abundance of the culture
medium, which contained 10 g L-1 of yeast extract;
this medium was, moreover, used by several authors
without a nutrient limitation [22, 28]; (b) low require-
ments of the microorganism for growth factors and
mineral components, as established in several pa-
pers [9, 11, 29]; (c) low quantity of produced bio-
mass, about 2 g L - l - - i n agreement with biblio-
graphic data--compared with the supply of
nutriments. Moreover, this hypothesis was verified
by comparison of cultures with 100 g L-~ of glycerol
and I0 or 20 g L-1 of yeast extract. In both cases,
the development of fermentation was the same (data
not shown) as the final biomass content (2.3 g L-1).
C. Claret et al.: Glycerol Inhibition of DNA Production 151

Biomass g.1.-' p g.g.-i .h.-'

2,5

0,3

~1. 0,2

0,1

o
0 20 40 60 80
TIME hours
0 20 40 60 80
qo g.g.-'.h. 1
Time hours 10

Glycerol g.1.- 1 8
140 6
b
120
4
10(

8(

6( 0 20 40 60 8O
40[ TIME hours
2o Fig. 2. Evolution of the specific growth rates (a) and specific rates
of DHA production (b) during cultures performed with different
o
0 20 40 60 initial glycerol concentrations: ([3) 31 g L -I, (O) 51 g L -x, (A) 76
g L -1, (O) 95 g L -I, or (*) 129 g L-l).
Time hours

DHA g.1. 1
2, Table l). The growth rate values obtained during
120 f cultures with 76 and 129 g L-~ of glycerol concentra-
100 tion represented respectively 60% and 30% of the
value determined for the culture at 31 g L -~ of
8O
glycerol.
12
60 A detailed and simultaneous analysis of growth
and residual glycerol concentration allowed us to
4O
make a distinction between the cultures containing
2 31, 51, and 76 g L - 1 of glycerol, for which the stop-
page of growth and the exhaustion of medium in
0 ~ 20 40 60 80 glycerol are concomitant; in the cultures with a
higher glycerol concentration (95 and 129 g L -1)
Time hours growth stopped, whereas glycerol still remained at
high levels in the fermentation broth: 30 g L -~ for
Fig. 1. Evolution of integral parameters: biomass (a), glycerol
the culture performed with 95 g L -1 of glycerol,
(b), and DHA (c) obtained during cultures of G. oxydans per-
formed with ([]) 31 g L -1, (~) 51 g L -1, (A) 76 g L -~, ((3) 95 g and 53 g L -1 for the one begun with 129 g L -1 of
L -I, or (*) 129 g L -1 of glycerol. substrate.
For glycerol concentrations lower than 80 g L - ~,
the decrease of the rates of biomass production, due
The effect of the increased initial content of glyc- to the increase of the initial substrate content, led
erol also appeared in a lag period for beginning of to a dominating utilization of glycerol from D H A
growth (Fig. 1). The biomass production rates and synthesis and to a total exhaustion of the medium
the growth-specific rate were strongly reduced (Fig. in glycerol, so creating the limitation o f biomass
152 CURRENT MICROBIOLOGY Vol. 25 (1992)

production. In the case of cultures with a high con- Table 2. Modelization of the inhibitory effect of glicerol on
growth: application of the models found in the literature to the
centration of substrate (up to 80 g L-l), the effect
description of the experimental observed p h e n o m e n o n
of the glycerol content on the growth rates always
occurred in a pronounced way, but the conversion Evaluation of
of substrate continued whereas the growth was Formulation of models constants i,2
stopped.
Haldane (1930)
The determination of DHA concentration in the
S K~s /Zmax = 0.88 h -~
culture (Fig. 1) showed that, at the moment growth K~s = 3 0 g L - 1 0.983
stopping, the DHA content in the medium was at 61 g = /Xm~x"(S + K S ) ' ( S + KIS) Ks = 4.2 g L -~
g L-~ for the two fermentations. The stopping of Andrews (1969)
biomass production could be imputed to the pres- 1 #max = 0.77 h
ence of DHA at a high concentration (up to 60 g /1, = /'~max" 1 + (Ks/S) + (S/KIs) Kis = 34 g L -I 0.983
L-~), for which the microbial development was com- Ks = 6.7gL
pletely blocked. Aiba (1968)
In order to dissociate the inhibitory effect due /Zmax = 0.61 h -1
to glycerol from the one linked to DHA, we com- S e(_S/Kis~ Kis = 80,5 g_L -I 0.983
/x=/Zma x (S + Ks) K s = 7.1 g
pared the growth rate determined at the 10th, for
which the DHA concentration in all the cultures Luong (1987)
did not exceed 25 g L-1. The observed decrease S Determination
of studied parameters with the increase of initial /~ = /Zmax" K
S ~+- " S " (1 - (S/SIn)~) impossible
glycerol content is attributed to an immediate sub-
strate effect on microbial development.
Glycerol effect on DHA production. The influ-
ence of the initial concentration of glycerol on the Table 3. Modelization of the inhibitory effect of glycerol on
the production of DHA: application of the models found in the
production of DHA was then studied (Fig. 1, Table
literature to the description of the experimental observed
1). For the cultures in which the initial substrate phenomenon
content varied from 31-95 g L - 1, the yield of conver-
sion of glycerol into DHA was constant (about 90%). Evaluation of
Glycerol concentration had no effect on this parame- Formulation of models constants r 2

ter. Whereas the yield was constant in this range of

Haldane (1930)
concentrations, Fig. 1 shows that the time necessary
S K~s qp max = 21,1 h - 1
for the production of 25 g L - Lof DHA significantly K~s = 27 g L-~ 0.982
increased when the initial substrate content in- qP=qPmax (S + K s ) ' ( S + KIs) Ks = 8.2gL-1
creased: 9 h were required for the culture with 31 g Andrews (1969)
L - 1of glycerol, 14.5 h for 76 g L - ~, and 33 h for 129 1 qpmax = 35.6 h -I
g L - I of substrate. From a kinetic point of view, L -1 0.968
qP = qPmax 1 + (Ks/S) + (S/KIs) KIs = 36 g L -I
a comparison between the cultures revealed that K s = 6.3 g
maximal productivities and specific rates of DHA Aiba (1968)
production were reduced by 31% and 65% respec- qPmax = 15.8 h -1
= S e( _ s/K~s/ Kls = 67.3 g L -~ 0.983
tively when the substrate content increased from qp qPmax (S + Ks) K s = 8 . 5 g L -I
31-129 g L -~ (Fig. 2, Table 1).
The characteristic parameters were determined Luong (1987)
at the beginning of the cultures, when the DHA S Determination
content in the medium was low; consequently, they qp = qPmax S + K s (1 - (S/Sin) a) impossible

are not subject to an effect of the product. These

results established that a high concentration in sub-
strate exerted a direct inhibitory effect on the kinet-
ics of conversion of glycerol into DHA, expressed
by a reduction of the levels of parameters re and qp.
The inhibition by glycerol also appears in the time
for the obtention of maximal levels of specific param-
eters: the specific rate of DHA production qv is maxi-
mal at the 8th hour for the culture with 31 g L-~ of
glycerol and at the 30th for 129 g L - 1 of substrate.
This result means that the higher the glycerol con-
centration, the more important is the time necessary
for the cells to reach their optimal level of activity.
In short, the glycerol effect on the production of
DHA is expressed by the decrease of the rate of
C. Claret et al.: Glycerol Inhibition of D N A Production 153

conversion of substrate into DHA without modifica- Table 4. Modelization of the inhibitory effect of glycerol on
growth: application of the simplified models to the description
tions of the yield of production.
of the experimental observed p h e n o m e n o n

Evaluation of
Modelization. The evidence of the double inhibi- Formulation of models constants r2
tory effect of glycerol on growth and on the cells'
Inverse equation:
ability to synthesize DHA led us to express these
phenomena in a mathematical form. The study of 1 /xm~ = 0.65 h -1
0.983
modelization concerns two specific parameters: /z = /Xrnax" 1 + (S/Kis) K~s = 44 g L -1

specific rate of growth/x and specific rate of produc- Exponential equation:

tion of DHA qp. Maximal values of parameters /~ = ]-~max " e ( - S / K l s ) /Xmax = 0.53 h -1
/x and qp were determined for each culture and 0.991
KIS = 93.6 g L -1
allowed us to calculate kinetic constants, which General equation:
are characteristic of several models proposed in
/z = b6ma x - (1 - (S/Sm) a) N o adapted form
the literature to express substrate inhibition (Tables
S m > 10,000 g L -I
2 and 3).
Models of an inverse and exponential type [20]
use a constant of inhibition by the substrate K~s.
This parameter is not involved in Luong's formula- Table 5. Modelization of the inhibitory effect of glycerol on
tion [17], which uses a critical concentration in sub- the production of DHA: application of the simplified models to
strate S m in use, corresponding to the stopping of the description of the experimental observed p h e n o m e n o n
biological activity and a factor a, expressing the
incurvation of the curves. Evaluation of
Formulation of models constants r2
For all the mathematical models studied, the
numerical values Ks are very low in regard to the Inverse equation:
substrate concentrations used in experiments, which
1 qPmax = 12.6 h -1
are higher than the limiting glycerol concentrations qP = qPmax " 1 + (S/K~s) KIS = 5 0 g L - 1
0.963
for the biological system. We propose to simplify
Exponential equation:
these equations by disregarding the term K s in front
of S, thus leading to another expression of each qP = qPmax " e(-S/KIs) qPmax = 11.7 h -~
0.994
Kis = 76.7 g L -1
model (Tables 4 and 5). Once the analogy of results
between theoretical equations and simplified expres- General equation:
sions was verified, we restricted our work to the qp = qPmax ' (1 - (S/SIn) ~) No adapted form
analysis of the latter. S m > lO,O00 g L -1
Luong's model was not suitable for the descrip-
tion of the phenomenon observed for the use of the
constant Sin. The curves /x = f(S) and qe = f(S)
approach the X-axis by an asymptotic way, which DHA production are:
makes the evaluation of Sm impossible (point of in-
tersection between the curves and the horizontal = 0.53 e(-S/93'6!
axis).
The inverse equation describes in a satisfactory forS > 3 0 g L -~
manner the experimental data for the substrate con-
centrations lower than 80 g L - ~. Beyond, especially qp = 11.7 e (-S/76'7)
for the parameter qp, the theoretical curves move
from the observed results (Figs. 3 and 4). On the The inhibitory constant K~s linked to DHA produc-
contrary, the theoretical curves obtained for the ex- tion was lower than the one calculated for growth.
ponential model express the experimental data for The maximal specific rate of DHA production
all concentrations studied (Figs. 3 and 4, Tables 4 seemed to be more affected by the initial content of
and 5). The maximal specific parameters (/z, qr) vary substrate than the microbial development. How-
in an inversely exponential way with the initial con- ever, the two constants have the same rough esti-
centration of substrate; the selected equations for mate, which shows the weak selectivity of glycerol
the description of the inhibition of growth and of the towards the two cellular activities.
154
p g.g.-'l.-'
0,6
0,4
0,2
i i
0 50 100 150
Glycerol g.l.-'
Fig. 3. Modelization of the evolution of maximal specific rate of
growth with initial concentration of glycerol: comparison between
experimental data (~), Aiba model (--), exponential equation
(--), and inverse equation (---).
qp g . g . - ' 1.-'
12,5
10
7,5
5 concentrations than at high concentrations. The
2,5
0 50 100 150
Glycerol g.l.-'
Fig. 4. Modelization of the evolution of maximal specific rate of
DHA production with initial concentration of glycerol: compari-
son between experimental data (~), Aiba model (--), exponential
equation (m), and inverse equation (---).
Discussion and Conclusion
In Acetobacter xylinum, Blazejak and Sobcjak [3]
pointed out that initial substrate contents higher than
100 g L-~ created a reduction of the yield of DHA
production. The same phenomenon was observed in
Gluconobacter oxydans by Underkofler and Fulmer
[28] and Yamada et al. [31] for different substrate
concentrations--respectively 60 and 200 g L - 1. Ya-
mada et al. [31] also indicated that the rate of DHA
production was strongly affected by an increase in
the initial content of glycerol. However, there is no
study detailing the influence of the initial concentra-
tion of glycerol on the kinetics of growth of G. oxy-
dans and of DHA production. Especially, the de-
crease of fermentative performance with an increase
of initial substrate content, observed by several au-
C U R R E N T MICROBIOLOGY V o l , 25 (1992)
thors [7, 27], could not be attributed to an immediate
effect of glycerol on biological activity or to an indi-
rect action, by accumulation of its product of con-
version, DHA.
Our work allowed us to demonstrate the exis-
tence of a direct inhibitory effect of glycerol. When
the initial concentration of substrate in the medium
increased, the maximal values of productivities (rx,
rs, rp) and specific rates (/x, qs, qe) respectively
decreased by 30% and 70%. The determination of
these parameters was realized at the beginning of
the cultures, when the DHA content was still low;
therefore, the inhibition phenomenon could be at-
tributed to the initial glycerol concentration. More-
over, the time lag observed for the maximal specific
rates tends to specify the mode of glycerol inhibition.
Thus, it seems that the substrate acts on the trans-
port of glycerol and/or nutriments from the medium
to the active sites of the enzymes involved in the
synthesis of biomass or DHA and does not exert an
immediate enzymatic inhibition.
The modelization study, which compared equa-
tions of inverse, exponential, and general forms,
shows that the inhibition phenomenon is best de-
scribed by exponential models. These express an
inhibition that is relatively more pronounced at low
value of the Kls constant involved in the modeliza-
tion of the formation of DHA was lower than the
one that characterized the growth inhibition. The
glycerol effect is thus more marked on the produc-
tion of DHA than on the cellular development. This
difference of sensibility can have two origins: on
the one hand, with the glycerol flow converted into
biomass lower than the one used for DHA produc-
tion, disturbances of the substrate supply will mod-
ify the product synthesis more highly than growth;
on the other hand, the different localization in the
cell of the enzymes implicated in the two biological
activities--glycerol dehydrogenase, which cata-
lyzes the glycerol oxidation into DHA, is membrane-
associated, while the enzymes responsible for the
formation of biomass are cytoplasmic--must be
considered for a better understanding of the phe-
nomenon.
While kinetic parameters are subject to a direct
inhibition linked to the substrate, the yield of DHA
production is not affected by the initial concentra-
tion of glycerol. Bibliographic data put forward a
decrease of the conversion yield for the high glycerol
concentrations, but the lack of kinetic data does
not allow us to differentiate an inhibitory effect of
glycerol from an inhibitory effect due to DHA. Ac-
cording to our previous work [4, 5] and the results
C. Claret et al.: Glycerol Inhibition of DNA Production
obtained during this study, DHA had an inhibitory
effect more pronounced than glycerol towards
growth; we may assume that the decrease of the
yield of production was caused by the accumulation
of DHA at the end of the cultures.
Nabe et al. [21] established that the contact of
G. oxydans cells with high concentrations of DHA
could modify the stability of the glycerol dehydroge-
nase, the key enzyme of the glycerol oxidation.
Moreover, a specific study dealt with the effect of
DHA on cellular activity and confirmed this hypoth-
esis. In consequence, this study allowed us to com-
plete the kinetic and physiological knowledge of
glycerol conversion into DHA by G. oxydans. The
results obtained were then applied to the determina-
tion of the fermentation process in order to improve
the performance of this bioconversion.
The fed-batch fermentation mode, the best ad-
justed system in the case of inhibition by the substrate,
was studied, and the fermentation performances were
thus improved. Because of the simultaneous existence
of an inhibition due to DHA, modifications of the fed-
batch process were then considered in order to take
into account the two inhibitions. Thus, an optimized
system of DHA production by means of a microbia/
way can be proposed.
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