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1. Introduction
0340--2118/78/0006/0029/~ 01.80
30 G. Braunegg et al.
exact regulation of external physical and chemical parameters such as the oxygen par-
tial pressure or the C/N ratio will be dependent on the speed and accuracy of the PHB
determination. The existing methods (Lemoigne, 1926; Williamson and Wilkinson,
1958; Slepecky and Law, 1969; Ward and Dawes, 1973; Jiittner et al., 1975) for PHB
determinations are either relatively inaccurate or extremely time-consuming. In most
cases either all cell material, with the exception of PHB, must be fully destroyed or the
PHB itself must be extracted from the cells prior to determination. Both procedures
possess considerable inherent sources of error.
The development of biotechnological processes on the basis of process kinetics de-
pends to a great extent on the quantitative determination of all fermentation parameters
(Moser and Lafferty, 1976). In order to analyze the kinetics of microbial PHB syn-
thesis for the optimization of the process, the analytic methods must be characterized
by a high degree of reproducible accuracy. The determination of the kinetics of PHB
synthesis necessitates the analysis of a large number of samples from the fermentation.
The amount of work and the time required for analyses must, therefore, be reduced
to a minimum. This aspect assumes a vital importance especially when the intermittent
analysis of fermentation products is to be employed for control purposes.
An extensive testing of all described analytic methods for determining PHB revealed
that these do not fulfill the previously mentioned requirements. Either poor accuracy
prohibited the exact determination of the process kinetics or the time required - up
to 24 h and more - made a process control almost impossible. The aim of this ex-
perimental work, therefore, was to design and perfect a method for the determination
of PHB in order to fulfill the previously described requirements. These seem to be ad-
equately met by using a gas chromatographic method.
The basis of the method of Slepecky and Law (1969) is that PHB is depolymerized
and dehydrated b y sulfuric acid. The reaction product, crotonic acid, could be further
converted to crotonic acid methylester, which can then be determined using gas chro-
matography. In the course of these investigations it was found though that this simple
method gave satisfactory results only with an undue amount of effort.
However, under mildly alkaline conditions PHB can also be depolymerized (Fukui
et al., 1976); whereas our experiments revealed that a depolymerization also occurs
even under mildly acidic conditions. In both cases 3-hydroxybutyrate is the product
of the reaction.
for cultivation. The temperature was 30oc, the pH maintained at 7.0 using an automat-
ic pH-measuring and control system (Jungkeit, Meredos, type PH/R, Gdttingen, FRG).
The oxygen partial pressure during cell cultivation in the fermentor was measured
using a polarographic oxygen electrode (Instrumentation Laboratories, type IL 530,
Ingold, Frankfurt am Main, FRG) and the concentration of dissolved oxygen was main-
tained between 3 and 5 rag/1 by regulating the sterile air flow and/or the impeller speed.
2.2. Cbemicals
Poly-[J-Hydroxybutyric Acid (PHB) was first extracted from acetone-dried cells of
Alcaligenes eutropbus strain H 16 with chloroform at 25oc. PHB was then precipi-
tated from the filtrate at low temperatures (+4oc) using cold acetone. This initial pre-
cipitate was subsequently purified three times by dissolving the PHB in chloroform
then reprecipitating. The precipitated PHB was washed each time using analytic grade
diethylether. The PHB obtained in this manner corresponded in purity to that de-
scribed by Jiittner et al. (1975).
Column Materials
The internal diameter of all glass columns used was 0.1 in. An 8-ft column was filled
with 2% Reoplex 400 (Merck, Darmstadt, FRG) on Chromosorb GAW 60 to 80 mesh,
whereas as a 6-ft column, filled with Carbowax M 20 TPA on Chromosorb WAW
DMCS 80 to 100 mesh was purchased directly from Hewlett-Packard, Vienna, Austria.
All samples to be analyzed were prepared in screw-capped borosilicate glass test tubes
with teflon seals (Coming Glassworks, New York, USA).
L_
l p ' ' ' I , ~ ,
0 5 10 0 5 10
Retention time(min) Retention time (rain)
Fig. la and b. GC analysis of 3-hydroxybutyric acid methylester obtained by the acidic meth-
anolysis method, a prepared from pure D,L-3-hydroxybutyric acid and/or from purified PHB.
b prepared from a suspension of Alcaligeneseutropbus strain H 16
A Rapid Gas Chromatographic Method 33
20 _ p
15
u
"6
Fig. 2. Calibration of the gas chromatog-
10 raphic determination of PHB. Different
n'l
T amounts of PHB were treated according
I1
to the acidic methanolysis method as
described. The amount of PHB per in-
5 jected sample was plotted versus PHB
detected (areas of peaks) and shows a
linear relationship with excellent correla-
H I I tion. Injection volume: 2 #1
5 10 15 20
/.z,g P H B / i n j e c t i o n
Quantitative determinations were done by evaluating peak areas. PHB was quanti-
tatively recovered to the extent of 100% in the form of the 13-hydroxybutyric acid
methylester when using/3-hydroxybutyric acid as a test substance. A linear relation-
ship between peak area and concentrations of up to 20/~g PHB in each sample was
determined: it exhibits a correlation coefficient: r = 0.999 (Fig. 2).
100
##
#
9O
8O
7O
60
o~
50
z~0
,{
A / 1 ~ 1 7 6
3O
20
J
10
__A / I I I J t
0 1 2 3 4
Incubation time (hours)
Fig. 3. Influence of reaction time at 100~ and H2SO 4 concentration (1% A, 2% O, 3% i, and
4% 9 v/v) on the yield of 3-hydroxybutyric acid methylester from PHB
cell suspension at 100oc reduces the sensitivity of the PHB determination; longer re-
action times, however, lead to the formation of secondary products. As such they
cause no difficulties, neither for the qualitative nor for the quantitative determina-
tion of the 3-hydroxybutyric acid methylester, but they do necessitate frequent heat-
ing of the columns.
worked out with strains H 16 and M 7 ofAlcaligenes eutropbus. To test the validity of
the method for determining PHB in other organisms, cells ofMycoplana rubra were
also used. With this organism there was an excellent correlation between the gas chro-
matographic method and the other methods for determining PHB.
3.4. Discussion
Although the determination of PHB using the method of residual optical density
(Williamson and Wilkinson, 1958) is a relatively rapid method, it does possess inherent
disadvantages. The results are dependent on the size, form, and number of PHB granula
and also on the homogeneity of the cell suspension. This method is, therefore, proba-
bly more suitable for routine checking and as an indicator of the approximate PHB con-
tent of the cell suspension. All other methods described for PHB determinations are
based on the preliminary extraction of PHB from the cell material. In addition, the
determination of PHB using either the method of Slepecky and Law (1969) or that of
Ward and Dawes (1973) can be negatively influenced by small amounts of impurities
in the reagents used. The quantitative extraction of PHB from cells using chloroform
requires in general a period of 24 h or longer. Furthermore, the reproducibility of the
PHB extraction depends to a great extent on the physical properties of the cell material,
10
oD
o~
10
7:3
>.
~J c
R
f 0.1 g
o
r~
o
a_
.v
/;
0.1
/
0.01
0 5 10 15 20 25
Fermentotion time [hours)
i.e., whether freeze dried, heat dried, finely ground, etc. This fact alone necessitates a
large number of parallel samples, which must be statistically evaluated in order to re-
ceive significant values. In the case of the infrared spectographic determination of PHB
(Jfitmer et al., 1975), lipids which could falsify the PHB determination must also be
removed from the sample prior to analysis. The gravimetric determination of precipi-
tated PHB is characterized by large inherent errors when the PHB concentration is low.
To compensate for this negative fact, large sample volumes are required. If a laboratory
fermenter is being employed, it is very questionable whether or not a gravimetric PHB
determination can be used at all for accurately determining the kinetics of the product
synthesis. One significant advantage of the described GC analysis of the PHB content
of cell suspensions is the fact that only relatively small sample volumes (0.5-5.0 ml)
are required. Furthermore, once the sample is collected, all subsequent operations
take place using the same screw-capped test tube. Sample preparation only involves
centrifugation and preparation of the methylester derivative. On the basis of these
facts, both the possible sources of error and the time for analysis are reduced to a min-
imum. At the same time both the accuracy and the reproducibility of the GC method
is better than in the case of the other methods quoted. It is apparent from Fig. 4
that the determination of low concentrations of PHB using gas chromatography is
very superior to other methods, whereas in the case of higher PHB concentrations
there is good agreement, for example, with the gravimetric method. In the case of
Slepecky and Law's method (1969), an accurate objective comparison between the GC
determination of PHB and the former method can be based only on a statistical evalua-
tion of the first method. The GC determination of PHB was elaborated using cells of
Alcaligenes eutropbus, strains H 16 and M 7. However, to demonstrate the reliability
of the method with a different bacterial strain, Mycoplana rubra was tested with fully
satisfactory results. One can therefore assert with a considerable degree of certainty
that this method should be applicable to other prokaryotic organisms.
Acknowledgements. Our thanks are due to the "Fond zur Fbrderung der wissenschaftlichen
Forschung" (project number 3296) for having supported this work.
References