European 9

European J. Appl. Microbiol. ,.....,o,A p p h e d

Biotechnol. 6, 2 9 - 3 7 (1978) Microbiology a n d
Biotechnology
9 by Springer-Veriag9 I978

A Rapid Gas Chromatographic Method for the

Determination of Poly-~-hydroxybutyric Acid
in Microbial Biomass

G. Braunegg, B. Sonnleimer, and R.M. Lafferty

tnstitut fiir Biochemische Techno~,ogieund Lebensmittelchemie der Technischen UnSversit~tGraz,

Schl6gelgasse 9, A - 8010 Graz, Austria

Summary. The gas chromatographic method for the determination of poly-

/3-hydroxybutyric acid (PHB) consists of a mild acid or alkaline methanolysis
of poly-~-hydroxybutyric acid directly without previous extraction of PHB
from the cells; this is followed by gas chromatography of the 3-hydroxybutyric
acid methylester. The method is characterized by high accuracy and excellent
reproducibility, permitting determinations as low as 10 -5 g/1. Only 4 h is re-
quired from sampling from the fermenter till completion of the PHB determina-
tion.

1. Introduction

Poly-/3-hydroxybutyric acid (PHB) is a carbon reserve material universally found in

many genera of bacteria particularly under certain growth conditions, such as a high
carbon-to-nitrogen ratio (C/N ratio) of the medium (Lundgren et al., 1965). Chemical-
ly it is a polyester of D (-)-3-hydroxybutyric acid occurring in microorganisms in the
form of crystalline granula, at amounts of 65% or more of the dry weight of the cells
(Cornibert and Marchessault, 1972; Dawes and Senior, 1973). If microbial biomass is
to be used as a source of single-cell protein, it is logical that the intracellular synthesis
of poly-/3-hydroxybutyric acid would directly decrease the biological value of the bio-
mass in the case of monogastric animals and indirectly in the case of ruminants. The
presence of PHB would, therefore, be a detrimental factor in evaluating the biological
value of microbial biomass. On the contrary, poly-/3-hydroxybutyric acid can be easily
extracted from bacterial cells and could possibly find application as a biodegradable
substance characterized by a number o f physical properties related to conventional
plastics now in use (Heinzle and Lafferty, 1977).
It is also possible to use poly-/3-hydroxybutyrate to make copolymerizates with
other substances which may not in themselves be biodegradable (Siapkas, 1976). In
tbis case PHB would be the desired product, the yield of which is to be optimized.
An accurate rapid method to determine the PHB content of biomass is, therefore,
of equal importance whether the microbial synthesis of PHB is desirable or not. The

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.

2. Materials and Methods

2.1. Organisms and Media

Alcaligenes eutropbus strain H 16 (Wilde, 1962) and a mutant strain H 16/M 7 (Srienc,
1977) which possesses a higher rate of PHB synthesis in comparison to the parent strain
when both strains are grown under chemoorganotrophic conditions were cultivated
using a mineral medium (Schlegel et al., 1961) with fructose or lactate as a carbon
source up to 2% w/v. Mycoplana rubra strain R 14 was grown in the same mineral me-
dium with 0.5%-1% (v/v) methanol as a carbon source. Both shake cultures in Erlen-
meyer flasks and a fermenter unit (Bioengineering t y p e L 1523, RiJti/Switzerland) of
18 1 total volume, equipped with two conventional turbine-type impellers, were used
A Rapid Gas Chromatographic Method 31

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).

3-Hydroxybutyric Acid. The sodium salt of D, L-3-hydroxybutyric acid (Fluka, Buchs,

Switzerland) was vacuum dried and kept over P205 . All other chemicals used were of
analytical grade quality (Merck, Darmstadt, FRG). Carrier gas (N2) , synthetic air and
hydrogen for the GC flame ionization detector system (FID) were of 99.999% purity
(Messer-Griessheim, FRG).

2.3. AnaIytical Metbod

A computer-controlled gas chromatograph (Model HP 5840 A, Hewlett-Packard, USA)
fitted as an all-glass system and equipped with a double FID was used for all analyses.
In addition, an autosampler (Model HP 7671 A, Hewlett-Packard, USA) equipped with
Hamilton syringes (Model 701 N, 10 gtl, Hamilton, USA) was employed to inject all
samples.

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).

2.4. Experimental Conditions

The 8-ft Reoplex column was used under the following conditions: Initial temperature
(T-l) = 90oc; final temperature (T-2) = 150oc. The holding time for the initial tem-
perature was 1 min, for the final temperature 5 min; the temperature rate of increase
was 8oC/min; the carrier gas flow rate was 30 ml N2/min. The 6-ft Carbowax-column
was employed at T-1 = 100oc with a carrier gas flow rate of 20 ml N2/min. All other
conditions corresponded to those used with the Reoptex-column.
32 G. Braunegg et al.

3. Results and Discussion

3.1. Preparation of Derivatives and Analysis of Pure Substances

All 3-hydroxybutyric acid samples are dissolved in 2 ml of acidified methanol, i.e.,
containing 3% (v/v) H2SO4, and 1 ml of chloroform in a screw-capped test tube. The
samples are then kept at 100oc for 60 rain. After cooling to ambient temperature,
1 ml distilled water is added and the whole sample is then shaken for 10 rain, after which
the two phases are permitted to separate. The organic phase is subsequently used for
the GC analysis. The methyl ester of 3-hydroxybutyric acid was prepared directly
from PHB by one of the two following procedures:
(i) PHB is added to a mixture of 2 ml methanol acidified with H2SO 4 (3% v/v) and
2 ml chloroform. The preparation is heated in a screw-capped glass tube at 100oc for
4 h and then cooled to ambient temperature; 1 ml distilled water is then added and
the whole sample shaken well for 10 rain. After separation of the two phases 2/21 of
the organic phase is injected for GC analysis.
(ii) PHB is mixed with 2 ml methanol which is 1 N with respect to KOH and held
at a temperature of 100oc for 1 h. After cooling to ambient temperature, 1 ml chlo-
roform and 1 ml H 2 0 are added and the sample intensively shaken for 10 rain. After
separation of the two phases 2/21 of the organic phase is used for sample injection. In
this case, the yield of the methylester of 3-hydroxybutyric acid reached an optimum
following heat treatment for 1 h. However, the absolute yield of the methylester was
considerably less in comparison to treatment of PHB with acidified methanol. The
results of the GC analysis are shown in Figure 1.

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).

3.2. Analysis of Cell Suspensions

The only pretreatment of cell samples is simply centrifugation, the time for which can
be considerably shortened by the addition of methanol to the suspension; this is par-
ticularly so when the cells contain large amounts of PHB. After decanting the liquid
phase, the cells can be directly subjected to the acidic methanol treatment prior to GC
analysis. Optimal conditions for this direct procedure were determined by parameter
variation (Fig. 3) and arc as follows. Centrifuged cells are suspended in a mixture of
2 ml methanol (3% H2SO4, v/v) and 2 ml chloroform, then heated at 100oc for 3 1/2 h.
After cooling to room temperature, 1 ml H 2 0 is added and the sample shaken vigorous-
ly for 10 min. The two phases are allowed to separate, during which cell detritus gath-
ers at the interphase. The organic phase which is to be analyzed is placed in vials and
can be stored unchanged at 4 o c for several weeks.
The accuracy and reproducibility of this method can be increased as usual by em-
ploying an internal standard. Benzoic acid was found to meet all the requirements of
an internal standard substance. The retention time of the benzoic acid methylester is
approximately 2 rain longer than the retention time of the 3-hydroxybutyric acid
methylester. For use as an internal standard benzoic acid is simply added to the acid-
ified methanol reagent.
Within the linear range of the detector used on the GC apparatus, the results of
this method for PHB determination depend neither on the volume of the cell suspen-
sion nor on the PHB content of the cells; the maximum standard deviation was -+ 0.5%.
The reliability of the method is determined only by the accuracy of the measurement
of the initial volume of the cell suspension. Shortening the time of treatment of the
34 G. Braunegg et al.

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.

3.3 Verification of the Method

It was found that there is no difference between the retention times on two different
columns using either 3-hydroxybutyric acid or pure PHB. The addition of known
amounts of 3-hydroxybutyric acid to pure PHB or vice versa, and subsequent GC
analysis using the previously described method (alkaline or acid methanolysis) resulted
in only a single peak with identical retention time. The peak area varied only in pro-
portion to the added amount of 3-hydroxybutyric acid or PHB. Peak areas show a
linear relationship to the volume of the cell suspension used (standard deviations
+ 0.2%) and are independent of the density of the cell suspension and the PHB con-
tent of the cells. Free 3-hydroxybutyric acid present in the cells prior to treatment
and analysis could theoretically lead to increased values for the determination of
PHB. To clarify this question, cells of Alcaligenes eutropbus (strain H 16) from various
growth stages were treated with lysozyme (Schwab, 1977) and 3-hydroxybutyric acid
was both enzymatically and gas chromatographically determined. In a series of experi-
ments it could be shown that in each case the maximum amount of 3-hydroxybutyric
acid was less than 1% of the PHB present in the same cells. The method was originally
A Rapid Gas Chromatographic Method 35

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)

Fig. 4. Time course of intracellular PHB during chemoorganotrophie growth of Alcaligenes

eutrophus strain H 16/M 7: PHB determination by different analytic methods, o - - e Gas chro-
matographic method; o Residual optical density (Williarnsonand Wilkinson, 1958); O Photo-
metric determination of crotonic acid (Slepecky and Law, 1969);v---* Modified gravimetric
method: 50-mlsample volumes were freeze dried and PHB determined according to Lemoigne
(1926). Growth conditions: mineral medium with lactic acid (SO = 30 g/I); concentration of
dissolved oxygen was maintained between 3-5 rag/1 by variation of air flow rate and impeller speed
(600-1500 rpm). Fermenter: L 1523, Bioengineering, Riiti/Switzerland, total volume 18 1, two
turbine-type impellers
36 G. Braunegg et al.

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

Cornibert, V., Marchessault, R.H. (1972): J. Mol. Biol. 7 1 , 7 3 5 - 7 5 6

Dawes, E.A., Senior, P.J. (1973): Adv. Microb. Physiol. 10, 1 3 5 - 2 6 6
Fukui, T., Yoshimoto, A., Matsumoto, M., Hosokawa, S., Saito, T., Nishikawa, H.,
Tomito, K. (1976): Arch. Microbiol. 110, 1 4 9 - 1 5 6
Heinzle, E., Lafferty, R.M. (1977): Chem. Rundschau 30 (41), 1 4 - 1 6
Jfittner, R.R., Lafferty, RM., Knackmuss, H.J. (1975): Europ. J. Appl. Microbiol.
1,233--237
Lemoigne, M. (1926): Bull. Soc. Chim. Biol. 8 , 7 7 0
Lundgren, D.G., Alper, R., Schnaitman, C., Marchessault, R.H. (1965): J. Bact.
89 (1), 245-251
Moser, A., Lafferty, R.M. (1976): 5th Intern. Ferment. Sympos., Berlin, Abstr. p.
103
A Rapid Gas Chromatographic Method 37

Schlegel, H.G., Gottschalk, G., Kaltwasser, H. (1961): Arch. Microbiol. 38,209-222

Schwab, H. (1977): Personal Communication
Siapkas, S. (1976): Diplomarbeit T.U. Graz
Slepecky, R.A., Law, J.H. (1969): Anal. Chem. 32, 1697-1699
Sfienc, F. (1977): Personal Communication
Ward, A.C., Dawes, E.A. (1973): Anal. Biochem. 52,607--613
Wilde, E. (1962): Arch. Microbiol. 43, 109-137
Williamson, D.H., Wilkinson, J.F. (1958): J. Gen. Microbiol. 19,198-209

Received May 2, 1978