Communication to the Editor

Optimization of Microbial
Poly(3-hydroxybutyrate)Recovery Using
Dispersions of Sodium Hypochlorite
Solution and Chloroform

Sei Kwang Hahn, Yong Keun Chang,* Beom So0 Kim, and Ho Nam Chang
BioProcess Engineering Research Center and Department of Chemical
Engineering, KAIST, 373- 1, Kusung-dong, Yusung-gu, Taejon, 305-70 1,
Korea
Received August 17, 1993/Accepted January 17, 1994

Optimization was carried out for the recovery of PHB synthesis are well understood. Using Alcaligenes
microbial poly(3-hydroxybutyrate) (PHB) from Alcali- eutrophus, PHA is now being produced industrially, on a
genes eutrophus. This process involved the use of a fairly large scale, by ZENECA (formerly ICI) in the United
dispersion made of sodium hypochlorite solution and
chloroform. The dispersion enabled u s to take advantage Kingd~m.~,"These polymers are commercially available
of both differential digestion by hypochlorite and solvent under the trade name Biopol. However, the use of bio-
extraction by chloroform. The PHB recovery (%) from logically produced PHAs as polymers is currently limited
cell powder was maximized using a 30% hypochlorite owing to their high production costs. The difficulty of
concentration, a 90-min treatment time, and a 1 : 1 (v/v) PHB recovery from microorganisms has been the primary
chloroform-to-aqueous-phase ratio. Under these optimal
conditions, the recovery was about 91% and the purity technical obstacle to its commercial exploitation. A number
of recovered PHB was higher than 97%. The number of different methods for the recovery of PHB, which is
average molecular weight, Mn, of recovered PHB was formed within a cell's cytoplasm as granular inclusions,
about 300,000 and the weight average molecular weight, have been s ~ g g e s t e d . ~ ~ ~ . ' ~ . ' ~ , ' ~
M,, was about 1,020,000, compared to the original M, of The majority of separation processes that have heretofore
530,000 and M, of 1,272,000. The moderate decrease in
both M,, and Mwmight be ascribed to the shielding effect been proposed involve the extraction of PHB from the cells
of chloroform. In addition, the relatively small decrease with solvent^.^,^ For example, PHB can be extracted from
in M, probably resulted from the loss of short PHB bacterial cells with methylene chloride, propylene carbon-
chains which might be water soluble. The crystallinity of ate, dichloroethane, or chloroform. The polymer solution
recovered PHB was in the range of 60 to 65%, although containing more than 5% (w/v) PHB is very viscous, so
a slightly higher crystallinity was observed when the
dispersion was used. 0 1994 John Wiley & Sons, Inc. the removal of cell residue becomes very difficult. Further-
Key words: poly(3-hydroxybutyrate) PHB recov- more, although the solvent is recovered for reuse, it incurs
ery sodium hypochlorite chloroform Alcaligenes a significant raw material cost."
eutroph us As an alternative to the solvent extraction, aqueous
enzymatic digestion methods have been developed by
ZENECA." These processes consist of thermal treatment
INTRODUCTION of PHA-containing biomass, enzymatic treatment, and
Poly(3-hydroxyalkanoate) (PHA) is a biodegradable, bio- washing with an anionic surfactant to dissolve non-PHB
compatible, microbial thermoplastic which is regarded as cell materials (NPCMs). They are very efficient when a
a potentially useful polyester replacing petroleum-derived less pure product can be tolerated for some applications
thermoplastic^.^,",'^ Poly(3-hydroxybutyrate) (PHB), which of PHB. However, they often require additional digestion
is the best known member of the PHA series of polyesters, or solvent extraction steps for increasing product purity,
shows similarities in its physical properties and even in rendering the recovery cost higher.
its molecular structure to isotactic polypropylene. It is Another separation process that has been proposed in-
accumulated by many species of bacteria as a carbon volves a differential digestion method employing sodium
or energy storage Alcaligenes eutrophus has hyp~chlorite.~,'~ Although this treatment is effective in the
been widely used for the production of PHB because it digestion of NPCM, at the same time it causes severe degra-
is easy to grow, it accumulates large amounts of PHB, dation of PHB, rendering the PHB unsuitable for many
and its physiology and genetic information leading to applications. Berger et al. reported that PHB of 95% pu-
rity with a weight average molecular weight of 600,000
* To whom all correspondence should be addressed. was recovered from Alcaligenes e ~ t r o p h u s The . ~ original

Biotechnology and Bioengineering, Vol. 44, Pp. 256-261 (1994)

0 1994 John Wiley & Sons, Inc. CCC 0006-3592/94/020256-6
molecular weight was 1,200,000. This was accomplished by was maintained at 15 g/L using an on-line glucose ana-
optimizing the conditions of sodium hypochlorite digestion lyzer. The pH was controlled with NaOH at pH 7. PHB
and by balancing the ratio of hypochlorite to NPCM. was accumulated under a nitrogen-limited condition. After
In order to take advantage of both differential digestion fermentation, the fermentation broth was concentrated by
and solvent extraction, we used dispersions of sodium centrifugation, washed twice, and then freeze-dried. The
hypochlorite and chloroform for efficient PHB recovery resulting cell powder was stored at 4°C until needed.
from Alcaligenes eutrophus. In a previous study, we
could see the possibilities of using the dispersion for PHB Recovery by Using Chloroform
PHB recovery.' The hydrophobicity of PHB and the
hydrophilicity of the cell membrane form the basis of Freeze-dried cell powder was washed with hot acetone for
this technique. Sodium hypochlorite isolates PHB from 20 min. After drying, the cell powder was mixed with
the cells in the aqueous phase. Then, the released PHB 50 vol chloroform for 48 hr at 25°C. A clear polymer
immediately migrates to the chloroform phase where the solution was recovered by centrifugation to remove the
chloroform, at least partially, protects the PHB molecules majority of the NPCM, followed by a polishing filtration.
from further destructive action by the hypochlorite. In Finally, pure PHB was obtained by nonsolvent (5 vol
addition, hypochlorite not only isolates PHB from the chloroform) precipitation and filtration. The nonsolvent
cells but also digests most of the NPCM including nucleic used was a mixture of methanol and water (7:3 v/v).
acids, so that the cell residue can be removed readily from This method was used only for analytical purposes in the
the solution of extracted PHB. This article describes the present study.
advantages of using the dispersions and the optimization of
treatment conditions. PHB Recovery by Using Sodium Hypochlorite
Sodium hypochlorite solution was diluted with distilled
MATERIALS AND METHODS water. The hypochlorite concentration was 30% (v/v). Af-
ter mixing PHB-containing biomass with the hypochlorite
Microorganism solution, PHB was separated from the aqueous fraction
Alcaligenes eutrophus (NCIMB 11599) was used for the containing debris by centrifugation. The PHB was
production of microbial PHB. rinsed with water, centrifuged again, and then rinsed with
acetone. The biomass concentration in the suspension was
4% (w/v) and the treatment time was 150 min at 30°C.
Medium
The composition of the growth medium was 40 g/L glu- PHB Recovery by Using Dispersions of
cose, 2 g/L (NH4)2S04,0.2 g/L MgSO, . 7H20, 1.5 g/L Sodium Hypochlorite and Chloroform
K&PO4,9 g/L Na2HP04 * 12H20,60 mg/L ferrous am- Eight grams of cell powder was treated with dispersions of
monium citrate, 10 mg/L CaC12 * 2H20, and 1 mL/L 100 mL chloroform and 100 mL diluted sodium hypochlo-
trace element solution which consisted of 0.3 g/L H3B03, rite solution of various concentrations. The hypochlorite
0.2 g/L CoC12 6H20,O.l g/L ZnS04 * 7H20,30 mg/L concentrations tested were 15, 20, 25, 30, and 35% (v/v),
-
MnC12 . 4H20,30 mg/L NaMo04 2H20, and 20 mg/L and the corresponding pH values were 11.66, 11.87, 12.04,
-
NiC12 6H20.14 12.15, and 12.20. After treatment at 30°C for 150 min, the
dispersion was centrifuged at 8000 rpm for 10 min at 30°C.
Chemicals We obtained three separate phases. The upper phase was
hypochlorite solution, the middle phase contained NPCM
Sodium hypochlorite solution was purchased from Junsei and undisrupted cells, and the bottom phase was chloroform
Chemical Co. in Japan and used as a stock solution for containing PHB. First the hypochlorite solution phase was
diluted sodium hypochlorite solutions. It contained 5.68 g removed with a pipette, and then the chloroform phase
active C1, 7.8 g NaOH, and 32 g Na2C03 in 100 mL. was obtained by filtration (if a very high purity is not
Reagent-grade acetone, methanol, and chloroform were also desired, then the chloroform phase can be directly obtained
purchased from Junsei Chemical Co. in Japan. High per- with a pipette after centrifugation). Afterward, the PHB
formance liquid chromatography (HPLC) grade chloroform was recovered from the chloroform phase by nonsolvent
was purchased from J.T. Baker in U.S. for analysis with precipitation and filtration.
gel permeation chromatography (GPC).
Determination of Purity and Recovery
Production and Storage of Purity of recovered PHB and recovery (%) were determined
PHB-Containing Biomass
using the methylation method of Braunegg _ _ et a1.6 The
Alcaligenes eutrophus was grown in a 5-L fermentor by amount of resulting methylester detected by gas chromatog-
fed-batch fermentation at 3OoC.l7 Glucose concentration raphy (GC) reflects the purity of recovered PHB. Benzoic

COMMUNICATION TO THE EDITOR 257

acid was used as the internal standard. PHB recovered by
the chloroform extraction method was further purified by
another extraction step using the dispersion and used as a
standard for PHB purity analysis. The purity of the standard h
C
PHB could be confirmed by the Lowry method for analysis
of residual protein content." Only trace amount of residual
z.
E -
proteins was detected from the standard PHB. The PHB .-m f
a
recovery (%) was calculated from the known amount of
PHB in the cell powder measured by GC and the total
amount of PHB recovered.
-0a
Determination of Molecular Weight and
Molecular Weight Distribution
s
Changes in the molecular weight and molecular weight
0
distribution of recovered PHB were analyzed using the 0 30 60 90 120 150
universal calibration method. The gel permeation chromato- Treatment time (minutes)
graph (Waters 150-CV models) was operated at 40°C for
the analysis. Three columns of pore size lo3, lo4, and los Figure 1. Effect of treatment time on the number average molecular
weight of PHB recovered from Alcaligenes eutrophus using a 30%
were connected serially. Monodisperse polystyrene was
sodium hypochlorite concentration: (H) sodium hypochlorite alone;
used as a standard and chloroform was used as a mobile ( 0 )dispersion.
phase. One hundred microliters of a 0.1% (w/v) solution
of the PHB sample was injected for each analysis.
Figure 2 shows the purity of PHB produced with in-
creasing treatment time. The zero treatment time data in
Determination of Crystallinity and
Melting Temperature Figure 2 indicate that the amount of PHB accumulated by
fermentation was about 70% (w/w) of the total cell mass.
To evaluate the effect of the dispersion on the thermal Although the purity of PHB recovered by using only sodium
property of the recovered PHB, we measured the melting hypochlorite solution was as high as 90%,it was considered
temperature and the enthalpy of fusion with a differential to be unsatisfactory. Using the dispersion, however, the
scanning calorimeter (Dupont 2000). We assumed the en- purity was higher than 97% as long as the treatment time
thalpy of fusion of a 100% crystalline (theoretical) sample was longer than 20 min.
was 146 J/g.3 From the enthalpy of fusion, we could The treatment time was optimized in terms of recovery
calculate the crystallinity of PHB. (%) for several hypochlorite concentrations, as specified
in Figure 3. For the first 30 min of mixing, a very large
RESULTS AND DISCUSSION fraction of PHB was released from the cells to chloroform.

After fermentation, the cell concentration was 100 g/L

l
and the amount of PHB accumulated was about 70%
(w/w) of the total cell mass. Using dispersions of sodium
hypochlorite solution and chloroform, we could effectively
recover PHB from freeze-dried cell powder. Hypochlorite
concentration, treatment time, and the ratio of chloroform
to aqueous phase were optimized to maximize the PHB n
"1 /
recovery (%).
The number average molecular weight (M,) of PHB
from chloroform extraction was 530,000 corresponding
P
to the zero treatment time data in Figure 1. It is as-
sumed that its intact M , was 530,000 because chloro-
70
form extraction was known to cause negligible degradation
of PHB.4 Using only sodium hypochlorite solution for
PHB recovery, the molecular weight decreased drastically
with increasing treatment time due to severe degradation
of PHB molecules. However, the treatment with disper-
6 0 ; f ' ' 30
' ' ' 60
' ' '

Treatment time (minutes)

90
I " 120
' '
'
150
sions of sodium hypochlorite solution and chloroform re-
sulted in a significant reduction in the degradation of Figure 2. Effect of treatment time on the purity of PHB recovered from
PHB molecules probably owing to the shielding effect by Alcaligenes eutrophus using a 30% sodium hypochlorite concentration:
chloroform (Fig. 1). (H) sodium hypochlorite alone; (0)dispersion.

258 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 44, NO. 2, JUNE 20, 1994
 Figure 4 shows the values of the recovery (%) according
1 I to the hypochlorite concentration for different treatment
times. Up to 90 min of treatment, the maximum recov-
ery (%) increased with increasing hypochlorite concen-
trations. However, when the treatment time was longer
than 120 min, the maximum recovery (%) decreased con-
siderably. From Figures 3 and 4, it can be seen that the
treatment time and the hypochlorite concentration in the
dispersion affected the recovery (%) in a similar manner.
We concluded that the optimal hypochlorite concentration
was about 30% and the optimal treatment time was about
90 min.

501
404.
10
-
40
1 I

70
- 100
7 a
130
r ' I
160
With a 30% hypochlorite concentration and a 90-min
treatment time, we optimized the ratio of chloroform to
the aqueous phase (Fig. 5). The recovery (%) increased up
to a ratio of 1, but ratios higher than 1 gave no increase.
Treatment time (minutes) This result shows that the optimal ratio of chloroform to
aqueous phase was about 1.
Figure 3. Effect of treatment time on the recovery (%) of PHB from
Figure 6 shows the number average molecular weight
Alcaligenes eutrophus for different sodium hypochlorite concentrations:
(m)15%; (+) 20%; (A)25%; (0)30%; (x) 35%. (M,) of PHB according to the treatment time. Its intact M,
was 530,000. With increasing treatment time, M, decreased.
With a 35% hypochlorite concentration, the decrease in M ,
It seemed that the hypochlorite action was very effective was significantly greater than those with 25% and 30% con-
and immediate. In the cases of 15,20, and 25%hypochlorite centrations. Interestingly, M , increased a little between 90
concentrations, the recovery (%) increased with increasing and 150 min when the hypochlorite concentration was 25%.
treatment time. This means that chloroform can protect the This result may imply that lower molecular weight chains
PHB molecules from being digested by hypochlorite if a with a similar degree of degradation to high-molecular-
moderate hypochlorite concentration is used. However, for weight chains will have a higher tendency to fractionate
a 30% hypochlorite concentration, the time profile of the into the aqueous phase and be further digested by the
recovery (%) showed a maximum point at 90 min. This hypochlorite.
means that the rate of PHB degradation became higher than The polydispersity index (PI; M,/M,) represents the
that of PHB release from the cells after 90 min. The optimal molecular weight distribution reflecting the structural degra-
recovery was about 91%. In the case of a 35% hypochlorite dation of PHB molecules. The initial PI value before
concentration, its tendency was similar to that of a 30% treatment was 2.4. Overall, PI increased with increasing
hypochlorite concentration, but the maximum recovery (%) treatment time. The higher the hypochlorite concentration
was slightly lower.

1001 1
' o o l
90.
90-
80- ee
h

z? 80-
70-
8
60- B

'O1
01'' 15 20 25 30 35 40 6
Hypochloriteconcentration (??) Ratio of chloroform to aqueous phase
Figure 4. Effect of sodium hypochlorite concentration on the recovery Figure 5. Effect of the ratio of chloroform to aqueous phase on the
(%) of PHB from Alcaligenes eutrophus for different treatment times: (0) recovery (%) of PHB from Alcaligenes eutrophus using a 30% sodium
20 min; (x)40 min; (A) 60 min; (m)90 min; (X) 120 min; (+) 150 min. hypochlorite concentration and a 90-min treatment time

COMMUNICATION TO THE EDITOR 259

 that by using sodium hypochlorite. This may be due to
the fact that, using the dispersion, PHB first goes into
500 the chloroform phase and is later recrystallized through
nonsolvent precipitation. On the other hand, using the
sodium hypochlorite, PHB is recovered as crude granules
400 from cells' cytoplasms, thus having no opportunity for
recrystallization. The crystallinity of recovered PHB was
300 roughly in the range of 60 to 65%.
As a whole, the roles of sodium hypochlorite and chlo-
roform in our PHB recovery process are summarized in
200
Table I. The new method we have proposed has some
advantages. It is simple and effective and easy to scale
up. Molecular weight can be controlled by changing the
hypochlorite concentration and treatment time. It gives a
high purity of over 97% and a high recovery of about 90%.
0 30 60 90 120 150 Direct solvent extraction of PHB from cell powder has
Treatment time (minutes) the drawback of a significant raw material cost incurred
due to the difficulties in cell residue removal and due to
Figure 6. Change in the number average molecular weight with increas- the high viscosity of the solution of extracted PHB.12 In
ing treatment time for different sodium hypochlorite concentrations: (0)
25%; (A) 30%; (M)35%. our method, the hypochlorite in the dispersion not only
isolates PHB from the cells but also digests most of NPCM,
making the separation of NPCM from PHB-dissolved chlo-
used, the greater increase in PI observed (Fig. 7). From roform far less complicated than that in direct solvent
Figures 6 and 7, it can be said that the molecular weight extraction processes. In addition, the digestion by sodium
and/or PI can be controlled by varying the treatment time hypochlorite gives a reduced viscosity of the chloroform
and the hypochlorite concentration in the dispersion. phase. Generally, as the molecular weight decreases, certain
To evaluate the effect of the dispersion on the thermal physical properties of a polymer become poor, but polymer
property of the recovered PHB, we measured the melting processing becomes much easier. Thus, depending upon
temperature and the enthalpy of fusion with DSC. There applications of PHB, the optimum range of the molecular
existed no difference in the melting temperatures (176°C) weight varies. Using our method, the molecular weight can
between PHB recovered by using sodium hypochlorite and be controlled by changing the hypochlorite concentration
that by using the dispersion. However, the enthalpy of of the dispersion.
fusion of PHB recovered by using the dispersion (96.8 J/g)
was slightly higher than that by using sodium hypochlorite
(85.0 J/g). This indicates that the crystallinity of PHB CONCLUSIONS
recovered by using the dispersion was slightly higher than In the present study, we identified the optimal treatment
condition for PHB recovery from Alcaligenes eutrophus
A E using the dispersions of sodium hypochlorite and chloro-
-?.cl I I
form. Under the optimal condition, we could obtain PHB
of a purity higher than 97% with 91% recovery. The
4- optimal condition for PHB recovery from cell powder
X
a, was a 30% hypochlorite concentration, a 90-min treatment
'c)
.-c time, and a 1 : 1 (v/v) chloroform-to-aqueous-phase ratio.
2 3.5-
.- Under the optimal condition, the number average molecular
2 weight M,, of the PHB recovered was about 300,000
a,
and the weight average molecular weight M , was about
.i? 3-
x-
0 Table I. Roles of sodium hypochlorite and chloroform in the dispersion
0
2.5- for PHB recovery.

1 I Sodium hypochlorite Chloroform

2 a Cell disruption Extraction of PHB
0 30 60 90 120 150 Molecular weight control Purification
Treatment time (minutes) Digestion of NPCM Protection of PHB molecules
Viscosity reduction of from hypochlorite
Figure 7. Change in the polydispersity index with increasing treatment solvent phase
25%; (A) 30%;
time for different sodium hypochlorite concentrations: (0) Bleaching
).( 35%.

260 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 44, NO. 2, JUNE 20, 1994
1,020,000. The original M , and M , were 530,000 and 5. Brandl, H., Gross, R.A., Lenz, R.W., Fuller, R.C. 1990. Plastics
1,272,000, respectively. The moderate decreases in both from bacteria and for bacteria: Poly(P-hydroxyalkanoates) as natural,
biocompatible, and biodegradable polyesters. Adv. Biochem. Eng.
M , and M , might be ascribed to the shielding effect of Biotechnol. 41: 78-93.
chloroform. In addition, the relatively small decrease in M , 6. Braunegg, G., Sonnleitner, B., Lafferty, R. M. 1978. A rapid gas chro-
probably resulted from the loss of short PHB chains which matographic method for the determination of poly-P-hydroxybutyric
might be water soluble. The melting temperature of PHB acid in microbial biomass. Eur. 3. Appl. Microbiol. Biotechnol. 6:
recovered was 176°C regardless of the treatment method 29-37.
7. Byrom, D. 1987. Polymer synthesis by microorganisms: Technology
used. However, the enthalpy of fusion of PHB recovered
and economics. TIBTECH 5: 246-250.
by using the dispersion was 96.8 J/g, whereas that by using 8. Doi, Y. 1990. Microbial polyesters. VCH, New York.
only sodium hypochlorite was 85.0 J/g. The crystallinity 9. Hahn, S.K., Chang, Y.K., Kim, B.S., Lee, K.M., Chang, H.N.
of PHB recovered was in the range of 60 to 65%. When it 1993. The recovery of poly(3-hydroxybutyrate) by using dispersions
comes to the recovery of microbial PHB from Afcafigenes of sodium hypochlorite solution and chloroform. Biotechnol. Techn.
eutrophus, the new method we have proposed seems to be 7: 209-212.
10. Harrison, S. T. L., Chase, H. A,, Dennis, J. S. 1991. The disruption of
superior to both the hypochlorite digestion method and the Alcaligenes eutrophus by high pressure homogenisation: Key factors
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The authors are grateful to the Korea Science and Engineering
biodegradable thermoplastic. Phys. Technol. 16: 32-36.
Foundation for its financial support.
12. Holmes, P.A., Lim, G.B. 1990. Separation process. U.S. Pa-
tent 4910145.
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COMMUNICATION TO THE EDITOR 261