9

Bioseparation 7: 915, 1997.

c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Pilot-scale extraction of PHB from recombinant E. coli by homogenization

and centrifugation
Y. Ling1, H.H. Wong1 , C.J. Thomas2, D.R.G. Williams1 & A.P.J. Middelberg1
1

Department of Chemical Engineering and 2 Department of Microbiology and Immunology, University of

Adelaide, S.A. 5005, Australia

Received 29 May 1997; accepted in revised form 19 November 1997

Key words: centrifugation, extraction, homogenization, poly- -hydroxybutyrate (PHB)

Abstract
A new method of poly- -hydroxybutyrate (PHB) extraction from recombinant E. coli is proposed, using homogenization and centrifugation coupled with sodium hypochlorite treatment. The size of PHB granules and cell debris
in homogenates was characterised as a function of the number of homogenization passes. Simulation was used to
develop the PHB and cell debris fractionation system, enabling numerical examination of the effects of repeated
homogenization and centrifuge-feedrate variation. The simulation provided a good prediction of experimental performance. Sodium hypochlorite treatment was necessary to optimise PHB fractionation. A PHB recovery of 80% at
a purity of 96.5% was obtained with the final optimised process. Protein and DNA contained in the resultant product
were negligible. The developed process holds promise for significantly reducing the recovery cost associated with
PHB manufacture.
Introduction
Poly- -hydroxybutyrate (PHB) is a bacterial storage polymer produced in various microorganisms in
response to nutrient limitation. PHB was first described
by Lemoigne in Bacillus megaterium in 1926 (Dawes
and Senior, 1973). Its copolymer P(HB-co-HV) has
been marketed as a biodegradable thermoplastic since
the middle 1980s.
The high production cost of PHB and its copolymer has restricted their use as bulk plastic materials.
Production economics are governed by several factors including product yield, product recovery, and the
techniques and strategy applied. It has been estimated that over half of the production cost of PHB is
associated with the recovery and purification process.
Moreover, the recovery process can significantly alter
the properties of the final product. While a number of
PHB recovery methods have been developed (Hahn et
al., 1994; Berger et al., 1989; Lee et al., 1995), most
have not been tested at pilot nor production scale.
Industrial methods for PHB release include enzymatic digestion (Holmes and Lim, 1990) and thermal

*157815*

treatment coupled with high-pressure homogenization

(Harrison, 1990). A high disruption efficiency for A.
eutrophus is obtained using high-pressure homogenization, and key factors affecting disruption have been
thoroughly investigated (Harrison et al., 1991). However, processes for removing cell debris from PHB
following cell disruption have received little attention.
The process reported by Harrison (1990) was based
on solubilisation of non-PHB components using detergent and enzymes coupled with multiple centrifugation
passes. This approach raises total production cost and
still yields a relatively low quality PHB, which is unacceptable for many applications.
For historic reasons, these extraction processes are
mostly focused on A. eutrophus. However, research
interest has progressively shifted to recombinant E.
coli. The advantages of using recombinant E. coli
strains as a host for PHB accumulation have been
detailed by Lee and Chang (1996). Effectiveness of
PHB recovery varies from one host to another due to
differences in cell-wall structure and PHB formation
factors. Consequently, differences in cell disruption
and the subsequent fractionation process are expected.

PIPS NO.:157815 (M) BIO2KAP

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For example, it has been reported that the accumulation
of high levels of PHB in recombinant E. coli facilitates
PHB granule release (Lee, 1996).
Centrifugation is one of the most common unit
operations in bioprocessing for the fractionation of
insoluble protein inclusion bodies from other cellular
material (Middelberg, 1996). Fractionation efficiency
can be enhanced by optimising centrifugation conditions (e.g. feedrate), even though perfect fractionation
is never achieved. Purity can be enhanced through multiple centrifuge passes, minimising the need for expensive chemicals and enzymes. Chemical treatment for
further purification can be considered if a high purity
product is required. Despite the widespread use of centrifugation for the recovery of inclusion bodies, this
technique has not been thoroughly investigated as a
method for collection of PHB granules.
Fractionation by centrifugation is dictated by the
relative sedimentation-velocity distributions of the
granules and cell debris (e.g. size and density) (Hoare
and Dunnill, 1989). A rational understanding of PHB
and cell debris characteristics is therefore required. The
size distribution of PHB granules is easily determined
by analytical disc centrifuge (CDS) (Middelberg et al.,
1995) and a mean PHB granule diameter of 1.131.25
m has been reported in recombinant E. coli, based
on a density of 1260 kg/m3. Cell debris is, however, more difficult to characterise. Various techniques
are available, but all have limitations (Wong et al.,
1996). Recently, Wong et al. (1996) introduced a new
analysis method (Cumulative Sedimentatian Analysis
or CSA) for characterising cell-debris size based on
cumulative sedimentation under centrifugal force, coupled with analysis of the sedimented fraction by SDSPAGE. This method provides a means to characterise
cell debris size and thus optimise centrifugal fractionation.
In this paper, CSA is used to characterise cell-debris
size distributions following the repeated homogenization of recombinant E. coli containing PHB granules.
Based on these experimental data, the fractionation of
PHB and cell debris by homogenization and centrifugation is simulated. A scaled up PHB extraction process combining homogenization, disc-stack centrifugation and chemical treatment is finally suggested and
tested. The process gives PHB with a high purity and
recovery, and is amenable to scale up.

Theoretical note
Boltzmann equation
The Boltzmann function, Equation (1), has been
applied to describe yeast and E. coli debris distributions
following homogenization (Siddiqi et al., 1996; Wong
et al., 1997). The median diameter D50 and parameter
w were obtained by regression of experimental data to
the equation.

W (D ) = 

1 + exp

D D50

w

(1)

Grade efficiency curve

The particle fractionation by a centrifuge can be evaluated using a grade-efficiency curve, which describes
the size-dependent particle classification. One of the
grade-efficiency curves for disc-stack centrifugation,
based on a complete radial (lateral) mixing model
assumption, is given by Equation (2) (Mannweiler,
1989).

T (D ) = 1

exp

n 
D
k D
c


(2)

The critical diameter, Dc , depends upon centrifuge

operating conditions and particle properties. Parameters k and n can be obtained by regression to experimental data.

Materials and methods

Bacterial strain and fermentation
A non-K-12 E. coli strain Topp1 [F, proAB, lacIq , Z
M15, Tn10, (tetR )) (Stratagene, CA, USA) was transformed with plasmid pJM9123 (Slater et al., 1992).
Transformants were selected using kanamycin (50 g
mL 1 ). A fed-batch fermentation using this strain
was conducted in modified R-medium, fed intermittently with a mixture of glucose, yeast extract, and
MgSO4
7H2 O. Detailed descriptions of the fermentation conditions used are given elsewhere (Ling et al.,
1997). The fermentation gave a total of 18 L of culture with 42.3 g L 1 cell concentration (DCW). The
culture was stored in the fermenter overnight at 10  C
before homogenization for debris size analysis (4 L) or
storage at 18  C for subsequent use (14 L).

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Figure 2. Size distributions for whole cells and PHB granules in

homogenates determined by photosedimentation. N is the number
of homogenization passes.

Figure 1. PHB recovery protocols.

Extraction process and analytical methods

Homogenization and debris size measurement
Fresh culture harvested from the fermentation was
diluted in PBS buffer (1.37 g L 1 KH2 PO4 , 6.5 g
L 1 NaCl, adjusted pH to 6.9 with 2 M NaOH) to give
an optical density of 1215 at 600 nm. Five discrete
homogenisation passes were conducted at 55 MPa in an
APV-Gaulin high-pressure homogenizer (15MR, CD
valve). The feed temperature was approximately 10  C
for each pass. 1 L of homogenate was collected after
each pass. CSA was conducted for the measurement of
cell-debris size distributions (Wong et al., 1997). Cell
disruption and PHB release by homogenization were
monitored by the use of an Applied Imaging DCF4
Disc Centrifuge (Gateshead, UK) with a standard water
spin fluid scheme (Middelberg et al., 1990). The density of whole cells and the resulting cellular debris
was taken as 1085 kg m 3 (Hwang, 1996), and PHBgranule density was taken as 1260 kg m 3 (Middelberg et al., 1995). These densities are approximate and
only used to transform distributions to a size basis for
presentation. In simulation, the settling-velocity distribution is the critical determinant of performance, and
this is not affected by the assumed density.

Frozen culture from the fermentation was thawed at

4  C and then diluted ten times with PBS buffer (as
above). Figure 1 summarises the three extraction processes and operating conditions employed. Centrifugation was performed with a Veronesi KLE-160 solidbowl disc-stackPcentrifuge with a fixed operating speed
of 8400 rpm ( =3775 m2 ). PHB collection efficiency during centrifugation was determined by comparing feed and supernatant samples using an analytical
disc centrifuge (CDS). Other analytical methods (e.g.
PHB quantitation by GC) were as previously described
(Ling et al., 1997).
Hypochlorite treatment
Sodium hypochlorite (NaOCl) (Fisons technical grade,
FSE Australia, 100 g active chlorine L 1 ) was diluted
with RO water to give a solution with 57 g active chlorine L 1 (Middelberg et al., 1995). This was added
to samples to give 0.85 g active chlorine L 1 corresponding to 1.5% NaOCl relative to the stock solution,
which were then incubated for 1 h at a temperature
between 15  C to 20  C, prior to centrifugation. The
centrifugation took about 40 minutes (Figure 1).

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Table 1. Median Diameters (D50 , m) and Boltzmann parameters (w) for whole cells, PHB granules, and cell debris in homogenates measured by
CSA or CDS (errors represent standard deviation
of the mean by regression to Equation (1))
Whole cells

D50
w

1.38
0.139

PHB in
homogenates

D50
w

0.58
0.084

D50
Cell debris in
homogenates
w

N=1
N=2
N=3
N=4
N=5
N=1
N=2
N=3
N=4
N=5

0.65
0.50
0.44
0.38
0.36
0.20
0.17
0.16
0.12
0.10

 0.010
 0.016
 0.017
 0.010
 0.010
 0.009
 0.016
 0.018
 0.010
 0.009

Results and discussion

Preliminary experimental investigation
The fermentation gave a cell broth with a dry weight of
42.3 g L 1 and a PHB content of 52% (w/w). Figure 2
presents the particle size distributions of homogenates
as a function of the number of homogenizer passes,
measured by CDS. PHB mean diameter after the third
homogenizer pass was centred at 0.62 m. The PHB
granules in this study are smaller than those reported previously (Middelberg et al., 1995), but are larger than recombinant IGF inclusion bodies recovered
successfully by centrifugation (Wong et al., 1996).
Obviously, high cell disruption was obtained after two
homogenizer passes. Further homogenization apparently caused some PHB granule breakage. This has also
been observed during the homogenization of frozen
cells containing PHB granules (Ling et al., 1997).
Cell debris is comminuted with each homogenization pass (Wong et al., 1997). Cell debris size after each
homogenizer pass was measured by CSA and regressed
to Equation (1). Figure 3 presents the cumulative oversize distributions of cell debris in homogenates as a
function of the number of homogenizer passes. Smooth
curves are regressions to Equation (1). The size distribution of whole cells measured by CDS is also included
for comparison. Table 1 lists the Boltzmann parameters (D50 and w) determined by regression. The Boltz-

Figure 3. Cumulative oversize distributions of cell debris in

homogenates as a function of the number of homogenizer passes (N) determined by cumulative sedimentation analysis. Smooth
curves were obtained by regression to equation (1) with parameters
given in Table 1.

mann equation describes the cell-debris distributions

for E. coli homogenates containing PHB granules very
well. The results confirm that repeated homogenization
causes micronisation of cellular debris. Median debris
size decreased from 0.65 m after the first homogenizer pass to 0.36 m after five homogenizer passes.
Median debris size and distribution width after each
homogenizer pass are comparable with those for E.
coli containing recombinant protein inclusion bodies
under similar homogenization conditions (Wong et al.,
1997).
Simulation study: design of an extraction process
Cell debris represents the insoluble contaminant in
PHB homogenates and thus is the major concern during fractionation by centrifugation. Based on the information on PHB granule and cell-debris sizes obtained
above, the settling characteristics of PHB granules and
cell debris in a disc-stack centrifugation can be studied. PHB collection efficiency and cell debris removal
by homogenization and centrifugation can be predicted by simulation, and a possible fractionation scheme
developed.
The settling behaviour of cell debris in a disc-stack
centrifugation is described by Equation (2), assuming
the parameters k and n are 0.13 and 2.1, respectively,
obtained for recombinant E. coli cell debris produced
by homogenization and fractionated in the same cen-

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13

Figure 4. Simulated PHB recovery and cell debris removal in a

centrifuge. N is the number of homogenization passes.

Table 2. Actual cell debris removal was 75% (A1) at

a feedrate of 3.09  10 9 m s 1 , which is comparable to the simulated value for 3 homogenizer passes
(70%). The small deviation could be due to several
factors. Firstly, cell material in the pilot scale tests was
frozen and stored before homogenization. Prolonged
storage and freeze-thaw cycles can promote changes
in cell wall characteristics and integrity, affecting the
actual debris size and also homogenate properties such
as viscosity. Secondly, an assumed viscosity of 1.2 
10 3 Pa.s was used in the simulation. Actual viscosity
could be higher, leading to a higher removal of debris.
The predicted PHB collection efficiency (87%) was
significantly higher than the experimental value (76%).
The errors indicated above will affect the prediction.
Furthermore, the parameters in Equation (2) used in the
simulation were determined for recombinant protein
granules with a median diameter of approximately 0.45
m. Extrapolation to the larger PHB granules in this
study may introduce errors.
Extraction processing

trifuge used in this study (Wong, 1996). The predicted effect of repeated homogenization and centrifugefeedrate variation on the removal of cell debris is presented in Figure 4. The simulation
P is limited to normalised centrifuge feedrates (Q/ ) between 1.32 
10 9 m s 1 to 3.97  10 9 m s 1 and to 1 to 5 homogenizer passes, as this is the range of model and parameter confidence. Cell debris removal depends strongly
on the centrifuge feedrate and the number of homogenizer passes as expected. Using the same number of
homogenizer passes, debris removal increases significantly as the centrifuge feedrate increases. PHB recovery decreases concomitantly. Clearly, there is a strong
interaction between the homogenization and centrifugation unit operations.
Figure 4 also includes the curve describing PHB
overall collection efficiency as a function of centrifuge
feedrate, using parameters k and n in Equation (2) of
0.16 and 2.6 (Wong, 1996) and the PHB size distribution measured after three homogenizer passes. Note
that the PHB size distribution is relatively insensitive
to the number of homogenizer passes as the extent
of granule breakage is low (Figure 2). Overall PHB
recovery remains high even at high feedrates (Figure
4), possibly due to the large granule size and high
density.
Based on the simulation, a fractionation process
including two centrifugations was tested experimentally (process A, Figure 1). The results are listed in

In process A, 72% PHB recovery and 94% debris

removal were achieved and a PHB purity of 90.2%
(w/w) was obtained by repeated centrifugation (A2).
Centrifugation was also efficient at removing protein
and DNA. However, the resultant protein and DNA
content were not below the limits reported to cause
PHB decolouration in thermal processing (Harrison,
1990), leaving scope for further improvement. Overall
PHB recovery in process A was also considered too
low.
To enhance PHB purity, sodium hypochlorite
(NaOCl) treatment was introduced (process B, Figure 1). The use of NaOCl at a relatively low concentration effects the degradation of non-PHB cellular
materials and has a negligible effect on PHB granules
contained in recombinant E. coli cells (Hahn et al.,
1954). Cell debris micronisation, probably coupled
with a reduction in viscosity, led to enhanced PHB
collection efficiency and debris removal (B1, Table 2).
Protein removal was also improved by NaOCl treatment. However, DNA removal did not follow the same
trend, possibly due to DNA denaturation by NaOCl
treatment and adsorption to the granule surface (Ling
et al., 1997).
To overcome the problem of DNA contamination, it
was decided to introduce the NaOCl treatment after the
first centrifugal fractionation (i.e. after the removal of
considerable DNA) (process C, Figure 1). Additional

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14
Table 2. Results for PHB fractionation by centrifugation
Extraction
process

PHB
(% w/w)

Protein
(% w/w)

DNA
(% w/w)

Cell debris
removal (%)

Cell broth

52.0

25

5.2

Collection
efficiency
(% per pass)

Process A

A1
A2

79.1
90.2

9.0
2.3

1.2
0.62

75.0
94.3

76
95

Process B

B1
B2

82.1
94.0

3.9
1.0

2.2
1.9

85.4
94.3

92
97

Process C

C1
C2
C3

70.2
90.8
96.5

9.1
0.54
<0.01

1.2
0.15
0.03

60.8
92.4
96.5

82
99
98

homogenization steps were introduced after centrifugation to enhance paste re-suspension and hence the
dispersion of granules and residual debris. This process gave both high PHB purity (96.5%) and recovery
(81%) (Table 2). High PHB recovery is attributed to
the reduction in feedrate (C1) and the effect of NaOCl
treatment on homogenate viscosity (C2). A comparison of C1 and A1 confirms that collection efficiency
was improved as the feedrate decreased from 3.09 
10 9 m s 1 to 2.21  10 9 m s 1 , but with a penalty
of decreased cell debris removal (75% to 61%). These
values match reasonably well with the simulated values of 70% and 62%, respectively. DNA removal was
enhanced in process C as expected.
The final recovery process yielded a very pure PHB
product with negligible DNA and protein contamination. This process is quite competitive in terms of
PHB purity and recovery with the industrial method
described by Harrison (1990), but does not require
enzyme, detergent and hydrogen peroxide washing.
The remaining protein and DNA contents are below
the levels reported to cause PHB decolouration in thermal processing (Harrison, 1990). This fractionation
process was conducted at pilot scale, and the results
obtained can be simply extrapolated to full-scale PHB
manufacture. Fundamental data on debris size was also
obtained. Further process simplification will be possible by using a continuous rather than solid bowl centrifuge, thus simplifying paste re-suspension and recentrifugation. Savings are also possible by optimising
the NaOCl treatment regime.

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Address for correspondence: A.P.J. Middelberg, Department of

Chemical Engineering, University of Cambridge, Pembroke Street,
Cambridge CB2 3RA, U.K.

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