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Bioseparation 7: 9–15, 1997. c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

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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 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.

1996). minimising the need for expensive chemicals and enzymes. however. but all have limitations (Wong et al. Cell debris is. Fractionation efficiency can be enhanced by optimising centrifugation conditions (e. 1996. coli containing PHB granules. One of the grade-efficiency curves for disc-stack centrifugation. even though perfect fractionation is never achieved. it has been reported that the accumulation of high levels of PHB in recombinant E. coli debris distributions following homogenization (Siddiqi et al. disc-stack centrifugation and chemical treatment is finally suggested and tested. A rational understanding of PHB and cell debris characteristics is therefore required. 1989). more difficult to characterise. 1996). Purity can be enhanced through multiple centrifuge passes. 1997).13–1. Various techniques are available. Wong et al. coli. Recently. In this paper.. size and density) (Hoare and Dunnill. is given by Equation (2) (Mannweiler. (1996) introduced a new analysis method (Cumulative Sedimentatian Analysis or CSA) for characterising cell-debris size based on cumulative sedimentation under centrifugal force. This method provides a means to characterise cell debris size and thus optimise centrifugal fractionation. based on a complete radial (lateral) mixing model assumption. Chemical treatment for further purification can be considered if a high purity product is required. the fractionation of PHB and cell debris by homogenization and centrifugation is simulated. A scaled up PHB extraction process combining homogenization. and is amenable to scale up. The median diameter D50 and parameter w were obtained by regression of experimental data to the equation. 1989). Based on these experimental data. W D  =  1 1 + exp D D50  w (1) Grade efficiency curve The particle fractionation by a centrifuge can be evaluated using a ‘grade-efficiency’ curve. Theoretical note Boltzmann equation The Boltzmann function. Despite the widespread use of centrifugation for the recovery of inclusion bodies. based on a density of 1260 kg/m3.g.g. feedrate).25 m has been reported in recombinant E. 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.. 1995) and a mean PHB granule diameter of 1. The size distribution of PHB granules is easily determined by analytical disc centrifuge (CDS) (Middelberg et al.10 For example. which describes the size-dependent particle classification. Fractionation by centrifugation is dictated by the relative sedimentation-velocity distributions of the granules and cell debris (e. coupled with analysis of the sedimented fraction by SDSPAGE. The process gives PHB with a high purity and recovery. CSA is used to characterise cell-debris size distributions following the repeated homogenization of recombinant E. has been applied to describe yeast and E. T D  = 1  exp n  D k D c . coli facilitates PHB granule release (Lee. this technique has not been thoroughly investigated as a method for collection of PHB granules.. Wong et al. Equation (1)..

Materials and methods Bacterial strain and fermentation A non-K-12 E. lacIq .3 g L 1 cell concentration (DCW). USA) was transformed with plasmid pJM9123 (Slater et al. The fermentation gave a total of 18 L of culture with 42. proAB. (tetR )) (Stratagene. A fed-batch fermentation using this strain was conducted in modified R-medium. Parameters k and n can be obtained by regression to experimental data. coli strain Topp1 [F’. Transformants were selected using kanamycin (50 g mL 1 ). CA. (2) The critical diameter. 1992).. . Dc . 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). Detailed descriptions of the fermentation conditions used are given elsewhere (Ling et al. and MgSO4 7H2 O. fed intermittently with a mixture of glucose. Tn10. Z∆ M15. yeast extract. 1997). depends upon centrifuge operating conditions and particle properties..

These densities are approximate and only used to transform distributions to a size basis for presentation.5 g L 1 NaCl.. . This was added to samples to give 0. Extraction process and analytical methods Homogenization and debris size measurement Fresh culture harvested from the fermentation was diluted in PBS buffer (1. 1996).. Frozen culture from the fermentation was thawed at 4 C and then diluted ten times with PBS buffer (as above).85 g active chlorine L 1 corresponding to 1. UK) with a standard water spin fluid scheme (Middelberg et al.g. Centrifugation was performed with a Veronesi KLE-160 solidbowl disc-stack centrifuge with a fixed operating speed P of 8400 rpm ( =3775 m2 ). The feed temperature was approximately 10 C for each pass. N is the number of homogenization passes. and this is not affected by the assumed density. FSE Australia. 6. In simulation. PHB collection efficiency during centrifugation was determined by comparing feed and supernatant samples using an analytical disc centrifuge (CDS).11 Figure 2. the settling-velocity distribution is the critical determinant of performance.. prior to centrifugation. Size distributions for whole cells and PHB granules in homogenates determined by photosedimentation. and PHBgranule density was taken as 1260 kg m 3 (Middelberg et al. Other analytical methods (e. Hypochlorite treatment Sodium hypochlorite (NaOCl) (Fisons technical grade. which were then incubated for 1 h at a temperature between 15 C to 20 C.. 1 L of homogenate was collected after each pass. Figure 1. CD valve). 1990). 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. adjusted pH to 6. PHB recovery protocols. The centrifugation took about 40 minutes (Figure 1).37 g L 1 KH2 PO4 . Figure 1 summarises the three extraction processes and operating conditions employed. 1997).5% NaOCl relative to the stock solution. Five discrete homogenisation passes were conducted at 55 MPa in an APV-Gaulin high-pressure homogenizer (15MR..9 with 2 M NaOH) to give an optical density of 12–15 at 600 nm. 1995). The density of whole cells and the resulting cellular debris was taken as 1085 kg m 3 (Hwang. 1995). CSA was conducted for the measurement of cell-debris size distributions (Wong et al. Cell disruption and PHB release by homogenization were monitored by the use of an Applied Imaging DCF4 Disc Centrifuge (Gateshead. 1997). PHB quantitation by GC) were as previously described (Ling et al.

.016  0. Results and discussion Preliminary experimental investigation The fermentation gave a cell broth with a dry weight of 42.010  0.139 0.12 Table 1.10 PHB in homogenates D50 Cell debris in homogenates w  0.084 0. and a possible fractionation scheme developed.009  0. The Boltz- mann equation describes the cell-debris distributions for E. m) and Boltzmann parameters (w) for whole cells.016  0.1. Further homogenization apparently caused some PHB granule breakage. 1997).44 0. The results confirm that repeated homogenization causes micronisation of cellular debris. obtained for recombinant E. This has also been observed during the homogenization of frozen cells containing PHB granules (Ling et al. The PHB granules in this study are smaller than those reported previously (Middelberg et al. 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.36 0. coli homogenates containing PHB granules very well.009 Figure 3. Figure 2 presents the particle size distributions of homogenates as a function of the number of homogenizer passes...12 0.010  0. Median debris size decreased from 0.017  0.65 m after the first homogenizer pass to 0.50 0. 1996). Based on the information on PHB granule and cell-debris sizes obtained above. Cumulative oversize distributions of cell debris in homogenates as a function of the number of homogenizer passes (N) determined by cumulative sedimentation analysis. the settling characteristics of PHB granules and cell debris in a disc-stack centrifugation can be studied. Smooth curves were obtained by regression to equation (1) with parameters given in Table 1.65 0. respectively. Obviously. Figure 3 presents the cumulative oversize distributions of cell debris in homogenates as a function of the number of homogenizer passes.16 0. but are larger than recombinant IGF inclusion bodies recovered successfully by centrifugation (Wong et al. PHB collection efficiency and cell debris removal by homogenization and centrifugation can be predicted by simulation. 1997). 1995). Cell debris size after each homogenizer pass was measured by CSA and regressed to Equation (1). coli containing recombinant protein inclusion bodies under similar homogenization conditions (Wong et al.3 g L 1 and a PHB content of 52% (w/w). 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 D50 w N=1 N=2 N=3 N=4 N=5 N=1 N=2 N=3 N=4 N=5 1.38 0. The size distribution of whole cells measured by CDS is also included for comparison.. Cell debris is comminuted with each homogenization pass (Wong et al.20 0. measured by CDS. Table 1 lists the Boltzmann parameters (D50 and w) determined by regression.018  0. high cell disruption was obtained after two homogenizer passes. Smooth curves are regressions to Equation (1).58 0. Median Diameters (D50 . coli cell debris produced by homogenization and fractionated in the same cen- .010  0. Median debris size and distribution width after each homogenizer pass are comparable with those for E.36 m after five homogenizer passes. PHB granules. assuming the parameters k and n are 0. 1997).62 m.. PHB mean diameter after the third homogenizer pass was centred at 0.13 and 2.38 0.010  0.17 0. The settling behaviour of cell debris in a disc-stack centrifugation is described by Equation (2).

Figure 1). Cell debris removal depends strongly on the centrifuge feedrate and the number of homogenizer passes as expected. To overcome the problem of DNA contamination. To enhance PHB purity. possibly due to the large granule size and high density. using parameters k and n in Equation (2) of 0. 1997). leading to a higher removal of debris.2  10 3 Pa. Extraction processing trifuge used in this study (Wong. . PHB recovery decreases concomitantly. Additional Figure 4. coli cells (Hahn et al. Based on the simulation. cell material in the pilot scale tests was frozen and stored before homogenization. Figure 1). a fractionation process including two centrifugations was tested experimentally (process A. Centrifugation was also efficient at removing protein and DNA. N is the number of homogenization passes. However. debris removal increases significantly as the centrifuge feedrate increases.. sodium hypochlorite (NaOCl) treatment was introduced (process B. The small deviation could be due to several factors. The predicted effect of repeated homogenization and centrifugefeedrate variation on the removal of cell debris is presented in Figure 4. 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. Secondly. Furthermore. DNA removal did not follow the same trend. an assumed viscosity of 1. leaving scope for further improvement. The simulation P is limited to normalised centrifuge feedrates (Q/ ) between 1. The results are listed in In process A.2% (w/w) was obtained by repeated centrifugation (A2).. 1954). Figure 1).32  10 9 m s 1 to 3. Using the same number of homogenizer passes. affecting the actual debris size and also homogenate properties such as viscosity. probably coupled with a reduction in viscosity. Simulated PHB recovery and cell debris removal in a centrifuge. 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). the resultant protein and DNA content were not below the limits reported to cause PHB decolouration in thermal processing (Harrison. However. The predicted PHB collection efficiency (87%) was significantly higher than the experimental value (76%).13 Table 2. 1996) and the PHB size distribution measured after three homogenizer passes. after the removal of considerable DNA) (process C. Overall PHB recovery remains high even at high feedrates (Figure 4). led to enhanced PHB collection efficiency and debris removal (B1. Extrapolation to the larger PHB granules in this study may introduce errors. it was decided to introduce the NaOCl treatment after the first centrifugal fractionation (i.16 and 2. 1990). as this is the range of model and parameter confidence. Cell debris micronisation. 72% PHB recovery and 94% debris removal were achieved and a PHB purity of 90. Clearly. Protein removal was also improved by NaOCl treatment. The errors indicated above will affect the prediction. Actual viscosity could be higher. Firstly. Prolonged storage and freeze-thaw cycles can promote changes in cell wall characteristics and integrity.45 m. Table 2). there is a strong interaction between the homogenization and centrifugation unit operations.97  10 9 m s 1 and to 1 to 5 homogenizer passes. 1996). the parameters in Equation (2) used in the simulation were determined for recombinant protein granules with a median diameter of approximately 0.6 (Wong.s was used in the simulation. Actual cell debris removal was 75% (A1) at a feedrate of 3. Figure 4 also includes the curve describing PHB overall collection efficiency as a function of centrifuge feedrate.e. which is comparable to the simulated value for 3 homogenizer passes (70%). Overall PHB recovery in process A was also considered too low. possibly due to DNA denaturation by NaOCl treatment and adsorption to the granule surface (Ling et al.09  10 9 m s 1 .

Lee IY.2 0.9 1. Trans.01 5. 3(4): 227–232. R. Hwang SO (1996) Effect of inclusion bodies on the buoyant density of recombinant Escherichia coli.1 0.2 90. Harrison STL. 1990). Bioeng. Ling Y. Mannweiler K (1989) The recovery of biological particles in highspeed continuous centrifuges with special reference to feed-zone break-up effects. coli.2 1.8 92. Biotechnol. Hahn SK.09  10 9 m s 1 to 2. detergent and hydrogen peroxide washing. Biotechnol. Adv. UK. Ramsay BA. PhD dissertation University of Cambridge.3 85.4 94. High PHB recovery is attributed to the reduction in feedrate (C1) and the effect of NaOCl treatment on homogenate viscosity (C2). Techn.5 25 9. PhD dissertation University of London.9 1. 14: 135–266. Kim BS and Chang HN (1994) Optimisation of microbial poly(3-hydroxybutyrate) recovery using dispersion of sodium hypochlorite solution and chloroform.21  10 9 m s 1 .03 0 75. Lond. J. Harrison ST (1990) The extraction and purification of poly. This fractionation process was conducted at pilot scale. but with a penalty of decreased cell debris removal (75% to 61%). Phil. Soc. These values match reasonably well with the simulated values of 70% and 62%.3 3. Physiol. Further process simplification will be possible by using a continuous rather than solid bowl centrifuge. DNA removal was enhanced in process C as expected. Patent 4910145. Chase HA and Dennis JS (1991) The disruption of Alcaligenes eutrophus by high pressure homogenization: key factors involved in the process.5 76 95 92 97 82 99 98 Process B Process C homogenization steps were introduced after centrifugation to enhance paste re-suspension and hence the dispersion of granules and residual debris. Biotechnol.0 79.0 2. Microbiol. This process is quite competitive in terms of PHB purity and recovery with the industrial method described by Harrison (1990). Hoare M and Dunnill P (1989) Biochemical engineering challenges of purifying useful proteins. Lee SY and Chang HN (1996) Characteristics of poly(3hydroxybutyric acid) synthesis by recombinant E. A comparison of C1 and A1 confirms that collection efficiency was improved as the feedrate decreased from 3.-hydroxybutyrate by alkaline solution treatment.1 90. This process gave both high PHB purity (96. Chang YK.A. J. Chang HN and Park YH (1995) A simple method for recovery of microbial poly. The final recovery process yielded a very pure PHB product with negligible DNA and protein contamination. Tech. U. 44: 256–261. Savings are also possible by optimising the NaOCl treatment regime. Microb.14 Table 2. but does not require enzyme.0 94. Tech. Results for PHB fractionation by centrifugation Extraction process PHB (% w/w) Protein (% w/w) DNA (% w/w) Cell debris removal (%) Collection efficiency (% per pass) Cell broth Process A A1 A2 B1 B2 C1 C2 C3 52. 5(4): 238–240. Biotechnol.hydroxybutyrate from Alcaligenes eutrophus. Microbiol.0 70.2 1. respectively. and the results obtained can be simply extrapolated to full-scale PHB manufacture.0 9. B324: 497–501. The remaining protein and DNA contents are below the levels reported to cause PHB decolouration in thermal processing (Harrison. Thomas CJ and Middelberg APJ (1997) Recovery of poly-3-hydroxybutyrate from recombinant Escherichia coli by homogenization and centrifugation. . Biotechnol.62 2.3 60. Lee SY (1995) Poly(3-hydroxybutyrate) extrusion by cells of recombinant Escherichia coli.2 82. Annals of the New York Academy of Science 782: 133–142. Fundamental data on debris size was also obtained. Williams DRG. References Berger E. 6(2): 147–149.5%) and recovery (81%) (Table 2). Dawes EA and Senior PJ (1973) The role and regulation of energy reserve polymers in micro-organisms.1 94.54 <0. thus simplifying paste re-suspension and recentrifugation.8 96. Bioseparation 2: 155–166. 10(3): 157– 160.4 96. Ramsay JA and Chavarie C (1989) PHB recovery by hypochlorite digestion of non-PHB biomass. Holmes PA and Lim GB (1990) Separation Process.2 0. Biotechnol.15 0. 11(6): 409–412.

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