Isolation and purification of bacterial poly(3-hydroxyalkanoates)

Biochemical Engineering Journal 39 (2008) 15–27

Review

Isolation and purification of bacterial poly(3-hydroxyalkanoates) Nicolas Jacquel a , Chi-Wei Lo b , Yu-Hong Wei c , Ho-Shing Wu b,c,∗ , Shaw S. Wang b,c,d a

Chemistry and Process Department, Ecole Sup´erieure de Chimie, Physique et Electronique de Lyon, France b Department of Chemical Engineering and Materials Science, Yuan Ze University, ChungLi, Taiwan c Graduate School of Biotechnology and Bioinformatics, Yuan Ze University, ChungLi, Taiwan d Departement of Chemical and Biochemical Engineering, Rutgers University, The State University of New Jersey, USA Received 9 October 2006; received in revised form 19 November 2007; accepted 25 November 2007

Abstract The isolation and the purification of bacterial polyhydroxyalkanoates are the key step of the process profitability in the fermentation system. That is why many scientists have studied this field for the production of this biodegradable polymer. The ideal method should lead to a high purity and recovery level at a low production cost. This paper reviews four isolation methods, i.e. solvent extraction of halosolvent and nonhalosolvent, digestion of non-polyhydroxyalkanoate cell material involving surfactants, sodium hypochlorite or enzyme, mechanical cell disruption methods like using bead mills and high pressure homogenization, and new methods like spontaneous liberation of poly(3-hydroxybutyrate), dissolved air flotation, air classification, or by using supercritical CO2 . The pretreatment of cell disruption and the purification methods and analytical methods of polyhydroxyalkanoates are also presented. © 2007 Elsevier B.V. All rights reserved. Keywords: Biopolymers; Polyhydroxyalkanoates; Recovery; Isolation; Purification; Bacteria

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extraction methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Solvent extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Digestion methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Chemical digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Enzymatic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Mechanical disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Bead mill disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. High pressure homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3. Disruption by using ultrasonication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.4. Centrifugation and chemical treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Supercritical (SC) fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Using cell fragility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Air classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Dissolved-air flotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗

Corresponding author at: 135, Yuan-Tung Road, ChungLi, Taoyuan 32003, Taiwan. Tel.: +886 3 4638800x2564; fax: +886 3 4559373. E-mail address: [email protected] (H.-S. Wu). 1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.11.029

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3.

4. 5. 6.

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N. Jacquel et al. / Biochemical Engineering Journal 39 (2008) 15–27

2.8. Spontaneous liberation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. Summary and comparison of PHAs extraction methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Heat pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Alkaline pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Salt pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Freezing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Large scale studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of polyhydroxyalkanoates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Yield determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Purity measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Molecular weight determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Nowadays, plastic materials are taking an important place in our every day life. Their physical properties make them very convenient in utilization. But these nondegradable plastics are accumulating in the environment at the rate of 25 million tonnes per year [1]. That is why replacement solutions like the using of biopolymers (biodegradable polymers) have been envisaged. On that purpose different kinds of polyesters – polyhydroxyalkanoates (PHAs), polylactides, aliphatic polyesters and polysaccharides – have been studied during the last 80 years. PHAs have some very interesting physical properties and biodegradable abilities. They could be as well used in packaging as in medical applications due to their biocompatibility and to their slow hydrolytic degradation [2]. The evolution of the number of publications and patents published in the last 25 years shows the increasing of interest for these polymers (Fig. 1). In order to produce these biopolymers, three main techniques have been studied: by chemical synthesis (ring-opening polymerization of ␤-lactones), by using transgenic plants cells, and by bacterial fermentation. Through the bacterial fermentation approach, PHAs are produced in the cytoplasm of the cell. In fact, some bacteria, such as Ralstonia eutropha (for-

Fig. 1. Evolution of the number of publication () and patents () concerning PHAs for the 25 last years. Research done on Scopus with the key words: polyhydroxyalkanoates, polyhydroxybutyrate, poly(3-hydroxyalkanoate) and poly(3-hydroxybutyrate).

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mer name Alcaligenes eutrophus), under limitation of nitrogen, phosphorus, magnesium or oxygen, metabolizes carbon sources into PHAs granules [3,4]. In 1962, a French microbiologist M. Lemoigne discovered the first variety of PHA in Bacillus megaterium: poly(3-hydroxybuyrate) P(3HB) (Fig. 2). Many studies have been done in order to produce bacterial PHAs at an industrial scale in Table 1, but results showed that these polymers were much more expensive to produce then usual petrochemical plastics (<1 US$/kg) [1]. So lots of improvements have been done to improve the extraction process. That is why, for PHAs producer, having a low cost recovery step has become a strategic asset to stay competitive. The major step of the separation process is the extraction of PHAs granules. But, in order, to get a better recovery a pretreatment step could be added. The purpose of this operation is to improve the cell disruption. Moreover to get a higher purity, a purification step could be added to the process. Fig. 3 shows an overview of the different strategies which were investigated for the polymer recovery. In the following parts, will be detailed most of the methods which were developed for the extraction of bacterial poly(3-

Fig. 2. Structure of PHAs. Since the discovery of Lemoigne, more than 80 different monomer units have been detected in various bacteria [5], HV, HB, and HX are the more common.

N. Jacquel et al. / Biochemical Engineering Journal 39 (2008) 15–27

Fig. 3. Purification strategy of PHAs. The recovery of PHAs could be composed of three steps pretreatment, extraction, and purification.

hydroxyalkanoates) (PHAs). Then most common pretreatment and purification methods will be exposed and finally some outlines about large scale process adaptations and PHAs analysis will be given. 2. Extraction methods 2.1. Solvent extraction The use of solvent to recover PHA is one of the oldest methods. The action of solvent can be divided in two, first it modifies the cell membrane permeability and then it solves PHAs [5]. The use of solvent was first described by Lemoigne

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(1923–1951) and Baptist (1967) [6], respectively on Bacillus megaterium and on Rhodospirillum rubrum. On that purpose they used several chlorinated hydrocarbon solvent such as chloroform, 1,2-dichloroethane, methylene chloride or some cyclic carbonates like propylene and ethylene carbonates [6]. These cyclic carbonic esters were also studied later by Lafferty et al. [7] (for 1,2 propylene carbonate, 95% of the PHB in the cell mass was extracted). Baptist (1967) also first used some solvent mixtures, like chloroform/methanol and dichloromethane/ethanol. The separation of P(3HB) from the solvent was performed by solvent evaporation or by precipitation in a non-solvent [6]. Later, Vanlautem and Gilain [8] investigated extractions from R. eutropha with liquid halogenated solvents, such as chloroethanes and chloropropanes. They found that the best results were obtained for solvents in which the functional carbon atoms are carrying at least one chlorine atom and one hydrogen atom [8], the use of diols (1,2-propandiol: recovery 79%, purity 99.1%, 140 ◦ C), acetalized triols (glycerol formal: recovery 85%, purity 99.7%, 120 ◦ C), di- or tricarboxylic acid esters (diethyl succinate: recovery 90%, purity 100%, 110 ◦ C), or butyrolactone (recovery 90%, purity 99.5%, 110 ◦ C), as extracting agents were studied by Traussnig et al. [9]. Other solvents like tetrahydrofuran methyl cyanide, tetrahydrofuran ethyl cyanide [10], and acetic anhydride [11] were also tested. Whereas these methods do not lead to very high recovery, it is possible to obtain a high level of purity. In fact, by using solvent extraction on poly(3-hydroxybutyrate-co3-hydroxyvalerate)(P(3HB-co-HV)) from R. eutropha biomass with methylene chloride, a purity above 98% can be observed after concentration by distillation, precipitation on ice-cold methanol and recrystallisation [12]. Noda [13] (Procter & Gamble) proposed a process involving, first, a treatment of biomass with a mixture of PHA solvent and non-solvent, then the insoluble biomass is separated, and finally the removal of PHA solvent led to a suspension of precipitated PHA in the non-solvent.

Table 1 Price and productivity of commercial PHAs: present and future Product name

PHA kind

Company

Price

Production (t/y)

Homopolymers Biomer® Biocycle® Biogreen® – – –

P(3HB) P(3HB) P(3HB) P(3HB) P(3HB) P(3HO)

Biotechnoly Co., Germany PHB Industrial S/A company, Brazil Mitsubishi GAS Chemical, Japan Metabolix, USA (BASF, ADM)b Jiangsu Nantian Group, China Metabolix, USA (BASF, ADM)b

20 D /kg (2003)a 3–5 D /kg (2010)a – 10–12 D /kg (2003)a 2.5-3 D /kg (2010)a ∼2.20 D /kg (2010)c – ∼2.20 D /kg (2010)a

50 (2003)a 60 1400 (2003) 30–60,000 (2010)a – – –

Copolymers Biopol® – ENMAT® Nodax – –

P(3HB-co-3HV) P(3HB-co-3HV) P(3HB-co-3HV) P(3HB-co-3HHx) P(3HB-co-3HHx) P(3HB-co-3HHx)

Metabolix, USA (BASF, AMD)b PHB Industrial S/A company, Brazil Tianan Biologic Material, China Procter & Gamble, USA (Kaneka) Jiangsu Nantian Group, China Lianyi Biotech, China

10–12 D /kg (2003)a 3–5 D /kg (2010)a – – 2.50 D /kg (2010)a – >5 US$/kgc

1100 (2003)a 50 (2003)a 10,000 (2006)a 1000 250 (2003)a 20–50,000 (2010)a – 2000c

D : Euro, US$: United States Dollars. a Techno-economic feasibility of large-scale production of bio-based polymers in Europe, Utrecht University and Fraunhofer Institute for Systems Innovation Research (2003). b The strategic alliance between ADM and Metabolix will lead to a production of PHAs of 50,000 t/year in 2008. c The fourth Knowledge Millennium Summit On Biotechnology & Nanotechnology, India, O’Bioer Technology Co., Ltd. (March 2006).

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The Austrian Company Chemie Linz GmbH also employed a solvent extraction method to recover P(3HB) from Alcaligenes latus cells with methylene chloride, the polymer-laden solvent was precipitated by adding water [14]. Like Chemie Linz the Imperial Chemical Industries (ICI) in the United Kingdom, initially used solvent extraction for PHA recovery, but that process was considered too expensive [15,16]. The extracted polymer solution containing more than 5% (w/v) P(3HB) is very viscous and the removal of cell debris is difficult. That is why, about 20 parts of solvent is required to extract 1 part of polymer [17]. This large quantity of solvent leads to a high production cost if no recycle of solvent is envisaged. Liddell [18] explored this idea by solubilizing P(3HB) into a solvent which is immiscible with water at high temperature (above 120 ◦ C). The polymer is then recovered by adding cool water, and the solvent may be reused several times before being distillated. Moreover, the use of solvents destroys the natural morphology of PHA granules that is useful in certain applications such as the production of strong fibers [19]. But, contrary to some other recovery methods, solvent extraction do not degrade the polymer and can be useful for some medical application by the elimination of Endotoxin which can be found in Gram negative bacteria. Lee et al. [20], showed, that the extraction of P(3HB) from Escherichia coli, by using chloroform decreased the Endotoxin level to an allowable limit in P(3HB) (the upper limit of the pyrogen level is 5.0 Endotoxin Units (EU)/kg (body weight)). Another problem connected with the use of solvents is that it creates hazards for the operators and for the environment. That is in part why solvent extraction is widely used in laboratories but with limited success in pilot-plant and large scale processing [21]. To alleviate this short coming, an extraction method using non-halogenated solvent has been introduced by Kurdikar et al. [22]. They used some long chain (4–10 carbons) alcohols, esters, amides, and ketones (both cyclic and acyclic compounds were studied). Some non-halogenated solvents were also used at high temperature (above 80 ◦ C), in those cases, the recovery of polymer is obtained by cooling the mixture [23]. Narasimhan et al. [24] (Procter & Gamble) investigated a new process involving first a solubilization at a high temperature (about 5 ◦ C under the PHA melting point) for 1 s to 15 min, then temperature is decreased before application of pressure (1–10 bar) and mixing (0.001–100 KW m−3 ) [25]. Other improvements can also be done, such as by building and using solubility prediction models of PHA’s in various solvents or solvent mixtures in order to select new green solvents [25]. Recently, Metabolix (USA) applies for a patent using organic solvents to extract PHA from wet biomass. This new technology could be a very important method to reduce PHA extraction costs. 2.2. Digestion methods 2.2.1. Chemical digestion 2.2.1.1. Digestion by surfactants. Surfactants, such as the anionic sodium dodecyl sulfate (SDS), disrupt cells by incorporating itself into the lipid bilayer membrane. As more surfactant is added, more of it enters the membrane to increase the volume of the cell envelope until it is saturated [26]. Further addition

breaks the membrane to produce micelles of surfactant and membrane phospholipids, this leads P(3HB) to be released into the solution surrounded by the cellular debris [26]. Another function of the surfactant is solubilization. Surfactant solubilizes not only proteins, but also other non-PHA cellular materials [27]. Some surfactants, such as synthetic palmitoyl carnitine, a widespread natural surfactant, have been shown to lyze R. eutropha and A. latus [28]. The lysis rate of R. eutropha exceeded 70%, whereas that of A. latus was over 85% (1 mM palmitoyl carnitine in 0.1 M Tris–HCl buffer, pH 7.0, 30 ◦ C, 60 min) [28]. The advantage of this method comes from the fact that surfactants lyzes cells without degrading polymer granules. Another method consists of recovering P(3HB) directly from high cell density culture broth, without pretreatments, just by addition of SDS, shaking, heating and washing [29]. On R. eutropha, a P(3HB) purity over 95% and a recovery rate >90% was obtained with a SDS/biomass ratio higher then 0.4. One main advantage of this method is the fact that it permitted to recover PHA directly from high cell densities: 50–300 g dry cell L−1 [29]. The use of surfactant alone cannot give a high PHA purity (>97%), other agents such as hypochlorite and sodium hydroxide are needed. Furthermore, a high surfactant dose (5 wt%) is increasing the recovery cost and causes problems in wastewater treatment and reuse. 2.2.1.2. Digestion by sodium hypochlorite. An other recovery method is the using of sodium hypochlorite for differential digestion of non-PHA cellular materials [30]. With this method, Hahn et al. [31] obtained get high purity levels of PHA: 86% with R. eutropha and 93% with recombinant E. coli. But, sodium hypochlorite causes severe degradations of P(3HB) resulting in 50% reduction in molecular weight [30]. This phenomenon was first described in 1963, by a synergy of researchers from Syracuse University, State University of New York and College of Forestry [32]. They highlight the degradation of the polymer by measuring the decreases of intrinsic viscosity and absolute molecular weight [33]. Native amorphous PHA granules are quite vulnerable to alkaline saponification and are quickly decomposed into soluble products such as monomers and oligomers. Hahn et al. [31], also observed that phenomenon in the case of R. eutropha. However, in the case of recombinant E. coli strain, the molecular weight did not change much. This stability during sodium hypochlorite treatment seemed to be due to its crystalline morphology. In fact, most of P(3HB) in R. eutropha is in a mobile amorphous state [31]. By using a heat pretreatment and a digestion with 4mL sodium hypochlorite at 50 ◦ C on Cupriavidus taiwanensis184, Lu [34], obtained a purity of 99% and a recovery of 94%. On Pseudomonas putidia KT2442, the action of 35 mL of sodium hypochlorite solution (4.1%) led to a purity and a recovery, respectively of 99% and 78% (Mw 53,000). In the case of Cupriavidus taiwanensis184 a dependence of the molecular weight on operating temperature and the amount of sodium hypochlorite was also noted in Fig. 4. To attenuate the effect of sodium hypochlorite on the P(3HB) from A. eutrophus, Roh et al. [35] proposed to add as an anti-oxidant some sodium bisulfite. By

N. Jacquel et al. / Biochemical Engineering Journal 39 (2008) 15–27

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purity of 98% and a recovery of 86.6%. The extraction was obtained by the treatment of 30 g/L biomass with 10 g/L of SDS at 55 ◦ C for 15 min and by exposing it to 30% NaClO for 3 min at 30 ◦ C. A freezing pretreatment was also used before the digestion in order to broke up cells [38]. The advantages of this method are its low operating cost and its limited degradation of PHA, as compared to Ramsay’s digestion method. By comparing, surfactant-hypochlorite digestion and chloroform-hypochlorite with different bacteria strains, the process employing R. eutropha with a surfactant-hypochlorite digestion resulted in the lowest price for P(3HB) recovery, US$ 5.58/kg (Annual production 2850 t) [1].

Fig. 4. Variation of the molecular weight with the operating conditions. The increasing of the volume of sodium hypochlorite (7.5%) (䊉), at 50 ◦ C, lead to an increasing of the polymer degradation. The molecular weight become also smaller with the increasing of the operating temperature () at constant amount of NaOCl (2 mL). (Experiments were done on 0.5 g of dry cells) [34].

this way, the molecular-weight-drop decreased from 30% to 14%. 2.2.1.3. Digestion by sodium hypochlorite and chloroform. To take advantage of both differential digestion by hypochlorite and solvent extraction, a new process to recover P(3HB) from R. eutropha was developed, in which a dispersion of a sodium hypochlorite solution and chloroform was used [31,36]. By this method, three separate phases are obtained, an upper phase of hypochlorite solution, a middle phase in which are accumulated non-PHA cell materials and undisrupted cells, and the chloroform phase containing P(3HB). The polymer is then recovered by precipitation in a non-solvent and filtration [31]. By using this method the degradation of polymer is significantly reduced. It was suggested that chloroform immediately dissolves the P(3HB) released by hypochlorite treatment, and thus protects the polymer from degradation [31,36]. Under optimal conditions – 30% (w/v) hypochlorite, 1:1 volume ratio of chloroform-toaqueous phase, 4% (w/v) cells in dispersion, 30 ◦ C, 90 min treatment – 91% P(3HB) could be recovered from R. eutropha with a high purity which exceeded 97% [31]. This method was also used by Ryu et al. [37] for the recovery of P(3HB) in R. eutropha, after a pretreatment by Al- and Fe-based coagulants (recovery 98–99% and purity 90–94%). Whereas the degradation of P(3HB) is significantly decreased, the use of sodium hypochlorite with chloroform requires a large quantity of solvent [31,36]. 2.2.1.4. Surfactant-hypochlorite digestion. Dong and Sun [38] studied a combination of surfactant and hypochlorite on Azotobacter chroococcum G-3. By using this method, they obtained a

2.2.1.5. Surfactant-chelate digestion. By adding a chelate to the surfactant the PHA releasing rate is increased. In fact, the role of the chelate can be explained by the fact that some Gramnegative bacteria like R. eutropha have divalent cations such as Ca2+ , Mg2+ in the outer membrane [27]. The addition of chelate can destabilize the outer membrane by forming complexes with divalent cations [27]. The changes in the outer membrane cause weakness in the inner membrane too. All these make the disruption of R. eutropha easier and give a higher purity of recovered P(3HB) [27]. By using this method for the recovery of P(3HB) from R. eutropha, Chen et al. [27] obtained a purity of 98.7% and a recovery yield of 93.3% under the following optimal conditions: 0.12:1 surfactant-dry biomass ratio, 0.08:1 chelate (EDTA disodium salt)-dry biomass ratio, pH 13, 50 ◦ C during a treatment time of 10 min. He also observed a decreasing of the molecular weight from 402,000 to 316,000 [27]. Although the surfactant and chelate method has the features of a convenient operation, high quality of product and low environmental pollution, a large volume of wastewater is produced during the recovery [39]. That is why, Chen et al. [39] proposed a continuous process which includes the using of recycle-wastewater. This involves small additions of surfactant and chelate to recover P(3HB) and treating the final waste water with hydrochloric acid and activated carbon [39]. By this method, a purity higher then 96% and a recovery yield near 90% was obtained with R. eutropha under the following conditions: 15 ◦ C, pH 13, and with a ratio surfactant and chelate to dry biomass of 0.0075 and 0.01, respectively [39]. The wastewater could be recycled for several rounds, and the number of recovery times in each round was five [39]. 2.2.1.6. Chelate-hydrogen peroxide treatment. In 1997, a combination of a chelate treatment with hydrogen peroxide was studied on a poly-3-hydroxybutyrate/3-hydroxyvalerate copolymer produced by R. eutropha [40]. The recovery of the PHA copolymer started with a heat pretreatment (150 ◦ C, pH 6.5 during 80 s), then a chelating agent (diethylene triamine penta methylene phosphonic acid) and hydrogen peroxide were added to the mixture. After 10 h, a copolymer with a purity of 99.5% was recovered by centrifugation [40]. 2.2.1.7. Selective dissolution of non-PHA cell mass by protons. Recently a new recovery method was introduced by Yu

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and Chen [41]. This new method, based on the selective dissolution of non-PHA cell mass (NPCM) by protons in aqueous solution and the crystallization of PHA biopolymers. This treatment is also followed by final decolorization in a bleaching solution. By using these green conditions on different PHAs from R. eutropha, high purity and high yield are observed [41]. Results are the followings: P(3HB): purity: 97.9 wt% and recovery: 98.7 wt%, P(3HB-co-3HV): purity: 98.5 wt% and recovery: 95.4 wt%, P(3HB-co-3HV-co-4HV): purity: 96.4 wt% and recovery: 94.8 wt% [41]. Extrapolation on large scale show that this method is much cheaper than conventional recovery, it reduce the chemical cost of PHA recovery by 90% [41]. 2.2.2. Enzymatic digestion The enzymatic digestion method was developed by Zeneca [42] (a former subsidiary of ICI), as an alternative to solvent extraction. In fact some varieties of enzymes such as, proteolytic enzymes have high activities on dissolution of proteins but little effects on PHA degradation. A typical processing of PHA-containing cell slurry (60 wt% PHA) starts with heat treatment followed by enzymatic hydrolysis, surfactant treatment and final decolorization with hydrogen peroxide [42]. Whereas the use of enzymes leads to good recovery levels, their high cost is a major drawback of this technology. Harrison et al. [43] reported the complete lysis of R. eutropha cells upon treatment (37.5 ◦ C, pH 7.3, 60 min) with lytic enzymes of Cytophaga sp. without any mechanical processing. The enzymatic recovery and purification of P(3HB) produced by R. eutropha was also investigated by Kapritchkoff et al. [44] in 2005. After evaluating the efficiency of different enzymes, best result of obtaining 88.8% P(3HB) purity was achieved with 2.0% of bromelain (enzyme mass per biomass) (US$ 84/kg-cost in 2002) at 50 ◦ C and pH 9.0 [44]. Experiments were also carried out with a pancreatin (three times cheaper than bromelain), leading to 90.0% of polymer purity [44]. A combined method involving enzyme and sodium hypochlorite was also carried out on Burkholderia sp. PTU9 [34]. A purity of 89% and a recovery of 78% were obtained by using papain. De Koning and Witholt [45] proposed a combined method involving consecutive treatment with heat, Alcalase (enzyme) and SDS assisted by EDTA for the recovery of PHA from Pseudomonas. The PHA content of the final solid fraction exceeded 95%. The choice of these optimal experimental conditions was done after testing different enzymes (Alcalase, Neutrase, Lecitase, Lysozyme), combined with hypochlorite, EDTA, heat, SDS, or with an alkali treatment [45]. Recently, another combined method, involving the use of Alcalase, SDS and EDTA, was studied by Yasotha et al. [46]. They showed by several designed experiments that the Alcalase contributed in 71.5% extraction of PHA from P. Putidia culture. Then, PHA granules are recovered in a water suspension by removing the solubilized non-PHA cell material through a crossflow ultrafiltration system and purified through a continuous diafiltration process [46]. Finally the purity of the PHA obtained in a water suspension was 92.6%, for a recovery yield of about 90%.

2.3. Mechanical disruption Mechanical cell disruption is widely used for recovering intracellular proteins [47]. This field is divided into two main categories of disruption: solid shear (e.g. bead mill) and liquid shear (e.g. high pressure homogenizer) [48]. 2.3.1. Bead mill disruption Bead mill consists of a vertical cylindrical grinding chamber having a concentric cylinder with variable speed rotor for agitation. Cell slurries entered the mills at its base, flows up the annular gap between the rotor and the stator, and exits near the top [47]. Operation of the mill generates heat which is removed by circulating cooling water in the jacket that surrounds the grinding chamber. Bead mill disruption is independent of the biomass concentration (8–66 kg DW m−3 ) and disruption performance is consistent and predictable (a first order disruption kinetics is usually observed), thus ensuring ease of scale up [47]. A complete disruption is achievable within eight passes with the following conditions, 52,800 rpm, 85% loading of 512 ␮m beads and 90 mL min−1 slurry flow rate. Tamer et al. [47] also showed that the diameter of the grinding beads does not affect the disruption rate, but the rate is strongly dependant on the bead loading. 2.3.2. High pressure homogenization High pressure homogenization (HPH), is one of the most widely known methods for large scale cell disruption [49]. This device consisted of an air driven positive displacement pump which forced the cell slurry through two parallel slots (2 × 100 ␮m) under high pressure. The resulting parallel fluid streams impinged on a vertical plate, flowed toward each other, recombined, and were forced out. Disruption occurred at ambient temperature (25 ◦ C) which was maintained by immersing the disruption chamber and the exit lines in ice throughout operation [47]. The performance of the homogenizer depends on biomass concentration. By comparison with the mill, the homogenizer performs quite poorly at low biomass levels, but at 45 kg DW m−3 of cell concentration the homogenizer is a somewhat better disrupter [47]. Moreover, homogenizer are frequently stopped by blockages which made the process difficult, but in the case of the mill-processed P(3HB), little micronization (reduction of the average diameter of particles to a few micrometer) can be observed after eight passes [47]. A method using high pressure homogenizer in the presence of SDS was also used in the P(3HB) recovery from Methylobacterium sp. V49. The maximum yield and purity (98% and 95%) were obtained at an operating pressure of 400 kg cm−2 after two cycles and by using a 5% (w/v) SDS solution [50]. 2.3.3. Disruption by using ultrasonication Recently the disruption kinetics with ultrasonication was studied by Hwang et al. [51]. This method was carried on the PHA extraction from Haloferax mediterranei. A theoretical model linking the cell survival fraction, the acoustic power, the size disruption index and the operation time was established.

N. Jacquel et al. / Biochemical Engineering Journal 39 (2008) 15–27

After disruption, cells were isolated by centrifugation [51]. Lu [34] also studied the use of sonificator as a pretreatment before chemical treatment on various bacteria strains such as E. coli, C. taiwanensis184 and Buskholderia sp. PTU9. 2.3.4. Centrifugation and chemical treatment Van Wegen et al. [52] investigated the recovery of P(3HB) by centrifugation in combination with chemical treatment for efficient removal of non-P(3HB) cell material. A purity of 98.5% (w/w) and a P(3HB) recovery of 80% were reached with a total of three centrifugation steps. Ling et al. [53] also reported an extraction method for E. Coli, by combining homogenization, centrifugation and sodium hypochlorite treatment. A P(3HB) recovery of 80% at a purity of 96.5% was obtained with an optimized process. 2.4. Supercritical (SC) fluid Supercritical fluids have unique physicochemical properties such as high densities and low viscosities that make them suitable as extraction solvents. In that purpose CO2 is most widely used because of its low toxicity and reactivity, moderate critical temperature and pressure (31 ◦ C and 73 atm), availability, low cost and nonflammability [54]. The use of supercritical fluids for disruption of microbial cells and extraction of intracellular materials such as proteins was developed few years ago [55–58]. Heijazi et al. [54] in their study of the recovery of P(3HB) from R. eutropha showed that the optimal conditions were obtained for 100 min of exposure time, at a pressure of 200 atm, a temperature of 40 ◦ C and 0.2 mL of methanol (as a polar modifier). The recovery obtained by using supercritical (SC) CO2 is quite similar to the recovery obtained by other methods: 89% [54]. Since the publication of Hejazi et al. [54], several investigations have been done by the combination of SC CO2 with NaOH, or salt (NaCl) pretreatments, in order to get higher disruption levels [59]. 2.5. Using cell fragility Some bacteria such as Azotobacter vinelandii and recombinant E. Coli become fragile after the accumulation of large amount of PHA [60,61]. Page and Cornish [61] showed this phenomenon on A. vinelandii in fish peptone medium. They exploited the fragility of these cells to get a simple procedure for the extraction of high-molecular-weight P(3HB). The cells were treated with 1N aqueous NH3 (pH 11.4) at 45 ◦ C for 10 min. This treatment removed about 10% of the non-P(3HB) mass from the pellet, of which 60–77% was protein [61]. The final product consisted of 94% P(3HB), 2% protein, and 4% nonprotein residual mass [61]. The polymer molecular weight (1.7 × 106 –2.0 × 106 ) and dispersity (1.0–1.9) were not significantly affected by this treatment [61]. Polymer recovery from recombinant E. coli is also suggested to be simple. In fact this clone of bacteria strains of E. coli have been developed by Fidler and Denis [62], for the synthesis of P(3HB) to levels as high as 95% of the cell dry weight. These clones have been further enhanced by the addition of a geneti-

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cally mediated lysis system, called phage lysis, that allows the P(3HB) granules to be released gently and efficiently [62]. Another method, proposed by Choi and Lee [63], using the fragility of the recombinant E. coli cells with a P(3HB) content of 77%. They treated the cells with 0.2N NaOH at 30 ◦ C for 1 h, then the polymer was recovered with a purity of 98.5% [63]. By working at 50 ◦ C and with 5 mL of 1N sodium hydroxide, Lu [34] obtained a purity and recovery of 99% and 96%, respectively (Mw 480,000). 2.6. Air classification This method was developed by Noda [64] of Procter & Gamble on recovering P(3HB) from R. eutropha. First cells are treated with an ultrasonic sonicator, to create a suspension of polymer granules. Then, the suspension is freeze dried and pulverized using a fluid energy mill. The milled sample is then air classified to produce a 38% fine fraction and a 62% coarse fraction [64]. The fine fraction is then subjected to chloroform extraction followed by methanol precipitation to produce P(3HB) particles having a purity of about 95% or higher, and a yield of about 85% or higher. Experiments done on E. coli led to a purity of ∼97%, and a yield of ∼90% [64]. 2.7. Dissolved-air flotation Recently Van Hee et al. [65] investigated the recovery of PHA inclusion bodies from Pseudomonas putida, using a dissolvedair flotation method. This method follows a pretreatment with enzymes (lysozymes and Novozymes) [66]. In fact, both P. putida cell debris and PHA inclusion bodies have an iso-electric point of about pH 3.5. Near this pH selective aggregation and then selective flotation can be done [65]. This flotation is controlled by an interplay of particle–particle interactions, particle–bubble interactions and hydrodynamics leading to the formation of aggregates [65]. To control these interactions, particle properties such as particle size, hydrophobicity and surface charge (zeta-potential) must be taken into account. A purity of 86% (w/w) for PHA was obtained in three consecutive batch flotation steps [65]. 2.8. Spontaneous liberation By using a new cultivation method, involving recombinant E. coli harboring Alcaligenes phbCAB genes, spontaneous liberation of intracellualar P(3HB) granules can be obtained [67]. This method was developed in 2005 by Jung et al. They used a low cell inoculum and 2× LB medium containing 21% glucose. The results showed that the recombinant MG1655 accumulated P(3HB) up to an efficiency of 99% from the glucose supplied, and up to 80% of cells spontaneously secreted P(3HB) granules outside of the cells [67]. These experiments indicated that the use of a simpler purification process could be possible, such as from one-step centrifugation/washing with distilled water [67]. Resch et al. [68] used in combination with the phaCAB genes, the expression of cloned lysis gene E of bacteriophage PhiX174 from plasmid pSH2. By these experiments they showed

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Table 2 Summary of polyhydroxyalkanoates isolation methods Extraction method

Comments

Bacteria strain

Solvent extraction

Chlorinated hydrocarbon, cyclic carbonates, solvents mixtures

Bacillus megaterium

Lemoigne (1923–1951)

Rhodospirillum rubrum Ralstonia eutropha

Baptist [6] Vanalutem and Gilain [8] Traussnig et al. [9]

Chloroethanes, chloropropanes 1,2-Propane diol lycerol formal diethyl succinate butyrolactone

Results

Purity: 99.1%; recovery: 79%

Reference

Purity: 99.7%; recovery: 85% Purity: 100%; recovery: 90% Purity: 99.5%; recovery: 90%

Digestion by hypochlorite

Ralstonia eutropha

Palmitoyl carnitine High cell density digestion by SDS

A. latus A. eutrophus Ralstonia eutropha

Release rate >85%; release rate >70% Puirty >95%; recovery >90%

R. eutropha Recombinant E. coli. Cupriavidus taiwanensis184 Pseudomonas putidia KT2442

Purity: 86%; purity: 93% Purity: 99%; recovery: 94% Purity: 99%; recovery: 78%

Sodium hypochlorite Sodium hypochlorite Sodium hypochlorite

Matsushita et al. [10]

Recovery: 95% Purity: 98%

Surfactant-hypochlorite treatment Chelate-surfactant

Enzymatic digestion

Sodium hypochlorite and chloroform Sodium hypochlorite and chloroform with Al- and Fe-based coagulants SDS-hypochlorite Surfactant-EDTA disodium salt Recycled-wastewater process Chelate-hydrogen peroxide Selective dissolution by protons Enzymes Lytic enzymes of Cytophaga sp. Bromelain; pancreatin Papain Enzyme combined with SDS-EDTA Enzyme combined with SDS-EDTA

Zinn et al. [12] Kurdikar et al. [23] Noda [13] Procter & Gamble Liddell [18] Terada and Marchessault [25]

Sodium hypochlorite and anti oxidant Dispersion of sodium hypochlorite in chloroform

Schmidt et al. [11] Lafferty et al. [7]

Lee et al. [28] Kim et al. [29] Berger et al. [30] Hahn et al. [31] Lu [34] Roh et al. [35]

R. eutropha R. eutropha

Purity: >97%; recovery: 91% Purity 90–94%; recovery 98–99%

Hahn et al. [31] Ryu et al. [37]

Azotobacter chroococcum G-3 R. eutropha R. eutropha R. eutropha R. eutropha

Purity: 98%; recovery: 86.6%. Purity: 98.7%; recovery: 93.3% Purity: 96%; recovery: 90% Purity: 99.5% Purity: >96.4%; recovery: >94.8%

Dong and Sun [38] Chen et al. [27] Chen et al. [39] Liddell [40] Yu and Chen [41]

Purity: 88.8%; purity: 90.0% Purity: 89%; recovery: 78% Recovery: 95% Puirty: 92.6%; recovery: 90%

Holmes and Lim [42] Harrison et al. [43] Kapritchoff et al. [44] Lu [34] De Koning and Witholt [45] Yasotha et al. [46]

R. eutropha R. eutropha Burkholderia sp. PTU9 Pseudomonas P. putidia

Mechanical treatment Bed mill disruption

Tamer et al. [47]

N. Jacquel et al. / Biochemical Engineering Journal 39 (2008) 15–27

Digestion method Digestion by surfactants

Tetrahydrofuran methyl cyanide, tetrahydrofuran ethyl cyanide Acetic anhydride Ethylene carbonate 1,2-propylene carbonate Methylene chloride Long chain alcohols, esters, amides, ketones Solvent mixture High temperature process Two temperature process

Jung et al. [67] Resch et al. [68]

Noda [64] Van Hee et al. [65]

Page and Cornish [61] Fidler and Dennis [62] Choi and Lee [63] Lu [34]

Hejazi et al. [54]

Tamer et al. [47] Ghatnekar et al. [50] Hwang et al. [51] Van Wegen et al. [52] Ling et al. [53]

Reference

N. Jacquel et al. / Biochemical Engineering Journal 39 (2008) 15–27

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that small PHB granules, which are in a semi-liquid state are squeezed out of the cells through the E-lysis tunnel structure. It was also shown that all envelope components remain intact after E-lysis [68]. Then, the released PHB granules are aggregated in an aqueous solution containing divalent cations and glassmilk [68].

Purity ∼97%; recovery ∼90% Purity: 86%

Table 2 shows the summary of polyhydroxyalkanoates concentration using different extraction methods with different bacteria. Table 3 shows the comparison of isolating polyhydroxyalkanoates, and lists the advantage and disadvantage proposed in this table. 3. Pretreatment Typically, after the fermentation step, the broth is harvested by centrifugation at a concentration of about 5–85 g L−1 [50,53,69]. This operation is usually followed by one or more pretreatments step in order to make the cell disruption more easily.

E. coli E. coli

3.1. Heat pretreatment E. coli Pseudomonas putida

Purity: 98.5% Purity: 99%; recovery: 96%

Azotobacter vinelandii UWD Recombinant E. coli Recombinant E. coli Recombinant E. coli

Recovery: 94%

Recovery: 89% R. eutropha

Purity: 98.5%; recovery: 80% Purity: 96.5%; recovery: 80% E. Coli

Purity: 95%; recovery: 98% Methylobacterium sp. V49 Haloferax mediterranei

High pressure homogenization High pressure homogenization-SDS Ultrasonification Centrifugation and chemical treatment High pressure homogenization-centrifugation and hypochlorite treatment

A preliminary heat treatment has an impact on the cell solidity. In fact, it denatures genetic material and proteins, and it also destabilizes the outer membrane [44]. Several temperatures and duration have been studied. For example, in the study of R. eutropha DSM545, Kapitchkoff et al. treated cell broths at 85 ◦ C for 15 min [44], whereas De Koning and Witholt, who used a treatment at 120 ◦ C for 1 min in the case of Pseudomonas [45]. According to Steinb¨uchel, in the case of R. eutropha DSM545 strain, this treatment also denature the PHB depolymerase, a PHB granule wall enzyme, capable of degrading the biopolymer [44,70]. 3.2. Alkaline pretreatment Usually, for this kind of treatment, a solution of sodium hydroxide is used. Tamer et al. showed that by using an optimal hydroxide concentration of 0.4 kg kg−1 as pretreatment on A. latus, most of the protein could be released within tree passes into a bead mill [69]. Whereas untreated cells required at least ten passes for the same level of disruption (biomass concentration: 4.85 kg DW m−3 and bad loading: 80%) [69].

Alkaline treatment Alkaline treatment Alkaline treatment Alkaline treatment

Comments

Bacteria strain

Results

2.9. Summary and comparison of PHAs extraction methods

Spontaneous liberation

Air classification Dissolved-air flotation

Recovery using cell fragility

Super-critical fluid

Extraction method

Table 2 (Continued )

3.3. Salt pretreatment As water is attracted by high salt concentrations, a high solution will make water go out from cells. That will lead to cells to shrivel and dehydrate. In that purpose the cell broth is treated by sodium chloride, for 1 h at 60 ◦ C (Salt concentration: 8 kg m−3 on A. latus (Tamer et al.) [69] and 140 mM on R. eutropha (Khosravi-Darani et al. [59]). As salt treatment is not so effective, this method is commonly combined with an alkaline pretreatment [59,69].

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Table 3 Comparison of polyhydroxyalkanoates extraction methods Isolation method

Advantages

Disadvantages

Solvent extraction

Elimination of Endotoxine/high purity No polymer degradation

Digestion by surfactants

Treatment of high cell densities No polymer degradation High purity Low polymer degradation high purity Limited degradation/low operating cost High purity/low environmental pollution

Break PHA granules morphology Hazards connected with halogenated solvents High price/Low recovery Low purity/Water waste treatment needed

Digestion by NaOCl Digestion by NaOCl and chloroform Digestion by NaOCl and surfactants Digestion by chelate and surfactants Selective dissolution of NPCM Enzymatic digestion Bead mill disruption High pressure homogenization

High recovery and high purity low operating costs Good recovery No chemicals used No chemicals used

Supercritical CO2 Using cell fragility Air classification Dissolved air flotation Spontaneous liberation

Low cost, low toxicity Use of weak extracting conditions High purity No chemicals used No extracting chemicals needed

Degradation of the polymer High quantity of solvent needed Large volume of wastewater Low degradation of the polymer

High cost of enzymes Require several passes Poor disruption rate for low biomass levels Low micronization Low recovery Low recovery Require several consecutive flotation steps Low recovery (∼80% cells secretes PHB granules spontaneously [67])

3.4. Freezing

5. Large scale studies

A freezing pretreatment also release PHA granules and cell contents to be easily digested by SDS and NaClO in a short time [38]. Usually the PHB containing biomass is free-dried after a deionized water washing [30,31,39], but due to the high energy which require this operation, it is difficult to consider it for a large scale production of PHA. The freezing is also used to store the culture for long periods (−20 ◦ C) [50,53], in fact a storage at 4 ◦ C could not be used for a period which exceed one month [69].

Scale up to medium-scale has been studied by De Koning et al. [72] on P. putidia, with digestion using Alcalase, EDTA and SDS, after a heat pretreatment at 121 ◦ C. The device is composed of a vessel (>200 L) launched with sterile filter recirculation loop. The final product was a PHA latex with a solide fraction amounting to up 30 vol%. The purity of the PHA usually exceeded 95%. Partial integrity of peptidoglycan was shown to have an important effect on the recovery process. For higher purity level <99%, the latex must be extracted further with chloroform. This additional step is preferred to the direct cell extraction due to its much lower consumption of chloroform [72]. Chen et al. [73] reported the recovery of P(3HB-co-3HHx) from Areomonas hydrophila 4AK4, directly precipitated cells in the 20,000 L fermentor by adding 1% Na2 HPO4 , 1% CaCl2 , and 100 ppm polyacrylamide. The water was removed with a filter press, and cakes of cells were processed in a rotary vacuum dryer, before being converted into powder in a grinder. Then the polymer was extracted from 200 to 500 kg of dry cell powder in a 30,000 L extraction tank containing 5000 L ethylacetate stirred at 60 ◦ C for 2 h [73]. The solution containing polymer was passed through a metal filter and centrifuged. Then, P(3HB-co3HHx) is recovered by adding 15,000 L of hexane or heptane [73]. Finally, flocculants of polymer are collected with a filter press and washed with ethanol before being vacuum-dried. By using this process, Chen et al. found that the cost of the recovery process represented more than 50% of the total PHA production cost. That is why the recovery of solvent by distillation was also studied. Another solution will be to use hot acetone instead of ethylacetate and precipitate the polymer in water [73].

4. Purification Common methods of purification are involving a hydrogen peroxide treatment combined with the action of enzymes or chelating agents (see enzymatic digestion and chelate-hydrogen peroxide treatment parts). Recently, a method of purification by using ozone was proposed by Horowitz and Brennan [71] in 2001. This method was studied as a step of the purification process, in order to increase the level of purity of the polymer. To this end, ozone was applied to the biomass or solution in an oxygen stream containing 2 and 5% of ozone. The ozone treatment has beneficial effects of bleaching, deodorization, and solubilization of impurities, and so facilitating their removal from aqueous polymer suspensions or latexes [71]. This method could replace the hydrogen peroxide treatment of PHA, which has many drawbacks such as: a high operating temperatures (80–100 ◦ C), an instability of peroxides in the presence of high levels of cellular biomass and a decrease of the polymer molecular weight [71].

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6. Analysis of polyhydroxyalkanoates To evaluate the efficiency of isolation methods several parameters such as the purity, the recovery and the polymer molecular weight must be tested. On that purpose, the following parts give a short overview of most common analytical methods. 6.1. Yield determination Yield of PHAs could be simply determined by a gravimetric method [26,47,74]: after the treatment, the remaining solids are recovered by centrifugation and thoroughly washed with deionized water. Then, the final pellet was dried at 55 ◦ C during 24 h to constant weight in preweighted aluminum dishes [47]. 6.2. Purity measurement The content and composition of PHA in original cell mass, separation intermediates and final product could be determined by methanolysis of the biopolyesters in methanol (3 wt% sulfuric acid) at 100 ◦ C for 15–18 h [41,75]. The methylhydroxyalkanoates are hydrolysed into the corresponding hydroxyakanoic acid by adding 10N NaOH solution till the solution pH was above 13.8 [41]. The hydroxyalkanoic acids were determined using HPLC equipped with an organic acid column at 65 ◦ C [41]. Other methods also based on transesterification, use the detection, by a non polar gas chromatography column, of esters produced by the addition of mixture of n-propanol and hydrochloric acid (20%, v/v, HCl, 100 ◦ C during 2 h) [44,76] or a solution of 15% H2 SO4 in methanol (100 ◦ C during 140 min (Lageveen et al. [77]) or 4 h (Huijberts et al. [78]). 6.3. Molecular weight determination The molecular weight of PHAs could be determined by several methods like the intrinsic viscosity, gel permeation chromatography (GPC) (ex: 40 ◦ C in THF [50]), or by using the distribution curves obtained by size exclusion chromatography (SEC), after the dissolution of the polymer in hot chloroform [41]. For this last method, a calibration with polystyrene standards must be done. 7. Conclusion Through the different research done by many research groups on the recovery of PHAs, lots of improvements have been done since the solvent extraction method used by Baptist [6]. The recovery by using solvent can lead to high purity and eliminate some Endotoxin present in Gram-negative bacteria. However, this method is also marked by high cost and the nonenvironmental friendly aspect of the use of some solvents. Other methods based on digestion of non-PHA cell material have also been envisaged, but by using hypochlorite a degradation of the polymer was observed. Best results were obtained by combining hypochlorite with chloroform or surfactant treatment. Enzymatic digestion method was also introduced, but the use

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of expensive enzymes und complex process makes it economically unattractive [42]. Mechanical cell disruption methods like using bead mills and high pressure homogenization appear to be more economical [47]. A clean recovery of P(3HB) have also been envisaged by the use of supercritical CO2 [54], but this method appears to be still expensive in comparison with other methods. Recently, new methods like spontaneous liberation of P(3HB) [67], dissolved air flotation [65], or air classification [64] are being investigated and are probably promised to have much more success. Improvement of these new extraction and purification methods should lead to an optimal recovery of P(3HB), with a high purity and recovery level at a low production cost. Acknowledgments The authors would like to thank the National Science Council of Taiwan (NSC 95-2622-E-155-001), and the R´egion Rhˆone Alpes for their financial support. The acknowledgments of Mr. Jacquel are also addressed to professors and students of Yuan Ze University Department of Chemical Engineering and Materials Science who helped him for his researches and in his every day life during his stay in Taiwan. References [1] J.I. Choi, S.Y. Lee, Process analysis and economic evaluation for poly(3hydroxybutyrate) production by fermentation, Bioprocess. Eng. 17 (1997) 335–342. [2] T.V. Ojumu, J. Yu, B.O. Solomon, Production of polyhydroxyalkanoates, a bacterial biodegradable polymer, Afr. J. Biotechnol. 3 (2004) 18–24. [3] G. Du, J. Yu, Green technology for conversion of food scraps to biodegradable thermoplastic polyhydroxyalkanoates, Environ. Sci. Technol. 36 (2002) 5511–5516. [4] S.Y. Lee, Review bacterial polyhydroxyalkanoates, Biotechnol. Bioeng. 49 (1996) 1–14. [5] Y. Chen, J. Chen, G. Du, S. Lun, PHB TDfx2 Chem. Ind. TD(advances in extraction of biodegradable PHB), Eng. Prog. 17 (1998) 41. [6] J.N. Baptist, Process for preparing poly-b-hydroxybutyric acid, U.S. Patent 3,044,942 (1962). [7] R.M. Lafferty, E. Hernzle, Cyclic carbonic acid esters as solvents for poly(beta)-hydroxybutyric acid, U.S. Patent 4,101,533 (1978). [8] N. Vanlautem, J. Gilain, Process for separating poly-beta-hydroxybutyrates from a biomass, U.S. Patent 4,310,684 (1982). [9] H. Traussnig, E. Kloimstein, H. Kroath, R. Estermann, Extracting agents for poly-d(-)-3-hydroxybutyric acid, U.S. Patent 4,968,611 (1990). [10] H. Matsushita, S. Yoshida, T. Tawara, JP, 07,79,788 (1995). [11] J. Schmidt, B. Biederman, H.D.E. Schmiechen, DE 223 (1985) 428. [12] M. Zinn, H.U. Weilenmann, R. Hany, M. Schmid, T.H. Egli, Tailored synthesis of poly([R]-3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/HV) in Ralstonia eutropha DSM 428, Acta. Biotechnol. 23 (2003) 309–316. [13] I. Noda, Solvent extraction of polyhydroxy-alkanoates from biomass facilitated by the use of marginal nonsolvent, U.S. Patent 5,821,299 (1998). [14] U.J. H¨anggi, Pilot scale production of P(3HB) with Alcaligenes latus, in: E.A. Dawes (Ed.), Novel Biodegradable Microbial Polymers, Kluwer, Dordrecht, 1990, pp. 65–70. [15] D. Byrom, Industrial production of copolymer from Alcaligenes eutrophus, in: E.A. Dawes (Ed.), Novel Biodegradable Microbial Polymers, Kluwer, Dordrecht, 1990, pp. 113–117. [16] Y. Poirier, C. Nawrath, C. Somerville, Production of polyhydroxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants, Biotechnology (NY) 13 (1995) 142–150.

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