Sie sind auf Seite 1von 10

Biochemical Engineering Journal 93 (2015) 250–259

Biochemical Engineering Journal 93 (2015) 250–259 Contents lists available at ScienceDirect Biochemical Engineering Journal j op aration and purification Corresponding author. Tel.: +34 881 816014; fax: +34 881 816 702. E-mail address: maria.lopez@usc.es (M. López-Abelairas). http://dx.doi.org/10.1016/j.bej.2014.10.018 1369-703X/© 2014 Elsevier B.V. All rights reserved. of PHA from PHA-containing cell mass are essential for produc- ing bioplastics from renewable resources in a cost-effective and environmentally friendly way. The ideal method would maintain polymer properties and achieve high purity and recovery levels with low production costs. On an industrial scale, the use of halo- genated solvents is the most common method for PHA extraction. However, this method has important drawbacks, such as its high chemical costs and its associated hazards [4] . Among the alternative recovery processes that have been proposed, digestion of the cells with chemicals or enzymatic cocktails and extraction with non- halogenated agents have received the greatest interest due to their simplicity and more affordable cost [5–8] . More recently, the use of chemicals that can selectively dissolve non-PHA biomass has also been proposed [9] . In this study, four separation processes that constitute promis- ing alternatives to the commonly applied processes on an industrial scale were studied using a PHB-containing biomass. For each pro- cess, the processing conditions that achieved the highest purity and polymer recovery were determined. Then, the different treatments were compared considering each treatment’s operational perfor- mance, economical and environmental criteria and final product characteristics. " id="pdf-obj-0-5" src="pdf-obj-0-5.jpg">

Contents lists available at ScienceDirect

Biochemical Engineering Journal

j o u r nal home p age: www.elsevier.com/locate/ bej

Biochemical Engineering Journal 93 (2015) 250–259 Contents lists available at ScienceDirect Biochemical Engineering Journal j op aration and purification Corresponding author. Tel.: +34 881 816014; fax: +34 881 816 702. E-mail address: maria.lopez@usc.es (M. López-Abelairas). http://dx.doi.org/10.1016/j.bej.2014.10.018 1369-703X/© 2014 Elsevier B.V. All rights reserved. of PHA from PHA-containing cell mass are essential for produc- ing bioplastics from renewable resources in a cost-effective and environmentally friendly way. The ideal method would maintain polymer properties and achieve high purity and recovery levels with low production costs. On an industrial scale, the use of halo- genated solvents is the most common method for PHA extraction. However, this method has important drawbacks, such as its high chemical costs and its associated hazards [4] . Among the alternative recovery processes that have been proposed, digestion of the cells with chemicals or enzymatic cocktails and extraction with non- halogenated agents have received the greatest interest due to their simplicity and more affordable cost [5–8] . More recently, the use of chemicals that can selectively dissolve non-PHA biomass has also been proposed [9] . In this study, four separation processes that constitute promis- ing alternatives to the commonly applied processes on an industrial scale were studied using a PHB-containing biomass. For each pro- cess, the processing conditions that achieved the highest purity and polymer recovery were determined. Then, the different treatments were compared considering each treatment’s operational perfor- mance, economical and environmental criteria and final product characteristics. " id="pdf-obj-0-16" src="pdf-obj-0-16.jpg">

Regular Article

Comparison of several methods for the separation of poly(3-hydroxybutyrate) from Cupriavidus necator H16 cultures

Biochemical Engineering Journal 93 (2015) 250–259 Contents lists available at ScienceDirect Biochemical Engineering Journal j op aration and purification Corresponding author. Tel.: +34 881 816014; fax: +34 881 816 702. E-mail address: maria.lopez@usc.es (M. López-Abelairas). http://dx.doi.org/10.1016/j.bej.2014.10.018 1369-703X/© 2014 Elsevier B.V. All rights reserved. of PHA from PHA-containing cell mass are essential for produc- ing bioplastics from renewable resources in a cost-effective and environmentally friendly way. The ideal method would maintain polymer properties and achieve high purity and recovery levels with low production costs. On an industrial scale, the use of halo- genated solvents is the most common method for PHA extraction. However, this method has important drawbacks, such as its high chemical costs and its associated hazards [4] . Among the alternative recovery processes that have been proposed, digestion of the cells with chemicals or enzymatic cocktails and extraction with non- halogenated agents have received the greatest interest due to their simplicity and more affordable cost [5–8] . More recently, the use of chemicals that can selectively dissolve non-PHA biomass has also been proposed [9] . In this study, four separation processes that constitute promis- ing alternatives to the commonly applied processes on an industrial scale were studied using a PHB-containing biomass. For each pro- cess, the processing conditions that achieved the highest purity and polymer recovery were determined. Then, the different treatments were compared considering each treatment’s operational perfor- mance, economical and environmental criteria and final product characteristics. " id="pdf-obj-0-25" src="pdf-obj-0-25.jpg">

M. López-Abelairas a, , M. García-Torreiro a , T. Lú-Chau a , J.M. Lema b , A. Steinbüchel c,d

a Department of Chemical Engineering, Institute of Technology, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain b Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain c Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany d Department of Environmental Sciences, King Abdulaiziz University, Jeddah, Saudi Arabia

a r t i c

l e

i n f o

Article history:

Received 28 January 2014 Received in revised form 21 October 2014

Accepted 23 October 2014 Available online 1 November 2014

Keywords:

Polyhydroxyalkanoates

Cupriavidus necator

Purification

Bioseparation

Cell disruption

Downstream processing

a b s t r a c t

Biopolymers, such as polyhydroxyalkanoates (PHA), are an environmentally friendly alternative to plas- tics derived from fossil fuels. However, producing PHA in a cost-effective way requires the development of highly efficient separation and purification treatments. In this study, one acid treatment (sulphuric acid combined with a subsequent bleaching step) and three alkaline treatments (sodium hypochlorite, sodium hydroxide and a combination of the latter with an halogenated solvent) were evaluated for recovering

poly(3-hydroxybutyrate) (PHB), a type of PHA, from Cupriavidus necator H16 cells with high biopolymer content (65%). Purity, percent of recovery and the properties of the PHB obtained after each treatment, together with the costs and environmental impacts associated with each treatment, were determined and compared. The lowest recovery costs were obtained with the sodium hydroxide and sulphuric acid treatments (1.02 and 1.11 D kg 1 , respectively). Estimated CO 2 emissions of these two treatments were 18% of those based on the use of sodium hypochlorite. However, the highest purity (98%) and lowest poly- mer degradation were achieved with the acid treatment. Consequently, the acid treatment was selected as the most effective choice for PHA recovery.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

A potentially interesting way to decrease the environmental impact of conventional petroleum-derived plastics is to replace them with biodegradable polymers. In this context, polymers of biological origin, such as polyhydroxyalkanoates (PHA) and among them polyhydroxybutyrate (PHB), play an important role. These polymers are accumulated inside bacterial cells as energy and carbon storage. Cupriavidus necator, the best-studied PHB- accumulating bacterium, stores PHB when there is an excess of carbon with respect to some other essential nutrient in the medium, such as nitrogen, phosphorous or oxygen [1,2]. To compete with conventional plastics, PHA production costs must be minimised. It has been estimated that more than 50% of the cost of PHB pro- duction are associated with the recovery and purification of the polymer [3]. Due to the large impact of the recovery step on pro- duction costs, efficient methods for separation and purification

Corresponding author. Tel.: +34 881 816014; fax: +34 881 816 702. E-mail address: maria.lopez@usc.es (M. López-Abelairas).

http://dx.doi.org/10.1016/j.bej.2014.10.018

1369-703X/© 2014 Elsevier B.V. All rights reserved.

of PHA from PHA-containing cell mass are essential for produc- ing bioplastics from renewable resources in a cost-effective and environmentally friendly way. The ideal method would maintain polymer properties and achieve high purity and recovery levels with low production costs. On an industrial scale, the use of halo- genated solvents is the most common method for PHA extraction. However, this method has important drawbacks, such as its high chemical costs and its associated hazards [4]. Among the alternative recovery processes that have been proposed, digestion of the cells with chemicals or enzymatic cocktails and extraction with non- halogenated agents have received the greatest interest due to their simplicity and more affordable cost [5–8]. More recently, the use of chemicals that can selectively dissolve non-PHA biomass has also been proposed [9]. In this study, four separation processes that constitute promis- ing alternatives to the commonly applied processes on an industrial scale were studied using a PHB-containing biomass. For each pro- cess, the processing conditions that achieved the highest purity and polymer recovery were determined. Then, the different treatments were compared considering each treatment’s operational perfor- mance, economical and environmental criteria and final product characteristics.

M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259

251

  • 2. Materials and methods

    • 2.1. PHB production

A mineral salt medium composed by 2.0 g L 1 (NH 4 ) 2 HPO 4 ,

  • 2.1 g

L 1 KH 2 PO 4 , 0.2 g L 1 MgSO 4 ·7H 2 O, 0.1 g L 1 CaCl 2 ·2H 2 O,

0.006 g L 1 FeCl 3 ·6H 2 O, 0.1 mL L 1 of trace element solution SL6 [10] and 30 g L 1 of sodium gluconate was used to produce the C. necator H16 inoculum in a Biostat DL-30 reactor (with 25 L of medium). Polymer accumulation with this strain was performed in a 400-L bioreactor (Biostat D650, Sartorius) for 68 h, using a fed-batch con- figuration into which gluconate was continuously fed to induce a nitrogen limitation [11]. A medium composed of 400 g L 1 of glu- conate, 5 g L 1 MgSO 4 ·7H 2 O, 57 g L 1 (NH 4 ) 2 HPO 4 and 257 g L 1 NH 4 Cl was fed during the first 40 h of operation at a rate that led to a constant gluconate concentration of approximately 17 g L 1 in

the reaction medium. Then, the cells were cultivated with an excess of carbon to attain nitrogen-limitation conditions.

  • 2.2. PHB separation

C. necator H16 cells were separated by centrifugation and then freeze-dried. The particle size of the resulting solid was first homogenised by milling using a conventional blender (maximum speed, 30 s). Then, the homogenised solid was subjected to one of the following treatments to recover the polymer accumulated inside the cells. All experiments were performed in triplicate in closed 500 mL glass bottles with a volume of liquid of 100 mL, except for chloroform extraction.

  • 2.2.1. Chloroform extraction

Chloroform extraction is a well-established reference standard procedure. 1 mg mL 1 of lyophilised cells was suspended in chloro-

form in closed glass tubes at 60 C for 36 h [12]. Then, the polymer was precipitated by adding ten volumes of methanol and dried at

  • 55 C.

    • 2.2.2. NaOH treatment (PHB-R1)

Alkaline digestion of biomass was performed using a solid con- tent of 2.5% w/v with NaOH solutions at different concentrations (0.25, 0.5, 1.0, 2.0 and 4.0 N) for 4 h at 37 C and 500 rpm. Then, the procedure was repeated with the optimal NaOH concentration selected in the previous step and various solids contents (2.5, 5, 7.5 and 10% w/v). Samples were centrifuged, and the solid phase was washed twice with water and once with ethanol and freeze-dried. In each washing step, approximately 0.8 mL of liquid per g of initial biomass was used.

  • 2.2.3. NaOCl treatment (PHB-R2)

Similarly, a commercial NaOCl solution (13% v/v) was used to perform biomass digestion at different solids contents (within the range 2.5–7.5%, w/v) at 37 C and 500 rpm for 4 h. The solid was separated as described above.

  • 2.2.4. NaOCl and dichloromethane treatment (PHB-R3)

At the same temperature and agitation conditions and with a solids content of 2.5% (w/v), a combination of NaOCl solution (13% v/v) with a halogenated solvent, dichloromethane, in a 1:1 (v/v) ratio was used to digest the biomass and to extract the polymer. The polymer in the organic phase precipitated when 10 volumes of ethanol were added. The resulting solid was washed with water and ethanol and freeze-dried.

  • 2.2.5. Acid treatment (PHB-R4)

H2SO4 solutions at several acid concentrations were used to digest the biomass (5%, w/v) at different temperatures and for dif- ferent times. Three levels were defined for each of these process conditions: (i) temperatures of 37, 65 and 100 C, (ii) treatment times of 1, 15 and 30 h; and (iii) acid concentrations of 2.5, 5 and 10% (v/v). After the acid treatment, the pH value was set to 10 using a 0.5 N NaOH solution, and the solid was washed with water. Finally, a mild bleaching step with sodium hypochlorite at 3% was applied for 1 h to remove residual protein. The mathematical relationships of the response variables (purity and recovery percentages) to the independent variables (temperature, acid concentration and time) were represented by quadratic model equations. Finally, response surfaces for purity and recovery percentages were plotted using the software MATLAB TM Version 7.3.0 (The Mathworks, Inc).

  • 2.3. Reuse of chemical solutions

After the biomass was chemically digested with alkali or acid and the resulting solution was centrifuged to recover the solids, different fractions of the liquid phase (20%, 40%, 60% and 80%) were mixed with fresh solution for reuse. This operation was repeated four times with each digestion agent.

  • 2.4. Polymer quantification

The polymer percentage in the samples was determined using gas chromatography (GC). Samples were subjected to methanol- ysis in a solution with 1 mL of chloroform, 0.85 mL of methanol and 0.15 mL of H 2 SO 4 [13]. The resulting esters were determined by gas chromatography in split injection mode with helium as the carrier gas (5 cm min 1 ) using GC equipment with an FID detec- tor and a PEG Permaphase column (60 m, 0.32 mm of diameter, 0.5 m, Restek GmbH, Bad Soden, Germany) [14]. A commercial P(3HB) (Ref. 363502, Sigma-Aldrich) was used to determine the calibration curve. Purity and recovery percentages of the polymer were calculated using Eqs. (1) and (2), respectively:

Purity (%) =

polymer

weight

total sample weight × 100

Recovery (%) =

W f × P f

W i × P i

× 100

(1)

(2)

where W i is the initial dry weight of the solid introduced into the recovery step (g); W f is the total dry weight of the solid recovered after the recovery step (g); P i , P f represent the purity of the solid before and after the recovery process, respectively (%).

  • 2.5. Polymer characterisation

    • 2.5.1. X-ray powder diffraction (XRD)

The crystalline structure of the samples was studied using an X-ray diffractometer (Philips X’Pert), which provides Cu K radia- tion (40 kV, 40 mA), employing the powder method. Every scan was recorded in the range of 2 = 2–50 in step-by-step mode with step

size 0.02 .

  • 2.5.2. Differential scanning calorimetry (DSC)

In DSC, samples were heated from room temperature to 650 C at a heating rate of 20 C min 1 in an N 2 atmosphere. The melting

temperature and enthalpy of fusion ( Hf) were calculated from the maximum and the area of the first endothermic peak, respectively, whereas decomposition temperature (Td) was calculated from the

  • 252 M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259

second endothermic peak. The degree of crystallinity (Xc) was cal- culated using by the following equation:

X c =

H f

H 0

f

(3)

where H

  • 0 corresponds to the enthalpy of fusion of 100% crys-

f

talline polymer (146 J g 1 ) [15].

  • 2.5.3. Gel permeation chromatography (GPC)

The polymer was analysed by GPC in order to determine the weight-average molar mass (M w ) and number-average molar mass (M n ). This analysis was performed using a Phenogel 5 10E5A col- umn and an IR detector. Chloroform was used as the eluent with a flow rate of 0.8 mL min 1 . Calculations were based on calibration curves obtained from monodisperse polystyrene standards ranging from 400 Da 1 up to 2106 kDa 1 (Fluka 81434). The polydisper- sion index (PI) and degree of polymerisation (DP) were calculated according to Eqs. (4) and (5), respectively.

PI =

M

w

M

n

DP =

M

n

m

(4)

(5)

where m is the molar mass of the repeat unit.

  • 2.6. Statistical analysis

Statistical analysis was conducted using the software SPSS Inc—PASW Statistics 18 (IBM) to determine the standard devia- tion (SD) of results obtained at different conditions, determine the existence of significant differences and conduct pair-wise multiple comparisons. First, a one-way analysis of variance, using a para- metric method (ANOVA, based on a linear model), was performed to determine whether values obtained using different treatment were significantly different. A post hoc analysis (Tukey HSD) was used to determine which pairs of treatments had significantly different values, at a significance level of 0.05.

  • 2.7. Economic and environmental analysis

Energy and mass balances were solved using the process mod- eling software Aspen PlusTM, version 7.3 (Aspen Technology, Inc, MA, USA). Some components for which the ASPEN library did not contain information, such as the biomass or the polymer, were specified using the compound structure from the CheBI database (http://www.ebi.ac.uk/chebi/). The simulated plant would have the capacity to treat 1000 kg h 1 of cellular biomass from the polymer accumulation step. Once the simulations were performed, mate- rial and heat flows were introduced using Aspen Process Economic Analyzer, Economic Evaluation V7.3 (ASPEN Tech, Cambridge MA). The costs considered in the economic analysis were those asso- ciated with the use of reagents and utilities (steam and electricity requirements). Costs for chemicals and utilities were calculated per kg of polymer. Greenhouse gas (GHG) emissions, expressed as equivalent kg of CO2, were calculated taking into account the reagents and utilities required for the different processes and the associated emissions reported for each of these inputs by the following equation:

GHG emissions kgCO 2 eq/kg PHA =

n i=1 F i × e i

P

(6)

where F i is the mass flow of reagent i(kg h 1 ); e i is the emissions associated with the production of reagent i(kg CO 2 -eq kg 1 ); P is the mass flow of product (PHA)(kg h 1 ).

  • 3. Results and discussion C. necator H16 cultivations were performed in a pilot bioreactor

at the Institut für Molekulare Mikrobiologie und Biotechnolo-

gie (IMMB) at the Westfälische Wilhems-Universität [11]. Carbon

source concentration was held constant during the entire pro- cess; nitrogen supplementation was stopped when the desired cell density was achieved. When ammonium concentration attained a critical level, the accumulation phase began. After approximately 68 h of operation, a total cell mass of almost 11 kg was obtained with a PHB content of 65%.

  • 3.1. Treatment PHB-R1

Alkaline treatment with NaOH, here termed PHB-R1, is com- monly used as a pretreatment step before the main separation process. However, its utility as a unique separation treatment has lately become an object of interest due to its attractive cost. In the first step of this study, the optimal NaOH concentration for alkaline digestion of the biomass was determined using a biomass percent- age of 2.5%. Solid purity after the treatment increased as the alkali concentration increased, up to 0.5 N (p < 0.001). Above this con- centration, no improvement in purity could be detected (p = 0.010) (Fig. 1a). This optimal value for alkali concentration agrees with that found by Lo et al. [6] to treat Escherichia coli cells. Moreover, as the concentration of NaOH solutions increased, it was more difficult to separate the biomass from the solution by centrifugation. There- fore, a 0.5 N NaOH solution was employed to digest the biomass obtained from C. necator fermentation. Additionally, temperatures over 37 C or treatment times longer than 5 h did not result in higher purity or recovery efficiency. Particularly extreme temperatures or treatment times actually had a negative effect on recovery (data not shown). Furthermore, purity and recovery efficiency were reduced when the initial solids concentration was increased (Fig. 1b). Specifically, at a solids concentration over 5%, purity decreased to 90% and recovery fell sharply (p < 0.001). The sharp drop in recovery effi- ciency may be explained by the increased difficulty in separating solid and liquid phases, which decreases the recovery ratio. This decrease in purity and recovery efficiency may be a drawback for the industrial application of this methodology, despite the advan- tage of its low cost. The best values of purity and recovery were obtained with a solids content of 2.5% in a 0.5 N NaOH solution (0.8 g NaOH g 1 solid).

  • 3.2. Treatment PHB-R2

The best-known alternative among the four treatments tested for PHB recovery is the sodium hypochlorite digestion method developed by Williamson & Wilkinson [16]. It has been com- monly used at the bench scale due to its high effectiveness and its simplicity [17–19]. Treatment conditions such as tempera- ture and hypochlorite concentration have been widely studied and optimised. High purity and recovery levels can be achieved at moderate temperatures (approximately 40 C) using only a commercial sodium hypochlorite solution [20]. Suspensions of C. necator cells with different biomass percentages were digested using this method. Similar percentages of the total solid (between 54 and 57%) were recovered from hypochlorite digestion of differ- ent solids loadings. However, the percentage of recovered non-PHB biomass increased as the solids loading increased (for example, it

rose from 3% at 2.5% of solids to 31% at 7.5% of solids), while the per- centage of recovered PHB decreased (at the same solid loadings, it fell from 82 to 70%). These results show that the alkali present in the solution was not enough to digest all of the biomass, which remained as an insoluble solid. The drop in PHB recovery may be

M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259

253

M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259 253 Fig. 1. Purity (grey
M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259 253 Fig. 1. Purity (grey
M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259 253 Fig. 1. Purity (grey

Fig. 1. Purity (grey bars) and recovery (white bars) percentages after 4 h at (a) several NaOH concentrations with a solids percentage of 2.5% w/v and (b) several solids percentages in a 0.5 N NaOH solution.

explained by the presence of some of the undigested biomass on the surface of the basic solution, which could not be recovered by centrifugation. This decrease in digestion performance led to a significant decrease in the purity of the final solid. For example, with a solids loading of 2.5%, the purity percentage achieved with hypochlorite was 97–98%; with a solids loading of 5%, the purity was close to 90% and, with a solids loading of 7.5%, the purity fell to 80% (data not shown).

  • 3.3. Treatment PHB-R3

PHA extraction using solvents is the oldest separation method. The solvent functions include modifying cell membrane per- meability and then dissolving the polymer inside the cells [4]. Halogenated compounds such as dichloromethane, chloroform or 1,2-dichloroethane are commonly used in these processes because of their high extraction efficiency. The combination of halogenated solvents with sodium hypochlorite for PHA separation has also been tested [5,18]. In this method, cells are degraded by the alkaline solution, and the polymer is released into the medium. Hahn et al. [18] have suggested that extensive polymer degradation is avoided by fast extraction of the released polymer into the organic solvent phase during the process of cellular digestion. In this method, a mixture of sodium hypochlorite and dichloromethane was applied for PHB isolation. PHB precipitation was performed using ethanol as a non-solvent. Precipitation with ethanol yielded a polymer with very high purity (over 99%) and achieved a good recovery per- centage (90%) (Fig. 2). Similar results for purity and recovery were

reported by Hahn et al. [18], who used chloroform as the solvent under optimised conditions. However, the hazards associated with these solvents, as well as their high cost, are important drawbacks for the application of this method on an industrial scale [4].

  • 3.4. Treatment PHB-R4

Acid treatment has been proposed as an alternative digestion method that would allow selective separation of the polymer from the cells. The acid solution degrades cells with a marginal hydroly- sis of the polymer stored inside, in contrast to alkaline treatments [9,21]. At moderate acid concentrations (0.1–4 N), Yu et al. [21] found that there was a dynamic balance between cleavage and repair of ester bonds, because protons from the acid are the catalyst for both hydrolysis and esterification. In contrast, hydroxyl anions from alkali are reagents in polymer hydrolysis because they can remove protons from the acid produced during ester bond cleav- age. Thus, alkaline treatments prevent re-esterification between the formed acids and alcohols, while acid treatments allow re- esterification. Because of this advantage over alkaline treatments, as well the novelty of this method with respect to the treatments previously described, an in-depth study of acid separation was performed. The influence of operational conditions on purity and recovery efficiency using sulphuric acid as a digestion agent was determined. Three variables were studied: temperature, acid concentra- tion and treatment time. A face-centred central composite design (FCCD) is applied to carry out optimisation. According to the FCCD, a total of 15 combinations of the levels of the three variables stud-

by Eqs. (7) and (8), respectively.

×T × t + 0.0006 × T × C × t

ied were selected as experimental points. The purity and recovery

percentages obtained in this assay are expressed as functions of

the process variables identified as significant by ANOVA analysis

Purity(%) = 63.03 + 0.55 × T + 1.86 × C + 1.08 × t 0.0117

0.0023 × T 2 0.17 × C 2

(7)

Recovery(%) = 49.29 + 0.71 × T 0.0041 × T 2 0.114 × C 2 + 0.0046 × t 2

(8)

M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259 253 Fig. 1. Purity (grey

Fig. 2. Purity (grey bars) and recovery (white bars) percentages for extraction meth- ods under optimised conditions (i.e., PHB-R1: 0.5 N NaOH solution and 2.5% w/v solids content, PHB-R2 and PHB-R3: 2.5% w/v solids content and PHB-R4: 0.64 M H 2 SO 4 solution, 5% w/v solids content and 6 h).

T is the treatment temperature ( C), C is the acid concentration (% v/v) and t is the treatment time (h). R 2 values were 0.988 for the purity equation and 0.997 for the recovery equation, indicating that the equations accurately reproduce the experimental data. The maximum recovery percentage calculated from these equations

  • 254 M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259

within the current working range was 79%. Likewise, maintain-

ing this recovery value, a high purity level (almost 98%) could

be achieved. This result is similar result to that obtained for the

hypochlorite treatments. The operational conditions at which these

purity and recovery percentages can be achieved correspond to a

temperature of 80 C, an acid concentration of 3.5% (v/v) (0.64 M)

and a treatment time of 6 h. Yu and Chen [9] suggested that the

high purity of the polymer is related to the selective degradation

of molecules such as peptidoglycan that form a protective mem-

brane that stabilises the granules. They found a high rate of protein

release from cells and a clear increase of crystalline fraction in the

granules. These authors connected these two observations because

the release of the proteins that participate in granule stabilisation

facilitates polymer crystallisation. Finally, because the obtained

structure has a higher degree of crystallisation than native PHB

granules, it has an increased resistance to the alkaline attack suf-

fered in the second step of purification with sodium hypochlorite

[9]. However, Eqs. (7) and (8) imply that, although more severe

conditions (i.e., higher temperatures and acid concentrations) lead

to higher purities, they also lead to lower polymer recovery levels,

perhaps due to partial degradation of the polymer.

  • 3.5. Recovery processes overview

    • 3.5.1. Product properties

The polymers extracted by the methods with the best results

under optimised conditions (PHB-R1, PHB-R2, PHB-R3, PHB-R4)

and those isolated using acid treatment under more severe con-

ditions (100 C, 10% v/v and 15 h), here termed PHB-R4*, were

characterised. GC analysis of these polymers indicated that they

were fully constituted by 3HB monomers, as expected for this

strain and the applied carbon source. Additionally, X-ray analy-

sis reflected diffractograms consistent with those generated by

poly(3-hydroxybutyrate) and corresponding to an orthorhombic

structure with a space group of P212121 and dimensions a = 5.76 A, ˚

b = 13.20 A ˚ and c = 5.96 A. ˚

The mechanical properties of amorphous polymers such as PHB

are significantly influenced by molar mass. Both weight-average

molar mass (M w ) and number-average molar mass (M n ) were

determined for each extraction method (Table 1). Molar mass dis-

tribution was also determined (Fig. 3). These parameters indicate

the degree of degradation suffered by the polymer during the

separation process. A reduction in molecular weight was observed

for all treatments compared with the chloroform-extraction con-

trol (Table 1 and Fig. 3). In this case, the most aggressive method

resulting in the lowest molar masses was treatment PHB-R2

(M n = 36 kDa, M w = 249 kDa). Due to their amorphous structure,

polymer granules are hydrolysed more easily than crystalline

polymers by alkaline solutions [21]. This fact is reflected in the

displacement of the molar mass distribution toward lower log M

values (Fig. 3a) and the increase in the range of molar masses

present. This latter outcome is quantified by the polydispersion

index (PI), whose value increased 4-fold over control. Dispersion of

molar mass was also high for treatment PHB-R4* (Fig. 3b) implying

a structure with short polymer chains among crystalline domains

consisting of chains of greater length. Another parameter calcu-

lated using the molar mass is the degree of polymerisation (DP),

which indicates the average number of repeat units that constitute

a polymer chain. The DP depended strongly on the separation

process: the DP values found after separation using treatments

PHB-R2 and PHB-R4* were less than half of those obtained using

the other two treatments. This result indicates the significant poly-

mer degradation caused by treatments PHB-R2 and PHB-R4*. The

molar mass distribution was unimodal for all procedures except

PHB-R4*, for which it was bimodal. This difference is similar to

that reported by Penloglou et al. [22] when long sonication times

254 M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259 within the current working

Fig. 3. Molar mass distributions (differential weight fraction versus molar mass) for the different recovery procedures (control with chloroform is represented by a grey dashed line): (a) basic treatments: PHB-R1 (black line), PHB-R2 (dotted black line) and PHB-R3 (grey line) and (b) acid treatments: PHB-R4 (black line) and PHB- R4*(grey line).

were applied to recover PHB from Azohydromonas lata cells. Severe

conditions lead to mechanical breakage of polymer chains as a

consequence of the high energy input [22].

Degradation of polymer structure when an alkaline digestion

method is used in the separation step has been reported by many

authors [4,23]. The decrease in molar mass observed after sepa-

ration using only hypochlorite (approximately 70%) is consistent

with that reported by Berger et al. [17]. In addition, Lo et al.

[6] reported that molar mass was reduced by 66% when NaOH

solutions with concentrations above 0.5 N were applied, which

is consistent with the results of the current work. These authors

proposed the use of a surfactant, SDS (sodium dodecylsulfate), to

avoid the polymer degradation caused by sodium hydroxide. How-

ever, a surfactant dosage over 5 wt% increases recovery costs and

causes problems in wastewater treatment and reuse [8]. By con-

trast, treatments with a dichloromethane/hypochlorite mixture or

acid under moderate conditions achieved better preservation of the

polymer structure (Fig. 3a and b, respectively) and less reduction

in the molecular mass of the polymer chains. For treatment with

dichloromethane/hypochlorite mixtures, the PI value is markedly

reduced in comparison with the value for the treatment with only

hypochlorite. Hahn et al. [18] suggest that the fast migration of

the polymer into the organic phase avoids prolonged contact times

with basic solutions, thereby decreasing the breakdown of polymer

chains. PHB-R3 and PHB-R4 treatments both resulted in a polymer

with high purity (greater than 98%), although recovery efficiency

M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259

255

Table 1 Characteristics of biopolymer extracted by the different recovery methods: enthalpy of fusion ( H), melting temperature (T m ), decomposition temperature (T d ), degree of crystallinity (X c ), weight-average molar mass (M w ) and number-average molar mass (Mn), polydispersion index (PI) and degree of polymerisation (DP).

Treatment

Conditions

T m ( C)

T d ( C)

H (J g 1 )

X c

M n (kDa)

M w (kDa)

PI

DP

PHB-R1

PHB-R2

PHB-R3

PHB-R4

0.5 N NaOH solution 2.5% w/v solids content 4 h, 37 C Sodium hypochlorite solution (13% v/v) 2.5% w/v solids content 4 h, 37 C Dichloromethane Sodium hypochlorite solution (13% v/v) 2.5% w/v solids content 4 h, 37 C H 2 SO 4 solution (3.5% v/v) 5% w/v solids content 6 h,

159–171

151–169

221–247

284–294

76.45

87.55

152–167

281–296

88.10

159–171

286–300

76.38

0.52

0.60

0.60

0.52

98 ± 5 36 ± 2

138 ± 5

130 ± 4

283 ± 1 249 ± 2

361 ± 2

395 ± 1

2.90 ± 0.15 6.82 ± 0.38

1140

420

2.61 ± 0.10

1605

3.03 ± 0.10

1510

 

80

C

PHB-R4*

H 2 SO 4 solution (10% v/v) 5% w/v solids content 15 h,

152–165

285–303

123.1

0.84

44 ± 3

321 ± 1

7.29 ± 0.50

510

100

C

Control

Chloroform extraction

149–165

283–310

87.15

0.60

521 ± 5

837 ± 2

1.61 ± .0.02

6058

was slightly higher when the combination of chemical digestion

with solvent extraction was applied.

No significant differences in melting temperature (T m ) were

found in PHB recovered by the different treatments (Table 1). How-

ever, the decomposition temperature (T d ) of the polymer recovered

by treatment PHB-R1 was markedly lower (221–247 C) than that of

the polymer recovered by the other treatments. This result implies a

drawback for treatment PHB-R1compared with the other methods

due to the reduction in the size of the processing window within

which the polymer could be melted without being degraded. This

result may be related to the lower purity of the polymer recovered

by this method. Hong et al. [24] have reported that the addition of a

small amount of impurities to PHB results in a significant decrease

in thermal stability due to a catalytic degradation effect.

The high degree of crystallinity (almost 85%) measured for

the polymer isolated by the PHB-R4* method is noteworthy; this

parameter was in the range of approximately 50 to 60% for the

other extraction processes and the control. This significant increase

in the degree of crystallinity for PHB-R4* was detected in both

DSC and XRD measurements. However, similar purities (98–99%)

were obtained for the acid procedures under severe and moderate

conditions, while different polymer recovery levels (79% at mod-

erate conditions and 65% at severe conditions) were found. These

different recovery levels may be explained by increased polymer

degradation under more severe conditions, which mainly affects

the amorphous fraction. Taking into account recovery, purity and

crystallinity data, the weight of amorphous and crystalline polymer

after the application of each method can be calculated. Treatment

under moderate conditions yielded 0.37 g amorphous polymer g 1

initial biomass and 0.40 g crystalline polymer g 1 initial biomass;

under severe conditions, treatment yielded 0.10 g amorphous poly-

mer g 1 initial biomass and 0.54 g crystalline polymer g 1 initial

biomass. The reduction in amorphous polymer weight under severe

conditions is most likely due to the fact that amorphous polymer

is more easily degradable than crystalline material. There was also

a net increase in crystalline polymer under severe conditions com-

pared with moderate conditions. Therefore, it can be concluded that

the use of more severe treatment conditions causes an increase in

native polymer crystallinity.

Previous studies have reported the amorphous nature of PHB

granules inside cells. Therefore, it seems that all extraction methods

lead to a rearrangement of polymer molecules toward the forma-

tion of crystalline structures. This result agrees with Lauzier et al.

[25], who indicated that polymer molecules within the amorphous

structure are in a metastable state, which can change with modifi-

cations in the conditions of the surrounding medium. These authors

found that small changes in the composition or an increase in the

temperature of the medium can induce surface crystallisation of

the PHB granules. This rearrangement of polymer molecules into

lamellar crystals may be explained by the diffusion out of the gran-

ules of small molecules that act as plasticising agents, which occurs

without the protection of the lipid–protein membrane that helps to

stabilise the granules. Taking this into account, it can be supposed

that the acid treatment studied in the current work leads to the

formation of a crystalline shell in the granules. In addition, it was

observed that the more severe the acid treatment conditions, the

higher the polymer crystallinity. This observation may be explained

by two phenomena affecting PHB granules:

(1) The partial degradation of the amorphous structure of the

granules, similar to the degradation that occurs in alkaline

treatments.

(2) The increase of the crystalline shell thickness, which depends

on the diffusion of plasticizing agents within the surface. The

increase in temperature contributes to increased diffusion and,

consequently, an increased crystalline fraction in the granules.

Concerning the presence of protein in recovered polymers, Yu

and Chen [9] have described the high vulnerability of proteins to

acid dissolution. The introduction of hypochlorite digestion after

the acid step provides further protein removal. However, Hahn et al.

[18] found a negligible protein content in the polymer extracted

using hypochlorite-organic solvent dispersion and hypochlorite

solution with a concentration higher than 10%. Therefore, a neg-

ligible protein concentration should be expected in the polymer

recovered by applying hypochlorite and acid digestions.

  • 3.5.2. Economic and environmental analysis

Notable similarities in M w , melting temperature and crys-

tallinity data were observed for the polymer commercialised

by Biomer (M w = 230 kDa, crystallinity = 0.60, T m = 175 C [26],

T d = 285 C [27]) and the polymer obtained after applying treat-

ments PHB-R2, -R3 and -R4. In addition, similar characteristics were

found for the PHB produced and commercialised by Copersucar

(M w = 220 kDa, T m = 173 C, crystallinity = 0.55 [26]). A significantly

higher M w (521 kDa) was found for the polymer produced by the

control chloroform treatment. However, it has been observed that

variation in molecular weight does not lead to changes in the

mechanical properties of this type of polymer at M n above 3.2 kDa

[28]. These characteristics imply that the polymers obtained from

treatments PHB-R2, -R3 and -R4 can be processed and applied sim-

ilarly to commercial PHB, at a similar commercial price. The selec-

tion of the most suitable recovery method should be then based on

criteria such as operational costs and environmental concerns.

In this investigation, it was experimentally demonstrated that

acid dissolution could be partially reused by replacing approxi-

mately 20% of the volume by fresh solution without losing efficiency

during the treatment (data not shown). Alkaline solutions could

also be partially reused; a replacement rate of 40% resulted in a min-

imal reduction in purity of the recovered polymer (approximately

2%). For hypochlorite solution, the pH was reset to its original

value with NaOH before reuse. According to these experimental

  • 256 M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259

a

256 M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259 a b c Fig.
256 M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259 a b c Fig.

b

c
c

Fig. 4. Schemes of extraction processes: (a) PHB-R1 and PHB-R2, (b) PHB-R3 and (c) PHB-R4.

M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259

Table 2

Operational costs associated to each isolation method.

257

 

Treatment

PHB-R1

PHB-R2

PHB-R3

PHB-R4

 

Input Cost

D h 1

Chemicals a NaOH

0.10

D

kg 1 b

42

19

19

20

H 2 SO 4

0.08

D

kg 1 b

36

NaOCl

0.11

D

kg 1 b

2288

2288

506

EtOH

0.76

D

kg 1 b

472

472

624

CH 2 Cl 2

2.36

D

kg 1 b

118

Electricity

0.017

D

MJ 1 c

5.51

5.51

18

5.51

Steam

13.80

D

t 1 c

7.11

6.76

759

11.87

Total costs

D kg 1 PHB

1.02

5.23

6.61

1.11

a Bi-distilled water was used to carry out the experiments at the bench scale. b http://www.sunivo.com/ennew/Products/Products list.asp.

  • c ASPEN database.

results, partial recirculation of these streams was considered dur-

ing the economic and environmental analysis of these processes.

Moreover, dichloromethane recirculation is a suitable alternative

process configuration, taking into account the hazards and high

cost of this halogenated solvent. With these criteria, the process

schemes represented in Fig. 4 were proposed for treatments with

better results. Basic treatments PHB-R1 and PHB-R2 follow the

same process configuration (Fig. 4a): after digestion, the polymer

is separated by centrifugation and then washed with water and

ethanol. In treatment PHB-R3 (Fig. 4b), separation of the polymer

from the solvent (dichloromethane) is performed by precipita-

tion with ethanol acting as a non-solvent. During centrifugation

for solid recovery, a cake with a moisture content of 30% was

obtained. This moisture content indicates that small quantities of

dichloromethane and ethanol are lost during the centrifugation

step, remaining in the separated solid. Therefore, these small quan-

tities of dichloromethane and ethanol must be replaced. Otherwise,

the polymer obtained after acid digestion (PHB-R4) was processed

as previously described (Fig. 4c). The costs of the chemicals asso-

ciated with each treatment were calculated from the process

simulation and were summarised in Table 2. The quality and quan-

tity of the chemicals used in this evaluation were included as

supplementary material (SM1). To compare processes that provide

similar polymer purity percentages and in view of the experimental

results, an initial solids percentage of 2.5% was considered for treat-

ments PHB-R1, PHB-R2 and PHB-R3 and an initial solids percentage

of 5% was considered for PHB-R4. PHB-R1 and PHB-R4 involved the

lowest chemical investment (1 D kg 1 PHB), although utility costs

were lower for PHB-R1 and PHB-R2 (0.02 D kg 1 PHB). By con-

trast, costs for treatment PHB-R3 were significantly higher (above

6 D kg 1 PHB), because utility costs were elevated by the higher

energetic requirements in the solvent recovery steps.

It should be noted that, in this study, the biomass was freeze-

dried to attain optimal preservation and homogeneity for use in

different experiments. However freeze-drying is not viable on an

industrial scale because of its high energy requirements. A centrifu-

gation stage followed by drying at mild temperature may be the

most feasible process. Chen et al. [29] found different PHB recovery

performances using a surfactant–chelate combination when differ-

ent drying procedures were applied. The best results were obtained

with freeze-drying compared with drying at 60 C or no drying at all.

However, the authors also found that the performance loss could be

offset by modifying the recovery process conditions. Consequently,

after the best strategy to dry biomass on an industrial scale is iden-

tified, recovery conditions should be reevaluated and readjusted as

necessary.

Another point that influences the recovery performance is the

polymer content in the initial biomass as well as its crystallinity

and composition. Yang et al. [30] have reported a clear influence

of initial PHA content on recovery yield after the treatment with

detergents. This effect can be explained by the digestion of non-PHA

biomass by these compounds, similarly to the acid and basic solu-

tions used in this study. From the side of crystallinity, a crystalline

polymer would show a high resistance to chemicals and it would

suffer a lower degradation during recovery process. According with

this fact, the copolymers of PHB/PHV would show a higher degrada-

tion due to its lower crystallinity in relation with the homopolymer

of PHB.

It should be remarked that distilled water has been used in the

current experiments. On an industrial scale, different qualities can

be found depending on water source and they can affect the process

performance. For example, a significant hardness would lead to a

low thermal stability and to the formation of precipitates in pipes

and vessels. Water consumption has not been contemplated in the

economic and environmental analyses but it has been calculated

and included in the supplementary material (SM1). The lowest

water consumption was found in treatments PHB-R2 and PHB-R3

in which water uptake is only contemplated during the washing

step after sodium hypochlorite digestion. The other two treatments

(PHB-R1 and PHB-R4) present an additional water uptake for the

preparation of the digestion solutions. The pH value of the output

streams must be adjusted to a neutral value as a part of wastewater

treatment. After organic matter removal, the reuse of the treated

water in the process may be performed.

The greenhouse gas (GHG) emission is a common metric used

to assess relative differences in the environmental impact of indus-

trial processes. GHG release is associated with the environmental

impact of global warming. Although direct GHG emissions were not

considered in the current isolation processes, emissions derived

from the production of the supplied chemicals and the energy used

as electricity or steam were taken into account. In this case, the

highest emissions resulted from the use of NaOCl (Table 3, SM2).

Therefore, the extraction methods requiring the largest amounts of

this chemical (PHB-R2 and PHB-R3) exhibit the greatest environ-

mental impact. The lowest emissions resulted from the use of acid

and sodium hydroxide.

In summary, PHB-R4 has a low cost (1.11 D kg 1 ) and achieves

similar purity and recovery percentages to PHB-R2 (p = 0.93 and

0.99, respectively). In addition, acid digestion leads to a lower

environmental impact, in terms of GHG emissions, than the other

separation alternatives. The lower polymer degradation observed

with the acid treatment is another advantage. PHB-R1 is another

  • 258 M. López-Abelairas et al. / Biochemical Engineering Journal 93 (2015) 250–259

Table 3

Greenhouse gasses emissions assigned to each process input and total emissions calculated for each separation process.

 

Treatment

PHB-R1

PHB-R2

PHB-R3

PHB-R4

 

Input emissions

 

kg CO 2 -eq/h

Chemicals a NaOH

0.47

kg

CO 2 -eq/kg b

196

90

90

96

H 2 SO 4

0.21

kg

CO 2 -eq/kg b

95

NaOCl

0.66

kg

CO 2 -eq/kg c

13728

13728

3036

EtOH

3.00

kg

CO 2 -eq/kg d

1863

1863

2463

CH 2 Cl 2

3.00

kg

CO 2 -eq/kg d

150

Electricity

0.128

kg

CO 2 -eq/MJ b

43

43

141

43

Steam

0.03

kg

CO 2 -eq/kg b

0.54

0.52

57.87

0.91

Total emissions

kg CO 2 -eq/kg PHB

4.08

29.46

28.71

6.27

a Bi-distilled water was used to carry out the experiments at the bench scale. b http://biograce.net/content/ghgcalculationtools/standardvalues/Bio-Grace addittional standard values - version 1 - Public.pdf.

  • c http://www.waterrf.org/PublicReportLibrary/4443.pdf.

  • d lca.ncms.org/VOC/CradleToSpray.xls.

economical alternative, with an operational cost of approximately

1.02 D kg 1 of polymer. However, it yields a polymer with more

impurities, which modify its initial properties, and most likely give

it less commercial value. The presence of impurities limits the

medical application if the polymer: medical applications require

a high-purity polymer, without the presence of bacterial proteins,

endotoxins and other contaminating chemicals and solvents.

4. Conclusions

Acid treatment is an effective choice for PHB recovery from

cells, with high recovery efficiencies and polymer purities. Acid

treatment also results in less polymer degradation and lower GHG

emissions than hypochlorite treatments. Digestion with NaOH is

also a low-cost treatment but this treatment achieves lower poly-

mer purity than hypochlorite or acid treatments. Treatments that

use sodium hypochlorite exhibit the most negative environmental

impacts.

Acknowledgments

This work was economically supported by the CDTI (Project

CEN-20091040) and also supported by the Ministry of Economy and

Competitiveness of Spain through the Local Investment Fund for

Employment (Government of Spain) and was carried out in collab-

oration with Abengoa Bionergía Nuevas Tecnologías. The authors

(MLA, MGT, TALC and JMLR) belong to the Galician Competitive

Research Group GRC 2013-032, programme co-funded by FEDER.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

References

[1] A. Steinbüchel, H.G. Schlegel, Physiology and molecular genetics of poly( - hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus, Mol. Microbiol. 5 (1991) 535–542. [2] A. Steinbüchel, H.E. Valentin, Diversity of bacterial polyhydroxyalkanoic acids, FEMS Microbiol. Lett. 128 (1995) 219–228. [3] Y. Ling, D.R.G. Williams, C.J. Thomas, A.P.J. Middelberg, Recovery of poly-3- hydroxybutyrate from recombinant Escherichia coli by homogenization and centrifugation, Biotechnol. Tech. 11 (1997) 409–412. [4] N. Jacquel, C.-W. Lo, Y.-H. Wei, H.-S. Wu, S.S. Wang, Isolation and purification of bacterial poly(3-hydroxyalkanoates), Biochem. Eng. J. 39 (2008) 15–27. [5] H.W. Ryu, K.-S. Cho, E.G. Lee, Y.K. Chang, Recovery of poly(3-hydroxybutyrate) from coagulated Ralstonia eutropha using a chemical digestion method, Biotechnol. Prog. 16 (2000) 676–679.

[6] C.-W. Lo, H.-S. Wu, Y.-H. Wei, High throughput study of separation of poly(3- hydroxybutyrate) from recombinant Escherichia coli XL1 blue, J. Taiwan Inst. Chem. Eng. 42 (2011) 240–246. [7] S.N.S. Anis, M.I. Nurhezreen, K. Sudesh, A.A. Amirul, Enhanced recovery and purification of P(3HB-co-3HHx) from recombinant Cupriavidus neca- tor using alkaline digestion method, Appl. Biochem. Biotechnol. 167 (2012)

524–535.

10.1021/bm4010244.

547–553.

391–394.

198–209.

[17] E. Berger, B.A. Ramsay, J.A. Ramsay, C. Chavarie, G. Braunegg, PHB recovery by hypochlorite digestion of non-PHB biomass, Biotechnol. Tech. 3 (1989)

227–232.

256–261.

13–21.

[24] S.-G. Hong, Y.-C. Lin, C.-H. Lin, Crystallization and degradation behav- iors of treated polyhydroxybutyrates, React. Funct. Polym. 68 (11) (2008)

1516–1523.

[25] C. Lauzier, R.H. Marchessault, P. Smith, H. Chanzy, Structural study of isolated poly( -hydroxybutyrate) granules, Polymer 33 (4) (1992) 823–827.

[26]

A.M. Abdel Ghaffar, Development of a Biodegradable Material Based on Poly(3- Hydroxybutyrate) (PHB), Martin Luther Universität Halle, 2002 (Ph.D. Thesis).

259

[27] C.R. Arza, P. Jannasch, F.H.D. Maurer, Network formation of graphene oxide in poly(3-hydroxybutyrate) nanocomposites, Eur. Polym. J. 59 (2014) 262–269. [28] J.M. Torres, C.M. Stafford, B.D. Vogt, Impact of molecular mass on the elastic modulus of thin polystyrene films, Polymer 51 (18) (2010) 4211–4217. [29] Y. Chen, Q. Xu, H. Yang, G. Gu, Effects of cell fermentation time and biomass drying strategies on the recovery of poly-3-hydroxyalkanoates from

Alcaligenes eutrophus using a surfactant–chelate aqueous system, Process Biochem. 36 (2001) 773–779. [30] Y.-H. Yang, C. Brigham, L. Willis, C. Rha, A. Sinskey, Improved detergent- based recovery of polyhydroxyalkanoates (PHAs), Biotechnol. Lett. 33 (2011)

937–942.