Use of Enzymes in Extraction of Polyhydroxyalkanoates Produced

by Cupriavidus necator

Andreia NevesDept. of Chemical Engineering, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil

Jose MullerDept. of Food Engineering, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil

DOI 10.1002/btpr.1624Published online in Wiley Online Library (wileyonlinelibrary.com).

Poly(3-hydroxybutyrate) (P(3HB)) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxy-valerate) (P(3HB-co-3HV), are biodegradable thermoplastic polymers. They are members ofthe polyhydroxyalkanoate (PHA) family, synthesized and accumulated as a carbon andenergy reserve by a variety of microorganisms. The aim of this study was to evaluate theuse of the proteases CorolaseV

R

L10, AlcalaseVR

2.4L, CorolaseVR

7089 and ProtemaxVR

FCand glycosidases CelumaxV

R

BC, RohamentVR

CL and RohalaseVR

Barley for the recovery ofP(3HB) and P(3HB-co-3HV) synthesized by Cupriavidus necator. The enzyme CelumaxV

R

BCprovided better lysis of the bacterial cell membrane and the results for the optimization ofthe operating conditions showed that this enzyme is most stable in acetate buffer at pH 4.0,bath at 60�C, hydrolysis time of 1 h and concentration of 0.02% (w/w). The optimization ofthe operating conditions showed that the enzyme CelumaxV

R

BC provided better lysis of thebacterial cell in acetate buffer at pH 4.0, bath at 60�C, hydrolysis time of 1 h and concen-tration of 0.02% (w/w). These conditions resulted in lysis of the membrane of the bacteriawith a recovery of 93.2% P(3HB-co-3HV) with 94% purity. The results showed that the useof enzymes for the polymer extraction is an efficient process that assists in the cell disruptionof Cupriavidus necator. VVC 2012 American Institute of Chemical Engineers Biotechnol. Prog.,000: 000–000, 2012Keywords: cupriavidus necator, polyhydroxyalkanoates, recovery, enzymes

Introduction

The environmental impact caused by the accumulation ofsynthetic plastics has been promoting greater interest in thedevelopment of biodegradable polymers such as polyhy-droxyalkanoates (PHAs). These polyesters are synthesizedby various bacteria and stored in the form of cytoplasmicinclusions as an energy reserve1 and reducing power.Cupriavidus necator is a well-known PHA producer thatmay accumulate up to 80% of the cell dry mass from vari-ous carbon sources.2,3 The PHA most studied and wellcharacterized is the homopolymer poly(3-hydroxybutyrate)[P(3HB)],4,5 the thermoplastic properties of which can bemodified by the production of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)].This copolymer has mechanical properties superior to thoseof P(3HB), being more flexible, which makes it moreattractive for industrial use.6

Being intracellular products, polymers need to be releasedfrom the cell to recover them. Most processes used to recoverPHAs from microbial cells use solvent extraction, mainlyincluding chloroform,7 methanol,8 1,2-propylene carbonate,9

ethyl acetate,10 methylene chloride, and 1,2-dichloroethane,11

and solvent mixtures.12 Because of the high viscosity of evendilute PHA solutions, large volumes of solvent are required torecover the polymer,13 and it makes this method economicallyunattractive, even after the recycle of the solvent.14 There arealso other methods in the literature, which use digestion withsodium hypochlorite,15 surfactants,16 supercritical fluid extrac-tion,17 detergent,13 and enzymatic digestion.18

It is important that the research about the application ofenzymes for PHAs cell lysis be extended because this methodhas the advantage of being a selective process and energy effi-cient, which requires milder operational conditions providingPHA films with higher purity and provides less risk of biofilmdamage when compared with other methods. Searching milderprocesses to cell lysis, some studies about the recovery by enzy-matic digestion treatment with proteases and/or glycosidases forPHAs extraction may be found in the literature.8,16,19–23

Researchers opt for enzymatic digestion because it facilitatescellular material removal due to heat treatment which assists incell lysis and denaturation of nucleic acids.

The first attempt to produce P(3HB) industrially was madeby the North American company W.R. Grace. in the1950s.24 Tsuge25 reported that the P(3HB-co-3HV) copoly-mer was produced from glucose and propionate on a com-mercial scale first by Zeneca BioProducts (Billingham, UK)and later by Monsanto (St Louis, MO).

Correspondence concerning this article should be addressed to A.Neves at deiapinho_@hotmail.com.

VVC 2012 American Institute of Chemical Engineers 1

This study aimed to evaluate the use of enzymes in the

extraction of PHAs synthesized by the Gram-negative bacte-

rium C. necator DSM 545. Because of the drawbacks associ-

ated with the use of chemicals, the efficiency of the enzymes

bacterial for cell wall lysis was evaluated. In this study, the

proteases CorolaseVR

L10, AlcalaseVR

2.4L, CorolaseVR

7089,

and ProtemaxVR

FC and the glycosidases CelumaxVR

BC,

RohamentVR

CL, and RohalaseVR

Barley were used because

these enzymes are capable of cleaving peptidoglycan which

is an insoluble polymeric constituent of the rigid cell wall of

Gram-negative bacteria26 such as C. necator. Initial experi-

ments were conducted to select the optimum temperature for

cell heat treatment, which is an important step in the poly-

mer extraction process because it destabilizes the bacterial

cell wall leaving the wall structure less rigid and susceptible

to enzyme action.

The extraction conditions have a major impact on PHAdegradation during the recovery process.27 Therefore, it isimportant to optimize the conditions so that the polymerrecovered has the appropriate characteristics for larger scaleproduction. Therefore, after the tests performed to select thebest enzyme, experiments were performed to determine thepH, temperature, enzyme concentration, and hydrolysis timeleading to greater efficiency in PHA extraction.

For the experimental evaluation, the investigators used afractional factorial design (FFD). In this study was per-formed a factorial 24�1, in which eight experiments (induplicate) were performed to analyze the effects of fourselected factors.

Materials and Methods

Microorganism and culture conditions

The microorganism used in this study was the bacteriumC. necator DSM 545. The strain was maintained at 30�C for48 h on nutrient agar (5.0 g L�1 meat peptone, 3.0 g L�1

meat extract, and 1.5% agar) and then stored in a refrigeratorat 4�C.

C. necator cells used in the extraction and recovery ofP(3HB-co-3HV) were provided by the research group of theLaboratory of Biochemical Engineering of the Department ofChemical Engineering and Food Engineering at the FederalUniversity of Santa Catarina State, southern Brazil, whichallows cultures to achieve high cell density in 5-L bioreac-tors (BIOFLO 110, New Brunswick Scientific), using aworking volume of 4 L. The culture medium had citrus mo-lasses as the carbon source and a supply of propionic acid atthe time of nitrogen limitation.

For the P(3HB) production, the cells were grown in cul-ture medium, as described by Khanna and Srivastava28 withmodifications, composed of (in g L�1) glucose 40.0; urea2.0; yeast extract 0.5; CaCl2�2H2O 0.01; KH2PO4 1.5;

MgSO4�7H2O 4.0; Na2HPO4 4.0; and trace metal solution 15mL L�1. The trace metal solution was composed of (in gL�1) (NH4)6Mo7O24�4H2O 0.6; H3BO3 0.6; FeSO4�7H2O 0.2;and ZnSO4�7H2O 1.3. The cultures were incubated in a ro-tary shaker (Certomat BS-1) at 30�C and 150 rpm for 24 h.

After both fermentations, the cells containing P(3HB-co-3HV) and the cells with P(3HB) were washed with distilledwater, separated by centrifugation and frozen.

Enzymes

To hydrolyze the cell wall structure of the C. necator bac-teria, experiments were performed with the following com-mercial enzyme products provided by the manufacturers:Alcalase

VR

from Novozymes (Brazil); CorolaseVR

L10, Corol-ase

VR

7089, RohalaseVR

Barley, and RohamentVR

CL from ABEnzymes (Germany); and Celumax

VR

BC and ProtemaxVR

FCfrom Prozyn BioSolutions (Brazil).

Preparation of cells for hydrolysis

After defrosting the biomass from each culture, suspen-sions were prepared in 250-mL Erlenmeyer flasks with 2%(w/v) of cells in 100 mL of buffer at a specific pH for eachtype of enzyme. Table 1 shows the buffer for each enzyme.

Heat treatment

Heat treatment is an important step in the polymer extrac-tion process because it destabilizes the cell wall of the bacte-ria. Different pretreatment conditions in the recovery ofP(3HB-co-3HV) were thus analyzed to evaluate whether it ispossible to obtain high extraction yields applying a mildthermal treatment. The different heat treatments tested wereperformed for 15 min. The pretreatments at 100 and 121�Cwere performed in an autoclave and the experiment at 80�Cwas conducted in a thermal bath. The enzymatic hydrolysiswas carried out in a thermal bath stirred at 100 rpm for 1.5h at 60�C. The cells used in this experiment contained 75%(w/v) of polymer, which was extracted with the enzymeCelumax

VR

BC at a concentration of 0.02% (w/w). The recov-ery of the polymer was carried out with 1 mL of chloroform.

Extraction of polymer

To perform the extraction of the polymer contained in thecells, 4 mL of material subjected to the thermal bath wascentrifuged in Eppendorf tubes for 3 min at 14,000 rpm. Af-ter discarding the supernatant, 1 mL of chloroform wasadded to dissolve the polymer. After centrifugation, the sol-vent in which the polymer was solubilized was removedwith a syringe. These solutions were weighed in Petri dishes,and after the evaporation of the solvent, the dishes wereweighed to calculate the percentage of recovered polymer.

Determination of recovery percentage and purity of thePHAs

The percentage of polymer extracted and recovered wasobtained through Eqs. 1 and 2.

%extracted polymer ¼ mfilm

mcell

100 (1)

where mfilm is the mass of the film obtained after extraction,and mcell is the total mass of cells used for the extraction.

Table 1. Buffer and pH for Each Enzyme

Enzyme* Buffer pH

AlcalaseVR

2.4L Tris-HCl 7.5Celumax

VR

BC Acetate 4.0Corolase

VR

7089 Tris-HCl 7.5Corolase

VR

L10 Acetate 5.5Protemax

VR

FC Tris-HCl 7.5Rohalase

VR

Barley Acetate 5.5Rohament

VR

CL Acetate 4.0

*Trade name of the enzyme.

2 Biotechnol. Prog., 2012, Vol. 00, No. 00

%recovery ¼ %ext:pol:

%pol:cell100 (2)

where %ext.pol. is the percentage of extracted polymer,obtained through Eq. 1, and %pol.cell is the percentage ofpolymer inside the cells measured by gas chromatography.

The purity of the films was calculated using Eq. 3.

%purity ¼ mPHA

mfilm

100 (3)

where mPHA is the mass of the biopolymer contained in thefilm, detected by gas chromatography, and mfilm is the totalmass of the film used for the chromatographic analysis.

The quantification of the biopolymer by gas chromatogra-phy was performed according to the method of methanolysisbased on Braunegg,29 with modifications proposed byBrandl.30

Gas chromatography

The biopolymers were quantified according to the methoddescribed by Squio.31 P(3HB-co-3HV) and P(3HB) (SigmaAldrich) of natural origin were used as the external standard.The resulting methyl esters of the polymers were quantifiedby injection of 2 mL in a gas chromatograph (CG-90)equipped with a semicapillary column (Supercowax-10 0.53mm � 30 m). The column temperature was 110�C, the flameionization detector was kept at 230�C, and the injection tem-perature was 185�C.

Data treatment

The extractions were performed in triplicate and the dataanalyzed by applying the Tukey’s test using the programStatistica (version 8.0), which is based on the honestly sig-nificant difference, with a significance level of 5%.

Evaluation of enzymes for lysis of cells

The enzymes mentioned in Enzymes section were eval-uated using specific buffers (Table 1). The cells used inthese tests were grown in a 5-L bioreactor, washed, sepa-rated by centrifugation, and frozen. With this biomass, sus-

pensions were prepared containing 2% (w/v) of cells. Thesuspensions were subjected to thermal treatment in an auto-clave at 121�C for 15 min. In these experiments, the 0.5%(w/w) concentration was adopted for all enzymes tested, andthe enzymatic hydrolysis was conducted for 1.5 h in a heatedbath at 60�C and shaken at 100 rpm. The polymers wereextracted with 1 mL of chloroform. The enzymatic hydroly-sis was performed in duplicate.

Optimization of conditions for activity of enzymeCelumaxV

R

BC

In this experiment, suspensions were prepared containing2% (w/v) of cells in acetate buffer. The cells in buffer werethermally treated by autoclaving for 15 min at 121�C. Enzy-matic hydrolysis with Celumax

VR

BC was conducted in athermal bath shaken at 60�C for 1.5 h. At the end of the hy-drolysis, the polymer was extracted as described in Extrac-tion of polymer section and after solvent evaporation in Petridishes the films of P(3HB-co-3HV) were weighed. The pu-rity of the films was obtained by gas chromatography.

A FFD was used to find the best operating conditions forCelumax

VR

BC. The specific combinations of factors weretested in eight experiments, where the hydrolysis was per-formed in duplicate, and the polymer extractions intriplicate.

In this factorial design 24�1, in duplicate, the pH of thebuffer solution, temperature, enzyme concentration in rela-tion to the mass of dry cells, and bath time for enzymatichydrolysis were varied. Table 2 provides data on the levelsof parameters used in the factorial design for the optimiza-tion of the operating conditions for the enzyme Celumax

VR

BC.

Results and Discussion

Evaluation of different heat treatment conditions

Table 3 shows the percentage of P(3HB-co-3HV)extracted applying different heat treatment conditions. Asshown in this table, the best results were obtained with thetreatment at 121�C where the percentage of P(3HB-co-3HV)extraction was 94.1%. This indicates that the higher tempera-ture contributes to the destabilization of the cell wall of C.necator. In the experiments at 80 and 100�C, the polymer re-covery was significantly lower compared to the test at121�C. Thus, the pretreatment at 121�C for 15 min wasadopted in the other experiments, because these conditionsassist the performance of the enzyme Celumax

VR

BC inincreasing the release of polymer to the environment where

Table 2. Levels of Parameters Used in the Factorial Design

Parameters Level þ1 Level �1

pH 4.5 4T (�C) 70 60[E] (%) (w/w) 0.1 0.02t (h) 1.5 1

Table 3. Average Percentage of P(3HB-co-3HV) Recovered Using

Different Pretreatment Temperatures

T (�C) % P(3HB-co-3HV)

121 94.1 � 0.7a

100 83.8 � 0.8b

80 79.5 � 0.5c

Averages with the same letters are not significantly different (P \0.05; Tukey’s test).

Table 4. Average Percentage of P(3HB) Recovered and Statistical

Analysis Obtained After Extraction With Different Enzymes

Enzyme* Average P(3HB) %

CelumaxVR

BC 87.6 � 0.4a

CorolaseVR

L10 85.0 � 0.2b

RohamentVR

CL 84.1 � 0.3bc

AlcalaseVR

2.4L 83.1 � 0.1c

RohalaseVR

Barley 73.8 � 0.2d

CorolaseVR

7089 66.4 � 0.3e

ProtemaxVR

FC 51.9 � 0.1f

Control 39.1 � 0.3g

Averages with the same letters are not significantly different (P \0.05; Tukey’s test).

*Trade name of the enzyme.

Biotechnol. Prog., 2012, Vol. 00, No. 00 3

it will be extracted. In studies on the use of enzymes torecover P(3HB) produced by C. necator, Kapritchkoff18 per-formed pretreatment of the cells at 85�C for 15 min. Accord-ing to the authors, the heat treatment denatures the proteinsand genetic material, destabilizing the bacterial cell wall,making it more sensitive to the action of enzymes. Heattreatment also inactivates the enzyme PHB depolymerasewhich degrades Poly(3-hydroxybutyrate).28

Evaluation of extraction of P(3HB) after enzymatichydrolysis

This study investigated seven enzymes in the extraction ofthe polymer present in the cells. The biomass used had 72%of P(3HB). Table 4 shows the average percentage of P(3HB)obtained in the extractions performed after the hydrolysisstep with the respective enzymes. A control test, withoutaddition of enzyme, was carried out for comparison ofresults.

Choosing the most appropriate enzyme and setting thebest operating conditions for the cell lysis process allowedgreater P(3HB) extraction of the cells of C. necator. TheTukey’s test (average of duplicates) showed that all enzymesimproved the yield of P(3HB) recovery. The extraction with-out addition of enzyme (control) led to significantly loweramounts of polymer recovered. Three of the enzymes testedled to notable P(3HB) extraction levels: Celumax

VR

BC, Cor-olase

VR

L10, and RohamentVR

CL. Through statistical analysisof the data in Table 4, it was observed that there was no sig-nificant difference between the extractions performed withthe enzymes Corolase

VR

L10 and RohamentVR

CL and betweenRohament

VR

CL and AlcalaseVR

2.4L.

The highest yield of 87.6% was obtained with CelumaxVR

BC, an enzyme system composed of mannanase, cellulase andhemicellulase, generally applied in the degradation of substan-ces derived from the cell walls of plants. With papain Corol-ase

VR

L10, a significant extraction of 85% polymer wasobtained. Ryan and Ward32 found that the enzyme b-1,3 gluca-nase from Basidiomycete aphyllophoroles has high lytic activityin yeast; however, when used in combination with papain, thesolubilization of yeast cells reached 95% of the dry mass. Theenzyme Rohament

VR

CL is obtained from the filamentous fun-gus Trichoderma reesei and degrades cellulose forming otheroligosaccharides. The biopolymer extraction with this glycosi-dase was high, reaching 84.1% of P(3HB) recovered.

AlcalaseVR

2.4L is an endoprotease used in many extractionprocesses. Santos and Ferrari33 used the protease Alcalase anda cellulase for the aqueous enzymatic extraction of soybeanoil, and the protease showed better extraction efficiency. How-

ever, in the lysis of the C. necator cell wall, it was observedthat Alcalase

VR

2.4L provided a statistically lower extraction(83.1%) than the enzyme composed of cellulase (Celumax

VR

BC). RohalaseVR

Barley contains predominantly the enzyme b-glucanase, besides cellulase and endoxylanase; however, inthe P(3HB) extraction, this enzyme was less efficient (73.8%)than Celumax

VR

BC. Although CorolaseVR

7089 is a versatileenzyme, capable of hydrolyzing proteins derived from varioussources such as milk, fish, and wheat gluten, its action on theC. necator cell wall was relatively low (66.4%). Of the sevenenzymes studied, the protease Protemax

VR

FC was least effi-cient at cell lysis, also leading to a relatively low extraction(51.9%) of P(3HB).

Based on these results, the enzyme CelumaxVR

BC wasselected for the P(3HB) extraction in the following experi-ments due to its higher efficiency in the lysis of the bacterialcell wall, which is shown in the results presented in Table 4.These data show that the use of enzymes is an efficient pro-cess, and thus, there is no need to use solvent mixtures, so-dium hypochlorite, surfactants, or detergent to assist in thedisrupting of C. necator cells.

Optimization of conditions for activity of enzyme CelumaxVR

BC

The optimum conditions for enzymatic hydrolysis (pH,temperature, enzyme concentration, and hydrolysis time)were sought to increase the efficiency of the enzyme in thecell lysis of the bacterium C. necator, and the recovery ofthe polymer. The enzyme concentration, pH, and temperaturewere chosen according to the optimal values for the enzymeCelumax

VR

BC indicated by the manufacturer. The exposuretime of the suspensions in thermal bath was varied from thatof the previous experiment, where the hydrolysis was con-ducted for 1.5 h.

In these experiments, the recovery of the copolymerP(3HB-co-3HV), which is synthesized by C. necator whensupplied with a carbon source and cosubstrates such as propi-onic acid, was evaluated. The cells contained 75% of P(3HB-co-3HV). Table 5 shows the experimental conditions used andthe average percentage of P(3HB-co-3HV) extracted.

The controlled conditions of pH, temperature, enzyme con-centration, and time contributed to the weakening of the cellwall and granules of P(3HB-co-3HV) were released into thebuffer solution for extraction. Hydrolysis with Celumax

VR

BCunder controlled conditions provided greater lysis in the pepti-doglycan layer, which resulted in higher release of the poly-mer contained within the cell, as also observed by Ratledgeand Kristiansen.34 Table 5 shows that the conditions evaluated

Table 5. Factorial Design for Optimization of Operating Conditions for CelumaxVR

BC in Terms of the Average Percentage of P(3HB-co-3HV)

Recovered

Experiments pH T (�C) [E] (%) (w/w) t (h) % P(3HB-co-3HV)

1 (�) 4.0 (�) 60 (�) 0.02 (�) 1.0 93.2 � 0.5a

2 (þ) 4.5 (�) 60 (�) 0.02 (þ) 1.5 86.8 � 0.4b

3 (�) 4.0 (þ) 70 (�) 0.02 (þ) 1.5 90.4 � 0.8cd

4 (þ) 4.5 (þ) 70 (�) 0.02 (�) 1.0 87.4 � 0.7be

5 (�) 4.0 (�) 60 (þ) 0.1 (þ) 1.5 88.7 � 0.4bd

6 (þ) 4.5 (�) 60 (þ) 0.1 (�) 1.0 89.4 � 0.5de

7 (�) 4.0 (þ) 70 (þ) 0.1 (�) 1.0 92.1 � 0.7ac

8 (þ) 4.5 (þ) 70 (þ) 0.1 (þ) 1.5 86.3 � 0.7b

Variables: pH, temperature (T), concentration of CelumaxVR

BC ([E]) and hydrolysis time (t).Averages with the same letters are not significantly different (P\ 0.05; Tukey’s test).

4 Biotechnol. Prog., 2012, Vol. 00, No. 00

in the first test of the design were optimum in terms of thepolymer recovery. All parameters were tested at the lowerlevel and a yield of 93.2% P(3HB-co-3HV) extraction, with94% of purity, was obtained. In studies performed by Hahn,35

where enzyme treatment was not used, the content of polymerwithdrawn from inside the cells was 62% with a purity of86%. In this study, besides the higher purity obtained (94%),it was observed that the use of enzymes in the recovery pro-cess can prevent polymer degradation due to the use of lowertemperatures in the process. The comparison between Experi-ments 1 and 7, showed no significant difference in the per-centage of copolymer extracted. These experiments differed interms of the bath temperature and enzyme concentration. Thisindicates that although the first test indicated a fivefold lowerconcentration of Celumax

VR

BC, this was sufficient to causeefficient lysis of the bacterial cell wall.

In Experiment 8, where all parameters were fixed at thetop level, the recovery of P(3HB-co-3HV) was lower(86.3%) compared with the first test, where all parameterswere tested at the lower level. It was also noted that underthe best conditions, there is a decrease in costs of the recov-ery, because the highest extraction was obtained in theexperiment with the lowest enzyme concentration (0.02%),lower temperature (60�C), and shorter heat exposure (1 h).

With the results shown in Table 5 and using the programStatistica, graphs showing the effect of each parameter (pH,temperature, enzyme concentration, and time) on the recov-ery of P(3HB-co-3HV) were obtained. With these data, it ispossible to determine which parameters have a statisticallysignificant influence, as observed in Figure 1.

The stability of enzymes is one of the most important fac-tors in industry because some are unstable under certainprocessing conditions of pH and temperature, quickly

becoming inactive.36 Thus, it is important to study the influ-ence of these parameters on the enzyme stability. As can beseen in Figure 1, the parameter that most influenced thepolymer extraction was pH and its optimum condition wasthat at the lower level (4.0).

Temperature is another factor that may influence enzyme

activity. The stability of an enzyme is dependent on the opti-

mum temperature of the medium. However, as shown in Fig-

ure 1, the temperature did not influence significantly the

extraction yield of P(3HB-co-3HV). The temperature chosen

for the polymer extraction was 60�C because this was the

optimum value recommended by the supplier and because

less energy is required during the enzymatic hydrolysis,

which is economically viable for the process.

According to information from Prozyn BioSolutions, the

optimal concentration of CelumaxVR

BC must be obtained

through practical tests, because it varies according to the

process and the quality of raw material. The manufacturer

recommends a concentration of between 0.02 and 0.1% on a

dry substrate. These two values were tested and the level of

0.02% (w/w) of enzyme resulted in a recovery of 93.2% of

P(3HB-co-3HV).

The results for P(3HB-co-3HV) extraction, with the sup-port of the FFD, showed that the enzyme Celumax

VR

BC pro-vided better lysis of the bacterial cell in acetate buffer at pH4.0, with the bath at 60�C, a hydrolysis time of 1 h, and a

Figure 1. Analysis of the effects of the parameters pH, temperature (T), enzyme concentration ([E]), and hydrolysis time (t) on theextraction of P(3HB-co-3HV).

Biotechnol. Prog., 2012, Vol. 00, No. 00 5

concentration of 0.02% (w/w). These conditions are suffi-cient to promote lysis of the bacteria cell wall and high poly-mer extraction, with a purity of 94%.

Conclusions

In this study, it was found that the heat treatment of cells,to inactivate nucleic acids and destabilize the cell wall, is acrucial step in terms of increasing the copolymer extraction.The biopolymer extraction without addition of enzyme wasstatistically lower than the extractions with the enzymes. Ofthe seven commercial enzymes evaluated, Celumax

VR

BC pro-vided the most efficient lysis of the bacterial cell membrane,indicating that the use of enzymes for the polymer extractionis an efficient process and that it does not require the use ofchemicals such as surfactant, detergent, or sodium hypochlo-rite to assist in the disruption of C. necator cells. The resultsobtained in the optimization of the conditions for cell lysis byCelumax

VR

BC showed that the conditions of pH 4.0, 60�C, 1h of hydrolysis, and concentration of 0.02% (w/w) resulted inhigh extraction yields and purity of the polymer.

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Manuscript received May. 3, 2012, and revision received Jul. 28, 2012.

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