A

hmcmC

K

1

maombisala

iTom(a

0d

Journal of Chromatography A, 1154 (2007) 473–476

Short communication

Rapid microwave assisted esterification method for the analysis ofpoly-3-hydroxybutyrate in Alcaligenes latus by gas chromatography

Aimesther Betancourt a,b, Abdessalem Yezza a, Annamaria Halasz a,Huu Van Tra b, Jalal Hawari a,∗

a Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal (Quebec) H4P 2R2, Canadab Chemistry Department, Universite du Quebec a Montreal, Case Postale 8888, succ. Centre-ville, Montreal (Quebec) H3C 3P8, Canada

Received 12 February 2007; received in revised form 5 April 2007; accepted 10 April 2007Available online 19 April 2007

bstract

In the present study, we used microwave energy instead of conventional heating to transform poly-3-hydroxybutyrate (PHB) into methyl 3-

ydroxybutyrate (Me-3HB) in acidified methanol (H2SO4, 10%, v/v) mixture in less than 4 min at 10% microwave power. The microwave assistedethod was then applied to analyze PHB produced by Alcaligenes latus. The PHB content in the biomass determined using microwave heating was

omparable to the amount found by conventional heating. Moreover, the new esterification method was at least 50 times faster than the conventionalethod, affording a significant saving of time and energy.rown Copyright © 2007 Published by Elsevier B.V. All rights reserved.

3-hy

Sb[t

artwmwosatkh

eywords: Microwave heating; Poly-3-hydroxybutyrate; Esterification; Methyl

. Introduction

Poly-3-hydroxybutyrate (PHB), the simplest and most com-only known poly-3-hydroxyalkanoate (PHA), is gaining

ttention as a substitute for petroleum-derived plastic becausef its competing thermoplastic properties with recalcitrant poly-ers derived from fossil fuel and above all because of its

iodegradability [1]. PHAs are produced by several microorgan-sms, such as Alcaligenes latus as intracellular energy and carbontorage materials. PHA granules are accumulated under unbal-nced growth (nitrogen, phosphorous, sulfur, or magnesiumimitation) or balanced growth (without limitation) conditionsnd in the presence of excess carbon source [2].

Presently, biomass is analyzed for its PHB content accord-ng to the widespread method developed by Braunegg et al. [3].he method involves hydrolysis and subsequent methanolysisf lyophilized PHB containing biomass followed by gas chro-

atography analysis of the 3-hydroxybutyric acid methyl ester

Me-3HB) produced. However, this method is time consumingnd requires more than 3 h of heating in a dry block heater.

∗ Corresponding author. Tel.: +1 514 496 6267; fax: +1 514 496 6265.E-mail address: jalal.hawari@nrc.ca (J. Hawari).

2

2

(

021-9673/$ – see front matter. Crown Copyright © 2007 Published by Elsevier B.V.oi:10.1016/j.chroma.2007.04.022

droxybutyrate; Alcaligenes latus

everal researchers attempted to improve Braunegg method [3]y changing the acid concentration and the dry-biomass weight4,5] or by changing the acid and the derivatizing agent [6] usinghe conventional heating method.

Recently, microwave technology has received considerablettention as a green process for sample extraction and prepa-ation in analytical chemistry [7–10]. The major advantage ofhe microwave digestion technique is its high heating efficiencyhich allows the occurrence of rapid breakdown of the sampleatrix [11]. In the present study, a microwave digestion methodas developed for the analysis of PHB in bacterial biomassbtained after fermentation of A. latus in sucrose. The effect ofeveral variables, such as heating time, microwave power, andcid concentration on the methanolysis of PHB was investigatedo quantify the biopolymer in the microbial biomass. To ournowledge, the use of microwave heating for PHB esterificationas not been reported before.

. Experimental

.1. Chemicals

Poly[(R)-3-hydroxybutyric acid] was purchased from FlukaBuchs, Switzerland). Methyl(S)-3-hydroxybutyrate (Me-3HB)

All rights reserved.

4 matogr. A 1154 (2007) 473–476

(Sl(oCAb

2

aocrp

2

2

Msrabptud

2

(cMsupw(emLdc

2

lU0t2

Table 1Methyl-3-hydroxybutyrate (Me-3HB) obtained at different microwave irradia-tion time and power from 20 mg pure PHB sample

Parameters Me-3HB mean ± SD (mg)

Power (%) Time (min)

10 2 Incomplete reaction3 7.6 ± 0.0724 12.6 ± 0.1145 11.8 ± 0.6446 Sample degradation

20 1 Incomplete reaction2 10.8 ± 0.0362.5 12.4 ± 0.1023 12.4 ± 0.2844 Sample degradation

3

E

5s

3

tfwFtreatment of pure PHB and biomass containing PHB with acidi-fied methanol (10%, v/v, of H2SO4) using a microwave heater at10% of power for 4 min (Fig. 1(A and C)) and 30% of power for2 min (Fig. 1(B)). We found that 4 min irradiation at 10% power

74 A. Betancourt et al. / J. Chro

99%), and benzoic acid (99.5%) were purchased fromigma–Aldrich (Oakville, Canada). Chloroform (CHCl3, stabi-

ized in 0.75% ethanol/certified ACS) was from Fisher ChemicalNepean, Canada), and methanol (CH3OH, HPLC grade) wasbtained from J.T. Baker Chemicals (Phillipsburg, NJ, USA).oncentrated sulfuric acid (H2SO4, 95–98%) was acquired fromnachemia (Montreal, Canada). Deionized water was obtainedy passing water through Milli-QUV plus (Millipore) system.

.2. Production of PHB by A. latus

PHB was produced by A. latus (ATCC 29714) using sucroses sole carbon source as described by Grothe et al. [12]. Aliquotsf fermentation broth (5–10 mL) were centrifuged and the pre-ipitated biomass was washed twice with distilled water toemove residual culture medium, frozen and then lyophilizedrior to methanolysis of PHB for subsequent GC analysis.

.3. PHB esterification

.3.1. Conventional heatingPure PHB reference material (20 mg) was treated with a 2 mL

eOH/H2SO4 (3% or 10%, v/v) solution in 150 mm × 20 mmcrew-cap Kimax tubes. Chloroform (2 mL) was added to theesulting mixture and then spiked with benzoic acid to serve asn internal standard. The tubes were sealed tightly and heated in alock heater at 100 ◦C for 3.5 h [3]. Samples were homogenizederiodically at 1 h intervals for 1–2 min. Bacterial biomass con-aining PHB was lyophilized and treated with acidified methanolnder conditions similar to the ones described for the PHB stan-ards.

.3.2. Microwave heatingA Sharp Carousel, Model R-430CSC, microwave oven

1.5 kW, 1100 W output, 2.45 GHz) was used for the esterifi-ation of PHB. A PHB sample (20 mg) was first esterified ineOH/H2SO4 (3% or 10%, v/v) solution in 150 mm × 20 mm

crew-cap Kimax tubes. Samples were heated for several min-tes (1–6 min) with occasional shaking every 1 min using aower ranging between 10 and 30%. The reaction mixtureas brought to room temperature and treated with chloroform

2 mL) and 1 mL of water. The mixture was vigorously shakenvery 1 min. The organic phase was separated and analyzed forethyl 3-hydroxybutyrate (Me-3HB) by gas chromatography.yophilized bacterial biomass was subjected to microwave irra-iation in acidified methanol (3% or 10%, v/v, H2SO4) underonditions similar to the ones described above for PHB standard.

.4. GC-flame ionization detection (FID)

Me-3HB was quantified by a gas chromatograph (Agi-ent 6890 GC-FID; Agilent Technologies Inc., Wilmington,

SA) equipped with a capillary column SPB-1 (15 m × 530 �m,.15 �m; Agilent J&W GC Columns) connected to an FID sys-em. The injector and detector temperatures were set at 265 and75 ◦C, respectively. The oven temperature was set at 50 ◦C for

Fpus

0 1 Sample degradation

ach value is the average of three measurements.

min and then increased at a rate of 30 ◦C/min until 270 ◦C. Aplit injector (3:1) and He carrier gas (7.1 mL/min) were used.

. Results and discussion

Table 1 summarizes the amount of Me-3HB as recovered inhe chloroform phase after methanolysis of PHB (20 mg) at dif-erent time intervals and microwave power. Generated Me-3HBas quantified using a reference standard material of Me-3HB.ig. 1 represents GC chromatograms of Me-3HB produced after

ig. 1. GC-FID chromatograms of the methyl 3-hydroxybutyrate (1.17 min)roduced during treatment of PHB with acidified methanol (10%, v/v, H2SO4)sing microwave oven: (A) PHB standard at 10% of power, 4 min; (B) PHBtandard at 30% of power, 2 min; (C) PHB in biomass at 10% of power, 4 min.

A. Betancourt et al. / J. Chromatogr. A 1154 (2007) 473–476 475

Fl4

goMtwt[

PmbHHaibmHma

tcicmipdtoa

yooia

Table 2PHB content in A. latus biomass after PHB esterification in conventional blockheater and microwave heating

Conventional heating Microwave heating

Inchloroform

Withoutchloroform

Withoutchloroform

PHB content (%, w/w) 61.73 60.64 61.49S a

R

dvgPbptmmehwenetoa

4

em4cTfi[

A

tr

R

ig. 2. Me-3HB amount quantified in PHB standard and PHB contained in A.atus after esterification in block heater (BH) for 3.5 h and microwave (MW) formin and 10% power at 3 and 10% sulfuric acid concentration.

ave the highest Me-3HB amount (12.6 mg), representing 56%f the theoritical yield (Table 1; Fig. 1(A)). The lower yield ofe-3HB was attributed to the partition of the chemical between

he organic phase and water [13]. Below 2 min, methanolysisas incomplete and prolonged irradiation to 6 min lead to the

ransformation of Me-3HB to Me-crotonate (Table 1; Fig. 1(B))6].

Fig. 2 shows the amounts of Me-3HB obtained from pureHB and from A. latus biomass after being subjected toicrowave irradiation (10% power, 4 min) or heated in a heater

lock (100 ◦C, 3.5 h) in acidified methanol (3 and 10%, v/v,2SO4). The two tested acidified methanol (3 and 10%, v/v,2SO4) solutions did not reveal any major difference in the

mount (9 mg) of Me-3HB produced using conventional heat-ng, but recovered amount increased from 6 to 9 mg, respectively,y changing the acid concentration from 3 to 10% (v/v) usingicrowave. Lower recovery of Me-3HB obtained at 3% (v/v)2SO4 was partially attributed to incomplete rupture of cellembrane and thus reduced efficiency of PHB depolymerization

nd methylation.We found that microwave assisted depolymeriza-

ion/methylation of PHB was 50 times faster than that ofonventional heating. According to Gedye et al. [9], microwaverradiation produces efficient internal heating due to directoupling of microwave energy with polar molecules, such asethanol leading to a temperature increase in the system. Such

ncrease in temperature drastically depends on the dielectricroperties of the medium [14]. Since chloroform is bound toecrease the dielectric properties of the reaction mixture wehus modified the process by adding chloroform at the endf esterification so that the microwave efficiency will not beffected.

After optimizing the microwave conditions for the methanol-sis of PHB standards we applied the method for the analysis

f A. latus containing PHB. Fig. 1(C) shows GC chromatogramf Me-3HB obtained after esterification of PHB from biomassn acidified methanol (10%, v/v, H2SO4) during 4 min irradi-tion at 10% microwave power. The PHB content (%, w/w,

D (%) 4.49 3.86 2.44SD (%) 7.27 6.37 3.96

a Standard deviation of five measurements.

ry biomass) obtained in bacterial biomass from both the con-entional heating method and the microwave-based method areiven in Table 2. PHB content in biomass was quantified usingHB reference standard material to reduce the bias createdy the partition of Me-3HB between the organic and aqueoushases [13] and benzoic acid as internal standard. We foundhat the presence and absence of CHCl3 has no effect on the

easured amounts of PHB in biomass using the block heaterethod (Table 2), indicating that the solvent is not needed in the

sterification step as previously reported [3]. Regardless of theeating method, similar PHB content (61.49%, w/w, biomass)ere obtained, thus confirming that the new microwave assisted

sterification method was suitable for PHB quantification. Theew microwave method affords a significant saving of time andnergy. For example, in block heater the energy consumed forhe esterification step is about 5800 kJ, however in microwaveven, the energy consumed does not exceed 360 kJ, which meansn energy saving of 94%.

. Conclusion

Use of microwave oven instead of block heater for PHBsterification proved to be rapid, quantitative and a competitiveethod. The PHB content produced by A. latus measured aftermin microwave treatment was comparable to that obtained byonventional esterification using a dry block heater for 3.5 h.he developed microwave method for PHB esterification maynd utility as an alternative to the currently available protocol3].

cknowledgements

The authors are thankful to Ms. Chantale Beaulieu for herechnical assistance and Dr. Fanny Monteil-Rivera for proofeading the revised manuscript.

eferences

[1] G. Braunegg, R. Bona, M. Koller, Polym-Plastics Technol. Eng. 43 (2004)1779.

[2] S.Y. Lee, Biotechnol. Bioeng. 49 (1996) 1.[3] G. Braunegg, B. Sonnleimer, R.M. Lafferty, Eur. J. Appl. Microbiol.

Biotechnol. 6 (1978) 29.[4] R.G. Lageveen, G.W. Huisman, H. Preusting, P. Ketelaar, C. Eggink, B.

Witholt, Appl. Environ. Microbiol. 54 (1988) 2924.[5] G.N.M. Huijberts, H. Van der Wal, C. Wilkinson, G. Eggink, Biotechnol.

Lett. 8 (1994) 187.

4 mato

[[

[132.

76 A. Betancourt et al. / J. Chro

[6] V. Riis, W. Mai, J. Chromatogr. 445 (1988) 285.[7] K. Srogi, Anal. Lett. 39 (2006) 1261.

[8] C.O. Kappe, Angew. Chem. Int. Ed. 43 (2004) 6250.[9] R.N. Gedye, F. Smith, K. Westaway, H. Ali, L. Baldisera, Tetrahedron Lett.

27 (1986) 279.10] R.N. Gedye, W. Rank, K.C. Westaway, Can. J. Chem. 69 (1991) 706.11] K.J. Lamble, S.J. Hill, Analyst 123 (1998) 103.

[

[

gr. A 1154 (2007) 473–476

12] R. Grothe, M. Moo-Young, Y. Chisti, Enzyme Microb. Technol. 25 (1999)

13] S. Jan, C. Roblot, G. Goethals, J. Courtois, B. Courtois, J.E. Nava Saucedo,J.-P. Seguin, J.-N. Barbotin, Anal. Biochem. 225 (1995) 258.

14] B.L. Hayes, Microwave Synthesis: Chemistry at the Speed of Light, CEMPublishing, Matthews, NC, 2002.