Oxidative degradation of poly(3-hydroxybutyrate). A new method of synthesis for the malic acid copolymers

Abstract

The thermal stability of poly(3-hydroxybutyrate) (PHB) in an oxidation environment was investigated in bulk at temperatures ranging from 100 °C to 140 °C. The process carried out in pure oxygen resulted in PHB backbone degradation with resulting non-volatile products typical for regular PHB thermal degradation while thermal treatment of PHB in an oxygen/ozone mixture resulted in increased rate of polymer backbone scission. The non-volatile degradation product contained macromolecules with several types of terminal groups but also a part of the 3-hydroxybutyrate repeating units was transformed into 3-malic acid units. NMR and multi-stage MS characterization revealed the random distribution of 3-malic acid units in the oligomeric products as well as the content of the malic acid units being dependent on oxidation conditions.

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1. Introduction

Poly([R]-3-hydroxybutyrate) (PHB) is natural origin polyester belonging to the group of poly(hydroxyalkanoate)s (PHAs) which are produced by a wide variety of microorganisms. Bacteria synthesize high molar mass PHB which serves as a carbon and energy source.^1,2 PHB complexed with polyphosphates forms ion channels in eukaryotic cells.³ Moreover PHB and its copolymers are synthesized by biotechnological processes on an industrial scale.^4–6

Generally, poly(hydroxyalkanoate)s can undergo hydrolytic,^7,8 enzymatic^9–12 and thermal degradation.^13–23 Depending on the degradation reaction mechanism and the process conditions the final product may be comprised of low-molar-mass polyesters possessing α-hydroxyl and ω-carboxyl end groups (enzymatic or hydrolytic degradation) or α-double bond and ω-carboxyl end groups (e.g., α-crotonate PHB) in the case of thermal degradation or hydrolysis in basic conditions; thermal degradation may also lead to cyclic products.²⁴ Chemical reduction of PHA as a degradation reaction was also presented as a method of PHA oligoesterdiol synthesis.²⁵

The influence of oxidative conditions, such as: oxygen atmosphere or treatment with ozone on PHA behavior was studied, however there are no data concerning its oxidative degradation, for example: the biomass containing PHB was purified with ozone/oxygen mixture, however the inventors determined only the purity of polyester characterizing the amount of ash after heating at 450 °C.²⁶ Thermo-oxidative degradations of polyethylene/PHB blends^27–29 or the degradation of PHB/segmented polyester–urethane³⁰ were also studied, though no changes of neither molar mass or structure of the polyester chain were reported. Recently, conversion of PHB crotonate terminal groups using ozone/oxygen mixture was described^31,32 but no changes in polyester structure were noticed. Apparently, the syntheses were performed in mild conditions applying relatively short time of exposure to oxidizing agent.

On the other hand, it is known esters can undergo oxidation according to free-radical chain mechanism (Scheme 1).³³ The reaction takes place in alkoxy and/or acyl fragment of the ester, however the highest reactivity was reported for α-C–H bond in ester alkoxy group with formation of acids, ketones and/or aldehydes. Simplified mechanism of methyl ester oxidation is shown in the Scheme 1. The structure of final products depends on structure of the initial ester as well as position of the oxidized carbon.³³

Scheme 1 Simplified mechanism of methyl ester oxidation.

The aim of this work was investigation of PHB oxidative degradation and possibility of applying the process to obtain new “green” material in one step. At first, the possibility of degradation of natural PHB in oxidative conditions was investigated. Next, detailed structure of resulting products was assigned supported by hypothesis of the oxidation mechanism. Finally, application of the oxidative degradation for manufacturing of useful materials without any additional agents such as solvents or catalysts was demonstrated.

2. Experimental

2.1. Materials

Poly([R]-3-hydroxybutyrate) (PHB) (ICI product – PHB G08; M_{n SEC} = 90 000, Đ_M = 3.01), oxygen, argon, air 5.0 (Messer), were used as received. Ozone was produced using an Ekotech Korona L (output stream: 90 L h⁻¹ O₂, 3 g h⁻¹ O₃) generator.

2.2. Oxidative degradation

2.2.1 Oxidative degradation of poly([R]-3-hydroxybutyrate). PHB (3.0 g) was introduced into the flow reactor equipped with glass frit on the bottom and heated to 140 °C. Then the polymer bed was purged with oxygen (90 L h⁻¹) for 60 h and characterized using SEC technique.

Degradation of PHB in air and argon (reference experiment) was carried out in the same manner.

2.2.2 Oxidative degradation of poly([R]-3-hydroxybutyrate) in presence of O₃/O₂ mixture. PHB (3.0 g) was placed in the flow reactor. The polymer bed was purged with ozone/oxygen mixture (90 L h⁻¹ oxygen containing approximately 3.0 g h⁻¹ of ozone, i.e., minimal flow rate for long-term experiments according to ozone generator manual). The reaction was carried out 8 h at temperature 100, 120, 125 and 135 °C, respectively. In case of PHB degradation carried out at 140 °C the reaction was finished after 5 h. Products of PHB degradation were characterized using ¹H and ¹³C NMR, ESI-MS and SEC techniques.

2.2.3 Thermal decomposition of PHB monooxalate. PHB monooxalate, prepared according to previously described procedure,³⁴ was thermostated at 140 °C in inert gas atmosphere (argon) for 2 h. The changes in molecular structure of the investigated sample were followed using ¹H NMR.

2.3. Instruments and measurements

Nuclear magnetic resonance (NMR) spectra were recorded at 600 MHz with the Avance II Bruker Ultrashield Plus spectrometer in CDCl₃ solution with TMS as internal standard. All spectra were obtained at ambient temperature. ¹H NMR spectra were obtained with 64 scans, 2.65 s acquisition time and 11 μs pulse width. The carbon proton decoupling and INVGATE (inverse gated decoupling for quantitative analysis) ¹³C NMR spectra were recorded at 150 MHz. The acquisition time was 0.9 s, the pulse width was 9.4 μs, the spectral width was 36 000 Hz with pulses delay 2 s or 5 s respectively with 80 000 scans.

Number-average, weight-average molar mass and molar mass distribution (Đ_M) were determined by size exclusion chromatography (SEC) conducted in chloroform at 35 °C with a flow rate of 1 mL min⁻¹, using an isocratic pump (VE 1122, Viscotek), a set of two PLgel 5 μm MIXED-C ultra-high efficiency columns and a Shodex SE 61 differential refractive index detector. Polystyrene standards (Polymer Laboratories) with narrow molecular weight distributions were used to generate a calibration curve.

Electrospray mass spectrometry (ESI-MS) analyses were performed using a Thermo Finnigan LCQ Fleet ion-trap mass spectrometer (Thermo Fisher Scientific Inc., San Jose, CA, USA). Methanol/chloroform (1 : 1 v/v) solutions of samples were introduced into the ESI source by continuous infusion with 3 μL min⁻¹ flow rate using an instrument syringe pump. The analyses were performed in negative-ion mode.

Acidic number (AN) was determined according to modified procedure PN EN ISO 2114:2000.³⁵ Briefly, sample of polymer (0.4 g) was dissolved in 20 mL of chloroform/ethanol mixture (1/1), and titrated with potassium hydroxide solution in ethanol (0.1 mol L⁻¹) in presence of 3–4 drops of phenolphthalein ethanol solution (1% w/w). Acidity (mg KOH) of chloroform/ethanol mixture (1/1) was subtracted from value obtained in the titration.

3. Results and discussion

Natural poly([R]-3-hydroxybutyrate) was exposed to air or oxygen stream at 140 °C through 60 h (Table 1). However, there were no significant differences in molar masses of such thermo-oxidized products in comparison with M_n of sample maintained in non-oxidative (argon) atmosphere at the same temperature.

Table 1 PHB thermo-oxidative stability towards chosen conditions

Entry	Atmosphere	Time [h]	Temperature [°C]	Molar mass [g mol^−1]	Acidic number (AN) [mg KOH per g]	Theoretical AN^a [mg KOH per g]
a Theoretical acidic number for PHB containing one carboxylic acid group per polymer chain of Mn determined with SEC technique.b Sample precipitates during titration.
0	—	—	—	90 000	n.d.	0.6
1	Argon	60	140	35 200	n.d.^b	1.6
2	Air	60	140	34 500	n.d.	1.6
3	O2	60	140	34 600	n.d.	1.6
4	O3/O2	8	100	28 100	n.d.	2
5	O3/O2	8	120	20 000	n.d.	2.8
6	O3/O2	8	125	9300	23	6
7	O3/O2	8	135	1400	101	40
8	O3/O2	5	140	800	261	70

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Based on this observation it was hypothesized that under investigated conditions the decrease of molar mass was rather the consequence of slow thermal degradation and not oxidation process. The suggestion was supported by formation of crotonate terminal groups as revealed by ¹H NMR analysis (very weak signal characteristic of crotonate end groups appearing in the spectrum of PHB after 60 h of heating in oxidative atmosphere).¹³

The question rises if PHB could undergo oxidative degradation. Trying to answer this question, the oxidation agent was switched to ozone/oxygen mixture and the thermo-oxidative treatment was conducted at temperatures ranging from 100 °C to 140 °C.

It was found that already after 8 h of thermo-oxidation of the poly(3-hydroxybutyrate) in presence of ozone the molar mass decreased significantly. In case of process carried out at 140 °C, similarly to the study described above, the M_{n SEC} of resulting product decreased to 800 g mol⁻¹ just after 5 h. Comparison of acidic number (AN) values determined for selected oxidation products with theoretical ANs calculated for macromolecules comprising one carboxylic group or even two such groups per oligomer chain, indicated that experimental AN is much higher than expected, e.g., AN of degradation product with M_{n SEC} = 800 should be around 70.2 mg KOH per g (for polymer with one COOH end group per chain) while 261 mg KOH per g was measured (Table 1 entry 8). Observed difference in experimental and calculated ANs indicates that the oligoester chains contain more than one carboxylic group, i.e., average of 3.7 per polymer chain in the case of oligomers with M_{n SEC} = 800.

¹H NMR (Fig. 1) analysis of non-volatile product of PHB oxidation with O₃/O₂ beside of proton signals characteristic for 3-hydroxybutyrate (3HB) repeating units of oligo(3-hydroxybutyrate) (a, b and c) did not reveal signals characteristic for crotonate terminal group which is usual end group of thermally-treated PHB. Instead signal d ascribed to methine proton of α-3-hydroxybutyrate (α-3HB) end group was observed. The assignment was confirmed via derivatization of the sample with trichloroacetyl isocyanate; in spectrum of the resulting product the d signal shifted downfield (ESI Fig. S1†).

Fig. 1 ¹H NMR (600 MHz, CDCl₃) spectrum of non-volatile product of ozone-induced oxidation of PHB at 140 °C after 5 h of reaction.

Moreover, in the spectrum there were also identified signals: e and f assigned to methyl and methylene group protons of α-acetylacetonate (α-AcAc), respectively and g, ascribed to α-monomalonate (α-mM) end group methylene protons.

It is worth to notice that signal c ascribed to methine group protons is quite broad which can suggest the presence of other overlapped methine proton signals. Unfortunately, not all of the signals in the spectrum were successfully ascribed.

Molar mass calculated based on integrals ratio of the identified end groups proton signals d, f, and g to signals a and b ascribed to polymer chain protons (Fig. 1) was M_{n NMR} = 1000 which was slightly higher than M_n determined using SEC analysis (M_{n SEC} = 800).

¹³C NMR spectrum of the non-volatile oxidative degradation products (see ESI Fig. S2†), beside signals corresponding to PHB main chain carbons (C1–C4), revealed also signals attributed to carbons of: α-3-hydroxybutyrate (α-3HB) (C5–C7) and ω-(1-methyl-2-carboxyethyl) (ω-3HB) (carbonyl carbon C8) end groups.

In the alkane carbon region of the ¹³C NMR spectrum (range δ = 19–70 ppm) (Fig. 2a) signals of α-acetylacetonate (α-AcAc) end group (C9, C10 and C11) were found.

Fig. 2 Aliphatic (δ = 18–70 ppm) and carbonyl (δ = 150–180 ppm) carbon region expansions of ¹³C NMR (150 MHz, CDCl₃) spectrum of non-volatile product of PHB oxidation at 140 °C after 5 h.

The presence of α-monomalonate (α-mM) end group identified in ¹H NMR spectrum was confirmed using ¹³C NMR spectroscopy (C25, C23 and C24). Additionally, to the above mentioned end groups, the signals were assigned to carbons of α-monooxalic (α-mOx) end group (C18 and C19). At the second extremity of the oligomer chains obtained via PHB oxidative degradation, beside ω-3HB carboxyl group (C8), ω-(1-carboxyethyl) end group (ω-LA) (C20–C22) was also identified. Unexpectedly, signals characteristic of 3-malic acid units were identified in the spectrum. Presence of such units was observed in resulting polymer backbone (3-malic acid (MA) units) (C12–C15) as well as ω-(1,2-dicarboxyethyl) end group (ω-MA) (C16 and C17).

Quantitative analysis based on the INVGATE ¹³C NMR spectrum revealed composition of terminal groups: α-side 75% of polyester chains contain α-3HB end groups, 18% α-mM, 5% α-mOx and 2% of α-AcAc ones. In case of the ω-end groups of the polymer chains the quantity of the identified carboxyl groups was as follow: 50% ω-3HB, 25% ω-LA and 25% ω-MA. In the same time, the non-volatile oxidation product contained 21% of malic acid units per oligomer chain (calculated based on the integrals ratio of MA units carboxyl carbonyl carbon signals (C12) to sum of integrals of ω-carboxyl end groups carbonyl carbon signals (ω-3HB, ω-LA and ω-MA)) clearly demonstrating conversion of PHB into poly(3-hydroxybutyrate-co-3-malic acid). Moreover, the calculation of M_n based on ¹³C NMR integral values of signals of carbonyl carbon of ω-end groups (ω-LA – C22, ω-MA – C16 and ω-3HB – C8) and ester carbonyl carbons from 3HB and MA units (C4 and C15) indicated that DP of the characterized oligomer was ten and the number-average molar mass was M_{n C NMR} = 920, which correlates with SEC results.

Comparison of the SEC and NMR data and the acidic number which for the investigated sample was 261 mg KOH per g allows to deduce the average amount of carboxyl groups in each polymer chain was in range 3.7 to 4.2.

Based on the AN and M_{n SEC} measurements of the reaction non-volatile products obtained at 125, 135 and 140 °C (Table 1, entry 6–8) it could be calculated that the HB-MA copolymers containing 3, 10 and 21 mol% of MA units were obtained.

The molecular structure of the products of poly(3-[R]-hydroxybutyrate) oxidation with ozone/oxygen mixture at 140 °C was also studied using multistage electrospray mass spectrometry (ESI-MSⁿ). In fact, the spectrum obtained in negative ion mode is complex (Fig. S3†) and there are more series of signals in the spectrum than expected based only on difference in terminal groups. Apparently, the observation of multiple series supports NMR-data-based hypothesis that the investigated reaction led to the mixture of oligomers having different composition not only in terms of end groups but also their backbone composition. The detailed analysis revealed six general fragmentation patterns among observed ions (Table 2.) confirming formation of 3-hydroxybutyrate and malic acid copolymer.

Table 2 Proposed structures of the obtained oligomeric product

Sequence	α-end groups^a	Structure	ω-end groups^b
a α-3HB – 3-hydroxybutyrate, α-mM – monomalonate, α-mOx – monooxalate, α-AcAc – acetylacetonate.b ω-3HB – (1-methyl-2-carboxyethyl) group, ω-LA – (1-carboxyethyl) group, ω-MA – (1,2-dicarboxyethyl) group.c 3-Hydroxybutyrate and monomalonate end groups possesses the same m/z 104 Da, and could not be differentiate using ESI-MS^n; due to the much higher amount of α-3HB end groups (75% to 18% α-mM), only α-3HB were drawn.
I^c	α-3HB or α-mM		ω-3HB or ω-MA
II^c	α-3HB or α-mM		ω-LA
III	α-mOx		ω-3HB or ω-MA
IV	α-mOx		ω-LA
V	α-AcAc		ω-LA
VI	α-AcAc		ω-MA or ω-3HB

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Focusing on a fragment of ESI-MS spectrum (Fig. S3b in ESI†) the sequence I was ascribed to products possessing α-3-hydroxybutyrate end group and polymer chain comprised of 3-hydroxybutyrate units and 3-malic acid units. Mass of 3-malic acid unit is 116 Da while mass of 3HB unit equals 86 Da therefore signals ascribed to the sequence I appear every 30 Da, as demonstrated based on structures with m (number of 3-malic acid units) equal to: zero (homooligomer) (Fig. S4,† m/z 533.4), one (Fig. S5,† m/z 563.1), two (Fig. S6,† m/z 593.1) and three (Fig. 3, m/z 623.6) which were identified in ESI-MS² spectrum. No signals ascribed to 3-malic acid homopolymer were found. The fragmentation profiles of these ions were similar to presented in literature fragmentation scheme for such structures.³⁶ In general, the ESI-MS² spectra of these ions revealed loss of fragment with mass of 104 Da characteristic either for 3-hydroxybutyric acid or malonic acid fragment (α-end group). Moreover, fragmentation of the peak m/z 533.4 indeed revealed fragments differing of 86 Da matching mass of 3-hydroxybutyrate units (Fig. S4†). In case of the peak ascribed to molecule containing three 3-malic acid units and two 3HB units at least three positional isomers of the backbone were evidenced based on the parent ion fragmentation (Fig. 3). Noticingly, the parent ion loses fragment of either 86 Da ascribed to ω-3HB terminal unit³⁷ (NMR analyses excluded presence of crotonates), or 116 Da (ω-MA unit)³⁸ and 104 Da corresponding to 3-hydroxybutyric acid α-terminal group, which demonstrates that malate units can constitute ω-terminal group of the oligomer. Fragmentation pattern of the parent ion suggests random distribution of malic acid units.

Fig. 3 The ESI-MS² spectrum of ion m/z 623.6 (negative ion mode) with proposed structures of oligomers; molecules with α-mM end groups are omitted for simplification.

The situation is even more complex for the sequences II and III which are isomers (the same m/z value). Differences were found in structure of their end groups: the sequence II has α-3HB (or α-mM) and ω-LA end groups (as evidenced by repulsion of the fragments 104 and 72 Da, respectively, Fig. 4a and S8†) while sequence III possessed α-mOx and ω-3HB (or ω-MA) end groups as it was proved by the parent ion fragmentation with loss of 90 Da and 86 Da or 116 Da, respectively (Fig. 4b and S8†). As it was shown in Fig. S8† ESI-MS² spectrum of this ion was very complicated therefore the spectrum was graphically separated into two patterns corresponding to sequences II (Fig. 4a) and III (Fig. 4b).

Fig. 4 Separated into two independent images ESI-MS² spectrum of ion m/z 579.2 (negative ion mode) (Fig. S8†) corresponding to sequence II (a) and sequence III (b), respectively, with proposed co-oligomer structures.

Sequence IV can be evidenced by the exemplary ion 767.1 Da which consists of three 3-hydroxybutyrate and three 3-malic acid units and possess α-mOx and ω-LA end groups (Fig. 5). This ion fragmentation shows three series of fragmentation ions, which allow to propose two different structures presented on Fig. 5.

Fig. 5 The ESI-MS² spectrum of sequence IV ion with m/z 767.1 (negative ion mode) and proposed structures of oligomer with α-mOx and ω-LA end groups containing three 3HB units and three units of MA.

Additionally fragmentation of ion with m/z 419.2, attributed to this sequence, containing no malic acid units is presented in Fig. S9 in ESI.†

The sequence V contains oligomers with α-AcAc and ω-LA end groups and the oligomer backbone consisting of 3HB and/or 3MA units. The structure of the ion with m/z 521.1 belonging to this sequence was confirmed by fragmentation experiment indicating presence of α-acetylacetonate (loss of 102 Da fragment) and ω-LA end groups and three MA units in oligomer chain (Fig. S10†).

The presence of sequence VI was identified in ESI-MS spectrum as a signals corresponding to ions with structure containing 3HB and/or MA repeating units and α-AcAc and ω-3HB or ω-MA end groups. However, probably due to relatively low amount of acetylacetonate end group (2% calculated from ¹³C NMR), presence of this sequence was not proved by fragmentation experiment.

According to the literature data esters undergo oxidation according to the free-radical chain mechanism and the α-C–H bond in alkoxy part of ester readily undergoes the reaction.³³ The products of ester oxidation are usually acids, aldehydes and/or ketones. In case of poly(3-hydroxybutyrate) oxidation can take place in methylene as well as methine group but methine group should be oxidized easier.³³ Two main paths resulting in products identified in the PHB oxidized with ozone are proposed (Scheme 2). The first one (i) proceeding via oxidation of the methine group with formation of 3-hydroperoxide 1 which undergoes the subsequent decomposition with cleavage of neighboring ester bond, resulting in formation of two shorter polymer chains possessing ω-3HB functionality (PHB-COOH) and α-AcAc end group 3, respectively. The oxidation of the methylene group ((ii) in Scheme 2) is believed to proceed via generation of hydroperoxide 2 followed by its decomposition leading to cleavage of nearest ester bond and formation of two new polymer chains possessing ω-LA 4 and formate 5 end groups, respectively. Products acetylacetonate-terminated (3) and formate-terminated (5) can undergo further decomposition under the oxidative conditions of O₃/O₂ in reaction mixture.

Scheme 2 Proposed pathways of PHB oxidative degradation in the presence of O₃/O₂ mixture.

Apparently, formate esters are known to decompose thermally in the presence of oxygen as well as in its absence³⁹ so the PHB with formate end group 5 is supposed to decompose easily in the reaction conditions with formation of several terminal groups. One of the possible decompositions (iii) can proceed with formation of carbon monooxide and radical 10, which can form the polymer chain with 3-hydroxybutyric end group after incorporation of hydrogen atom. Other decomposition reaction of 5 (iv) results in radical 11 and PHB crotonate 12, which is readily converted in presence of ozone/oxygen mixture to monooxalate end group and acetic acid.³² Third route (v) results in a formyl radical 13, and PHB terminated with acetylacetonate group 3.

Acetylacetonate end group can be oxidized subsequently according to the mechanism of ozone oxidation of ketones.⁴⁰ The two simplified possible decomposition routes of PHB with acetylacetonate end group 3 in the reaction conditions were presented in Scheme 2, path (vi) and (vii). Generally, in the first step a radical is generated either at methyl (path (vi)) or at methylene carbon (path (vii)). In the path (vi) generated radical 6 undergoes further transformations with formation of formic acid and polymer chain with monomalonate end group 8. In path (vii), formed radical 7 is expected to decompose into acetic acid and PHB with monooxalate end group 9.

According to the presented mechanistic explanations the main product of PHB oxidation in the presence of ozone should be copolymers with monooxalate end group but the amount of polymer chain with monooxalate end group was around 5% (based on ¹³C NMR spectrum). However, according to the literature the monooxalate esters may decompose at relatively high temperature (140 °C).⁴¹ Unfortunately, the literature gives no detailed information on the decomposition mechanism of such esters. To prove that PHB monooxalate decomposes at the reaction conditions, PHB with monooxalate end group (M_{n SEC} = 1600) was prepared³⁴ and heated at 140 °C in inert atmosphere. After 1 h of heating, in ¹H NMR spectrum of this sample the signal at δ = 4.24 ppm ascribed to methine proton of 3-hydroxybutyrate end group was revealed⁴² (Fig. S11†). After 2 h of reaction time the signal of methine proton was more intensive and the low intensity signal corresponding to crotonate end group was appeared. Moreover, after 2 h reaction time the molar mass of used oligomer was still identical with that of starting material i.e., M_{n SEC} = 1600, which enable to exclude the presence of hydrolytic as well as thermal degradation of polyester. Comparison of sample M_{n SEC} = 1600 and the M_n determined using ¹H NMR spectroscopy based on the integrals of methine protons of end groups allows to calculate that around 75% of monooxalate groups was decomposed after 2 h.

Unexpected result of PHB oxidation with oxygen/ozone mixture in 140 °C was identification of the 3-malic-acid-origin units in the obtained polymer backbone. Such groups can be generated only via oxidation of methyl group of 3-hydroxybutyrate repeating units, i.e., in the less probable oxidation path, albeit in literature there are examples of oxidation of methyl group in esters³³ or methyl group adjacent to methine one, e.g., in oxidation of i-propyl acetate in presence of hydroxyl radical.⁴³ Taking into account this data it was proposed that in the first step the abstraction of hydrogen from methyl group by ROO˙ or ˙OH radical with generation of radical at methyl group 14 (ref. 44) which undergoes subsequent transformation to carboxylic acid group which results in generation of the 3-malic acid unit 15 in polymer chain (Scheme 2, (viii)).

Proposed reaction pathways allow to explain thermo-oxidative transformation of 3-hydroxybutyric unit to all identified structures.

4. Conclusions

It was found that poly(3-hydroxybutyrate), microbial polyester, undergoes thermo-oxidative degradation. The exposure of PHB to ozone/oxygen mixture at temperature above 100 °C decreased its molar mass drastically in relatively short time (up to 8 h). Moreover, the ozone-induced degradation of this polyester carried out in bulk follows green chemistry idea of synthesis of the copolymers of 3-hydroxybutyric acid and 3-malic acid, using biodegradable material from renewable resources. Presented process is an alternative method to obtain poly(3-hydroxybutyrate-co-3-malic acid) to the methods employing ring-opening polymerization of appropriate lactones; the methods already presented in literature.⁴⁵ Moreover, the starting polymer was natural origin poly([R]-3-hydroxybutyrate) with one configuration and the same configuration (R) should retain at the stereogenic carbon of 3-malic acid units generated via oxidation of methyl groups of PHB. Nevertheless, as the detailed analysis proved that depending on the reaction temperature the content of 3-malic acid units varies from 3 to 21%. Noticingly, the resulting products do not require any purification process.

Poly(3-hydroxybutyrate-co-3-malic acid) may be applied as biorenewable and biodegradable cross-linker in synthesis of polyesters. Moreover, this copolymers can find potential use in drug delivery systems⁴⁶ or as biodegradable emulsifier.

Acknowledgements

This research was supported by Polish NCN, Project DEC-2012/05/N/ST5/00221.

References

1. Y. Doi, Microbial Polyesters, VCH Publishers, Weinheim, Germany, 1990.
2. R. W. Lenz and R. H. Marchessault, Biomacromolecules, 2005, 6, 1.
3. R. N. Reusch, M. E. Bryant and D. N. Henry, Acta Diabetol., 2003, 40, 91.
4. B. Hazer and A. Steinbuchel, Appl. Microbiol. Biotechnol., 2007, 74, 1.
5. G.-Q. Chen, Chem. Soc. Rev., 2009, 38, 2434.
6. I. Taniguchi, K. Kagotania and Y. Kimura, Green Chem., 2003, 5, 545.
7. Y. Doi, Y. Kanesawa, Y. Kawaguchi and M. Kunioka, Makromol. Chem., Rapid Commun., 1989, 10, 227.
8. M. I. A. Majida, J. Ismailb, L. L. Fewa and C. F. Tanb, Eur. Polym. J., 2002, 38, 837.
9. M. Scandola, M. L. Focarete and G. Frisoni, Macromolecules, 1998, 31, 3846.
10. D. Jendrossek and R. Handrick, Annu. Rev. Microbiol., 2002, 56, 403.
11. Y. Tokiwa and B. P. Calabia, Biotechnol. Lett., 2004, 26, 1181.
12. Y. Osanai, K. Toshima and S. Matsumura, Green Chem., 2003, 5, 567.
13. H. Nishida, H. Ariffin, Y. Shirai and M. Hassan, Precise depolymerization of poly(3-hydoxybutyrate) by pyrolysis, Biopolymers, ed. Magdy Elnashar, InTech, 2010, pp. 369–386, http://www.intechopen.com/books/biopolymers/precise-depolymerization-of-poly-3-hydoxybutyrate-by-pyrolysis.
14. N. Grassie, E. J. Murray and P. A. Holmes, Polym. Degrad. Stab., 1984, 6, 47.
15. N. Grassie, E. J. Murray and P. A. Holmes, Polym. Degrad. Stab., 1984, 6, 127.
16. F. D. Kopinke, M. Remmler and K. Mackenzie, Polym. Degrad. Stab., 1996, 52, 25.
17. F. D. Kopinke and K. J. Mackenzie, J. Anal. Appl. Pyrolysis, 1997, 40–41, 43.
18. S. Nguyen, G. Yu and R. H. Marchessault, Biomacromolecules, 2002, 3, 219.
19. P. Kurcok, M. Kowalczuk, G. Adamus, Z. Jedlinski and R. W. Lenz, J. Macromol. Sci., Part A: Pure Appl.Chem., 1995, 32, 875.
20. J. K. Kim, Y. Doi and H. Abe, Polym. Degrad. Stab., 2006, 91, 769.
21. M. Kawalec, G. Adamus, P. Kurcok, M. Kowalczuk, I. Foltran, M. L. Focarete and M. Scandola, Biomacromolecules, 2007, 8, 1053.
22. M. Kawalec, M. Sobota, M. Scandola, M. Kowalczuk and P. Kurcok, J. Polym. Sci., Part A: Polym. Chem., 2010, 48, 5490.
23. M. Kwiecień, M. Kawalec, P. Kurcok, M. Kowalczuk and G. Adamus, Polym. Degrad. Stab., 2014, 110, 71.
24. M. Melchiors, H. Keul and H. Hocker, Macromolecules, 1996, 29, 6442.
25. M. Kwiecień, G. Adamus and M. Kowalczuk, Biomacromolecules, 2013, 14, 1181.
26. M. E. Brennan and D. M. Horowitz, EP1070135 A1, 1999.
27. S. M. Marteli, Environmentally compatible polymeric blends and composites based on oxo-biodegradable polyethylene, University of Pisa, 2011.
28. Yu V. Tertyshnaya, L. S. Shibryaeva and A. A. Popov, Russ. J. Phys. Chem. B, 2012, 6, 38.
29. Yu V. Tertyshnaya, A. A. Ol'khov and L. S. Shibryaeva, Russ. J. Phys. Chem. B, 2015, 9, 498.
30. S. G. Karpova, A. L. Iordanskii, A. A. Popov, N. G. Shilkina, S. M. Lomakin, M. A. Shcherbin, S. N. Chvalun and A. A. Berlin, Russ. J. Phys. Chem. B, 2012, 6, 72.
31. M. Michalak, M. Kawalec and P. Kurcok, Polym. Degrad. Stab., 2012, 97, 1861.
32. M. Michalak, A. A. Marek, M. Kawalec, J. Zawadiak and P. Kurcok, Eur. Polym. J., 2013, 49, 4149.
33. E. T. Denisov, N. I. Mitskevich and V. E. Agabekov, Liquid-phase oxidation of oxygen-containing compounds: The oxidation of esters, Springer, US, 1977, p. 255.
34. M. Michalak, Badanie nad procesami utleniania biopoliestrów alifatycznych i ich syntetycznych analogów, Silesian University of Technology, 2013, in Polish.
35. PN EN ISO 2114:2000, Plastics (polyester resins) and paints and varnishes (binders)-Determination of partial acid value and total acid value.
36. I. Kwiecień, T. Bałakier, J. Jurczak, M. Kowalczuk and G. Adamus, Rapid Commun. Mass Spectrom., 2015, 29, 533.
37. G. Adamus, I. Kwiecień, M. Maksymiak, T. Bałakier, J. Jurczak and M. Kowalczuk, Anal. Chim. Acta, 2014, 808, 104.
38. S. R. Leadley, M. C. Davies, M. Vert, C. Braud, A. J. Paul, A. G. Shard and J. F. Watts, Macromolecules, 1997, 30, 6920.
39. P. Osswald, U. Struckmeier, T. Kasper, K. Kohse-Höinghaus, J. Wang, T. A. Cool, N. Hansen and P. R. Westmoreland, J. Phys. Chem. A, 2007, 111, 4093.
40. S. K. Rakovsky, M. P. Anachkov, V. I. Iliev and A. E. Eliyas, J. Adv. Oxid. Technol., 2013, 6, 31.
41. G. J. Karabatsos, J. M. Corbett and K. L. Krumel, J. Org. Chem., 1965, 30, 689.
42. P. Kurcok, Z. Jedliński and M. Kowalczuk, J. Org. Chem., 1993, 58, 4219.
43. B. Picquest-Varrault, J.-F. Doussin, R. Durand-Jolibois and P. Carlier, J. Phys. Chem. A, 2002, 106, 2895.
44. E. Briner and H. Biedermann, Helv. Chim. Acta, 1932, 15, 1227.
45. C. G. Jaffredo and S. M. Guillaume, Polym. Chem., 2014, 5, 4168.
46. G. Barouti, K. Jarnouen, S. Cammas-Marion, P. Loyer and S. M. Guillaume, Polym. Chem., 2015, 6, 5414.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra27041c

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