ENVIRONMENTAL BIOTECHNOLOGY

Conversion of post consumer polyethyleneto the biodegradable polymer polyhydroxyalkanoate

Maciej W. Guzik & Shane T. Kenny & Gearoid F. Duane & Eoin Casey & Trevor Woods &Ramesh P. Babu & Jasmina Nikodinovic-Runic & Michael Murray & Kevin E. O’Connor

Received: 9 October 2013 /Revised: 20 December 2013 /Accepted: 22 December 2013# Springer-Verlag Berlin Heidelberg 2014

Abstract A process for the conversion of post consumer(agricultural) polyethylene (PE) waste to the biodegradablepolymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in theabsence of air (pyrolysis) generated a complex mixture of lowmolecular weight paraffins with carbon chain lengths from C8to C32 (PE pyrolysis wax). Several bacterial strains were ableto grow and produce PHA from this PE pyrolysis wax. The

addition of biosurfactant (rhamnolipids) allowed for greaterbacterial growth and PHA accumulation of the tested strains.Some strains were only capable of growth and PHA accumu-lation in the presence of the biosurfactant. PseudomonasaeruginosaPAO-1 accumulated the highest level of PHAwithalmost 25% of the cell dry weight as PHAwhen supplied withthe PE pyrolysis wax in the presence of rhamnolipids. Thechange of nitrogen source from ammonium chloride to am-monium nitrate resulted in faster bacterial growth and theearlier onset of PHA accumulation. To our knowledge, thisis the first report where PE is used as a starting material forproduction of a biodegradable polymer.

Keywords Polyethylene . Polyhydroxyalkanoate . PHA .

Pyrolysis . Pseudomonas . Rhamnolipids

Introduction

Petrochemical based plastics, produced worldwide on a mul-timillion tonne scale, pervade modern society as a result oftheir versatile and highly desirable properties (Alexander1975). Polyethylene (PE) is produced more than any otherpolymer type, making up 29.1 % of worldwide polymerproduction (European Commission DG ENV 2011). PE hasa broad range of physico-chemical properties, which enable itsuse in a variety of products. These include heavy-duty com-modities, such as water pipes, food containers, toys and chem-ical storage containers. PE is also widely used in film appli-cations such as plastic bags, agricultural films, bubble wrap ormultilayer and composite films (EUPC and EuPR 2011;European Commission DG ENV 2011; rx3 2011). Like otherpetrochemical plastics, the success of PE as a conveniencebulk commodity polymer has led to post consumer PE prod-ucts becoming a major waste problem (rx3 2011). Current

M. W. Guzik : S. T. Kenny : J. Nikodinovic-Runic :K. E. O’Connor (*)School of Biomolecular and Biomedical Sciences, UniversityCollege Dublin, Ardmore House, Belfield Dublin 4, Irelande-mail: kevin.oconnor@ucd.ie

M. W. Guzik : S. T. Kenny :K. E. O’ConnorBioplastech Limited, NovaUCD, Belfield Innovation Park, UCD,Dublin, Ireland

G. F. Duane : E. CaseySchool of Chemical and Bioprocess Engineering, Engineering andMaterials Science Centre, University College Dublin,Belfield Dublin 4, Ireland

T. Woods :R. P. BabuSchool of Physics, Trinity College Dublin, Dublin Dublin 2, Ireland

R. P. BabuCentre for Research, Adoptive Nano Structures and Nanodevices,Trinity College Dublin, Dublin Dublin 2, Ireland

M. MurrayCynar PLC, Euston Fitzrovia, 85 Tottenham Court Road,London W1T 4TQ, UK

Present Address:J. Nikodinovic-RunicInstitute for Molecular Genetics and Genetic Engineering, Universityof Belgrade, Vojvode Stepe 444a, 11000 Belgrade, Serbia

Appl Microbiol BiotechnolDOI 10.1007/s00253-013-5489-2

recycling technologies focus mainly on mechanical recyclingto low value products (Al-Salem et al. 2009) or energy recov-ery by incineration (Butler et al. 2011).

We have previously reported the conversion of other pet-rochemical polymers — polystyrene (Ward et al. 2006) andpolyethylene terephthalate (Kenny et al. 2008) topolyhydroxyalkanoates (PHA). However, the conversion ofPE to PHA has not been reported so far. The pyrolysis ofdifferent polymers generates distinctive pyrolysis products,which are composed of a mixture of hydrocarbons (Wardet al. 2006; Kenny et al. 2008). While bacteria are capableof growth on a broad range of hydrocarbons, the distinctlydifferent types of hydrocarbons (aromatic, aliphatic, substitut-ed) present in a range of plastic pyrolysis products means thateach one provides significant and different challenges forbacterial growth and PHA accumulation.

The research presented here examines the development andimplementation of a two-step chemo-biotechnological con-version of PE to PHA. The approach uses a chemical step,which converts agricultural PE waste to a mixture of hydro-carbons (PE pyrolysis wax) by means of pyrolysis. PE pyrol-ysis wax is supplied directly to microorganisms for growthand PHA accumulation. Firstly we examined the ability ofmicroorganisms to convert PE pyrolysis wax — which iscomprised of a mixture of hydrocarbons— to a biodegradable

polymer. While the conversion of single aliphatic hydrocar-bons to medium chain length PHA (mcl-PHA) has beenreported (de Smet et al. 1983; Sabirova et al. 2006), neitherthe conversion of mixtures of these hydrocarbons nor a pyrol-ysis derived mixture to mcl-PHA has been reported. Finally,different inorganic nitrogen sources along with biosurfactant(rhamnolipid) supplementation were investigated in order toenhance growth and PHA production.

Materials and methods

PE pyrolysis

PE pyrolysis wax was supplied by Cynar plc Ltd (Portlaoise,Ireland). The PE pyrolysis wax was generated from wasteagricultural PE films using a commercially sensitive pyrolysisprocess.

Bacterial strains and growth medium

Bacterial strains used in this study are listed in Table 1. Theminimal salt medium (MSM) (Schlegel et al. 1961) was usedas the growth medium for all PHA production cultures. TheMSM contained 9 g l−1 Na2HPO4⋅12H2O, 1.5 g l−1 KH2PO4,

Table 1 Bacterial strains used inthis study Strain Accession number Reference

Acinetobacter calcoaceticusBD413 ATCC 33305 Juni and Janik 1969

Acinetobacter sp. DSM 17874 Throne-Holst et al. 2007

Acinetobacter sp. NBRC 100985 Yamamoto and Harayama 1996

Alkanivorax borkumensis SK2 DSM 11573 Yakimov et al. 1998

Marinobacter hydrocarbonoclasticus DSM 11845 Doumenq et al. 2001

Pseudomonas sp. GO20 NCIMB 14927 Kenny et al. 2008

Pseudomonas aeruginosa NBRC 3924 Hori et al. 2002

Pseudomonas aeruginosa NCIMB 14923 Yuste et al. 2000

Pseudomonas aeruginosa NCIMB 14924 Yuste et al. 2000

Pseudomonas aeruginosaGL-1 CIP 104590 Arino et al. 1998

Pseudomonas aeruginosa PAO1 ATCC 47085 Stover et al. 2000

Pseudomonas aeruginosaRR1 NCIMB 14928 Yuste et al. 2000

Pseudomonas aeruginosaUCD260 ATCC 9027 Zhang and Miller 1992

Pseudomonas fluorescensCHA0 DSM 19095 Smits et al. 2002

Pseudomonas frederiksbergensisGO23 NCIMB 41539 Kenny et al. 2008

Pseudomonas oleovorans NRRL B-14682 Freitas et al. 2010

Pseudomonas putidaCA-3 NCIMB 41162 O'Connor et al. 2005

Pseudomonas putidaGO16 NCIMB 41538 Kenny et al. 2008

Pseudomonas putidaGO19 NCIMB 41537 Kenny et al. 2008

Pseudomonas putidaGPo-1 ATCC 29347 Schwartz and McCoy 1973

Rhodococcus fascians DSM 20669 van Beilen et al. 2002

Rhodococcus fasciansRR12 NCIMB 14925 Yuste et al. 2000

Rhodococcus fasciansRR14 NCIMB 14926 Yuste et al. 2000

Appl Microbiol Biotechnol

0.2 g l−1 MgSO4⋅7H2O, 20 mg l−1 CaCl2, 1.2 mg l−1

Fe(III)NH4 citrate, 4 mg l−1 ZnSO4⋅7H2O, 10 mg l−1

FeSO4⋅7H2O, 1 mg l−1 CuCl2⋅2H2O, 1 mg l−1 MnCl2⋅4H2O,1 mg l−1 Na2B4O7⋅10H2O, 0.2 mg l−1 NiCl2⋅6H2O, 0.3 mg l−1

Na2MoO4⋅2H2O and nitrogen source (see below). Strainswere maintained on MSM solidified with 15 g l−1 agar andsupplemented with 20 mM sodium gluconate as a carbonsource. PE pyrolysis wax was used as a carbon source in allPHA production shake flask experiments.

Rhamnolipid production and supplementation to shake flaskcultures

Cultures were supplemented with 0.05 % w/v rhamnolipidsproduced by Pseudomonas aeruginosaGL-1 as described byArino et al. (1998). Briefly, P. aeruginosaGL-1 was grown ina synthetic medium (Arino et al. 1998) supplemented with30 g l−1 of glycerol for 5 days shaking at 30°C. Culture brothsupernatant was clarified by centrifugation (2,600×g),deproteinised at 100 °C for 10 min and acidified to pH 2 with6 M HCl. This was followed by an overnight extraction ofrhamnolipids with ethyl acetate. Organic solvent was evapo-rated under reduced pressure in order to obtain a honey-likerhamnolipid mixture. Composition of rhamnolipids producedwas determined according to Arino et al. (1998) by means ofthin layer chromatography (TLC) and gas chromatography–mass spectrometry (GC-MS) methods. This crude extract(~5 g from 1 l culture), containing mono- and dirhamnolipidmixture with predominantly 3-hydroxydecanoic acid compo-nent, filter sterilised with a 0.2-μm filter, was supplemented tothe bacterial cultures at a concentration of 500 mg l−1.

Growth conditions for PHA accumulation

For screening purposes, all strains were grown in 250-mlErlenmeyer flasks containing 50 ml of nitrogen limitedMSM and various carbon sources at a final concentration of1.95 g of carbon per litre (gC l

−1, an equivalent to 0.05%w/v).The flasks were incubated at 30 °C with shaking at 250 rpmfor 48 h. To screen for organisms capable of PHA accumula-tion, the inorganic nitrogen source ammonium chloride(NH4Cl) was supplied at 0.25 g l−1 (65 mg of nitrogen perlitre, mgN l−1). In order to establish the effect of carbonconcentration on growth and PHA accumulation, PE pyrolysiswax concentration was increased from 0.05 % to 2 % w/v andincubation time was extended up to 6 days. In addition weinvestigated the effect of another nitrogen source-ammoniumnitrate (NH4NO3, supplied at 0.19 g l−1=65 mgN l−1) ongrowth and PHA accumulation. At the end of each growthexperiment, bacterial cultures were spun down in 50 ml tubesin an Eppendorf 5810R centrifuge at 3,220×g at 4 °C. At theseconditions PE wax and/or mixture of PE wax+rhamnolipidsformed a layer on top of supernatant, which were decanted

leaving clean cell pellet, subsequently submitted forlyophilisation.

PHA content and monomer composition determinationfrom bacterial cultures

The polymer content of lyophilized whole bacterial cells wasdetermined by subjecting cells to acidic methanolysis accord-ing to previously described protocols (Brandl et al. 1988;Lageveen et al. 1988). 3-Hydroxyalkanoic acid methyl esterswere assayed by GC using an Agilent 6890 N chromatographequipped with a BP21 capillary column (25 m × 0.25 mm ×0.32 μm; SGE Analytical Sciences, Australia) and a flameionisation detector (FID) with a temperature program of120 °C for 5 min; followed by ramp of 3 °C min−1 until180 °C and held for 10 min. For the peak identification,commercially available 3-hydroxyalkanoic acids (BioplastechLimited, Ireland) were methylated as described above for PHAsamples. PHA monomer determination was confirmed usinganAgilent 6890NGC fittedwith a 5973 series inertMS, a HP-5 column (12 m × 0.2 mm × 0.33 μm; Agilent Technologies,Ireland) was used with an oven method of 50 °C for 3 min,increasing by 10 °C min−1 to 250 °C and holding for 1 min.

Nitrogen determination assays

The concentration of nitrogen (supplied to the medium asammonium chloride) in the growth media was monitored overtime using the previously described indophenol method,which detects NH4

+ (Scheiner 1976). Additionally, whenammonium nitrate was used as the source of nitrogen in thegrowth medium, it was assayed using colorimetric dip strips,which detect nitrate (Merck Millipore, Ireland).

Determination of PE pyrolysis wax compositionand hydrocarbon utilisation during growth

PE pyrolysis wax was dissolved in chloroform (4 g l−1) andanalysed as described below. Bacterial cultures grown in25 ml liquid media, in Pyrex conical flasks, on PE pyrolysiswax were extracted at various intervals with chloroform inorder to track the substrate utilisation. At each interval, dupli-cate flasks containing the bacterial culture were extracted withchloroform by shaking for 1 min (1:1 v/v). After the separa-tion of the layers, the organic layer was analysed on anAgilent6890 N series GC fitted with a 5 m × 0.53 mm × 0.15 μmDB-HT SimDis column (Agilent Technologies, Ireland) using asplit mode (split ratio 2:1). The oven method employed was30 °C for 1 min, ramping at 7.5 °C min−1 to 100 °C, followedby a ramp of 10 °C min−1 to 300 °C and held at this temper-ature for 2 min. For peak identification, single hydrocarbonsand two alkane standard solutions, the first with C8 to C20and the second with C21 to C40 (Sigma, Ireland), were used.

Appl Microbiol Biotechnol

Hydrocarbon determination was confirmed using an Agilent6890 N GC fitted with a 5973 series inert MS, an HP-5column (12 m × 0.2 mm × 0.33 μm; Agilent Technologies,Ireland) was used with an oven method of 60 °C for 6 min,increasing by 10 °C min−1 to 300 °C and holding for 10 min.

PHA polymer analysis

Differential scanning calorimetry (DSC) was performed withPerkin Elmer Pyris-Diamond Calorimeter calibrated to Indiumstandards. The samples, weighing 7–8 mg, were encapsulatedin hermetically sealed aluminium pans and heated from−70 °C to 100 °C at a rate of 10 °C min−1. To determine theglass transition temperature (Tg) the samples were held at100 °C for 1 min and rapidly quenched to −70 °C. The sampleswere then re-heated from −70 °C to 100 °C at 10 °C min−1 todetermine the melting temperature (Tm) and Tg. The Tm wastaken at the peak of the melting endotherm, while the Tg wastaken as the midpoint of heat capacity change.

To determine the thermal stability and decomposition pro-file of the samples, thermogravimetric analysis (TGA) wascarried out on a Perkin Elmer Pyris 1 thermogravemetricanalyser calibrated using Nickel and Iron standards. Eachsample was weighed to ca. 7 mg and placed in a platinumpan and heated from 30 °C to 700 °C at the heating rate of10 °C min−1 under an air atmosphere.

Molecular weight distributions were obtained by gel per-meation chromatography (GPC) using PL gel 5 mm mixed-C+PL gel column (Perkin Elmer) with PELV 290 UV–Visdetector set at 254 nm. Spectroscopic grade tetrahydrofuran(THF; Sigma) was used as the eluent at flow rate of1.0 ml min−1. Sample concentration of 1 % w/w and injectionvolumes of 500 μl. A molecular weight calibration curve wasgenerated with polystyrene standards with low polydispersityusing the Turbochrom 4.0 software.

X-ray diffraction (XRD) was performed at room tempera-ture and diffraction patterns were collected on a SiemensD500 difractometer fitted with a Cu-Kα radiation source.The X-ray beam was Cu-Kα (λ=0.1514 nm) radiation oper-ated, at 40 kVand 30 mA. Data was obtained from 2° to 60°(2θ) at a scanning speed of 0.1° min−1.

Results

Polyethylene pyrolysis product

The pyrolysis of waste agricultural PE film gave rise to a waxysubstance. The composition of the PE pyrolysis wax wasdetermined by GC and GC-MS. The analysis revealed thatthe PE pyrolysis wax is a mixture of low molecular weightparaffins and is composed of a broad range (C8–C32) ofaliphatic hydrocarbons (>99 %), 90 % of which were alkanes

and 10 % were alkenes (Fig. 1). The remaining impurities(<1 %) were of aromatic nature as determined by NMRanalysis (data not shown).

Conversion of PE pyrolysis wax to PHA in shake flasks

In total 23 bacterial strains, which were previously reported toeither degrade hydrocarbons (single or multiple) or producePHA and which were reported to have a broad spectrum ofsubstrate utilisation (Table 1), were screened for their ability toconvert PE pyrolysis wax to PHA. Of the 23 strains selected,only one has previously been reported to convert long chainhydrocarbons to PHA, i.e., octadecane by Alkanivoraxborkumensis SK2 (Sabirova et al. 2006). Surprisingly, thisstrain, which was previously reported to use a broad rangeof hydrocarbons (Sabirova et al. 2006), was unable to grow onthe PE pyrolysis wax.

Nine of the strains were able to grow on PE pyrolysis wax;however, only two strains accumulated PHA under the screen-ing conditions tested (0.05 % w/v PE pyrolysis wax and0.25 g l−1 ammonium chloride) (Table 2). Pseudomonas

Fig. 1 Distribution of saturated and unsaturated hydrocarbons in PEpyrolysis wax

Table 2 Growth and PHA production by bacterial strains using0.05 % w/v PE pyrolysis wax as the sole source of carbon and energy,in presence of NH4Cl, during 48 h

Strain CDW (g l−1) PHA (% CDW)

A. calcoaceticusBD413 0.20±0.02 ND

P. aeruginosa 3924 0.18±0.07 ND

P. aeruginosaGL-1 0.23±0.02 9.8±0.2

P. aeruginosa PAO1 0.19±0.02 ND

P. aeruginosaRR1 0.29±0.05 ND

P. aeruginosa 14923 0.12±0.03 ND

P. aeruginosa 14924 0.11±0.01 ND

P. oleovoransB-14682 0.32±0.10 3.1±0.3

Pseudomonas sp. GO20 0.15±0.09 ND

All data is representation of at least three independent experiments

ND not detected

Appl Microbiol Biotechnol

aeruginosa GL-1 accumulated three times more PHA thanP. oleovoransB-14682 (Table 2). Both strains accumulatedmedium chain length PHA (mcl-PHA), i.e., monomerswi th a carbon chain length ≥6 carbons . (R) -3-Hydroxydecanoic acid was the predominant monomer ac-cumula ted by P. aeruginosa GL-1, wi th (R) -3-hydroxynonanoic acid as the second most prevalent mono-mer (Table 4). PHA accumulated by P. oleovoransB-14682 contained monomers ranging from (R)-3-hydroxyheptanoic acid to (R)-3-hydroxydodecanoic acid,

with (R)-3-hydroxydecanoic acid as the predominantmonomer (Table 4).

P. aeruginosa GL-1, which accumulated the most PHAfrom PE pyrolysis wax, was subjected to PE pyrolysiswax hydrocarbon utilisation studies in shake flasks. Thestrain was grown in MSM with limited nitrogen to pro-mote PHA accumulation (0.25 g l−1 NH4Cl). Growth onPE pyrolysis wax was characterised by a lag period of21 h. However, 58.2 % of the PE pyrolysis wax substratewas removed from the growth media by that time (Fig. 2).

Fig. 2 Utilisation of PE pyrolysiswax hydrocarbons byP. aeruginosaGL-1 in shake flaskover a 48-h period with a startingconcentration of 1.95 gC l−1

(0.05 % w/v)

Appl Microbiol Biotechnol

During a 48 h experiment, this strain utilised 84.4 % ofthe hydrocarbons supplied, producing 0.23 g l−1 CDW ofwhich 9.8 % was mcl-PHA. This corresponded to a yieldof biomass to substrate (YX/S) of 0.14 g gC

−1 and yield ofPHA (YPHA/S) of 0.013 g gC

−1.

Enhancing PE pyrolysis wax conversion to PHA

We altered several parameters in the experimental set-up in anattempt to increase biomass and PHA production.While strainPAO1 did not accumulate PHA from 0.05 % PE pyrolysiswax, it is a well-known PHA accumulating strain (Stover et al.2000; Timm and Steinbüchel 1992) and was thus also broughtforward to see if altered conditions could induce PHAaccumulation.

Increasing the PE pyrolysis wax concentration to 2 % (w/v)did not result in better growth of P. aeruginosaGL-1 or PAO1and no PHA accumulation was observed for either strain after48 h of growth (Fig. 3a). P. oleovoransB-14682 failed to growwith 2 % (w/v) PE wax after 48 h of incubation (data notshown). An extended incubation of strain GL-1 with 2 %(w/v) PE wax resulted in similar growth but 1.6-fold morePHA accumulation (4 days) compared to growth with 0.05 %wax (2 days) (Fig. 3a). A dramatic improvement in PHAaccumulation was observed for strain PAO1 with almost

25 % of the cell dry weight as PHA (5 days) (Fig. 3a).P. oleovorans B-14682 failed to grow when 2 % w/v PEpyrolysis wax was supplied as substrate even after 6 days ofincubation (data not shown).

To further improve the PE wax to PHA process, wechanged the nitrogen source from ammonium chloride(NH4Cl; Fig. 3a) to ammonium nitrate (NH4NO3; Fig. 3b),as presence of NO3

− is known to increase surfactant(rhamnolipid) production in P. aeruginosaGL-1 (Arino et al.1998), which could aid growth on long chain hydrocarbons.For both strains, this inorganic nitrogen source triggered PHAaccumulation 1 day earlier for strain GL-1 and 2 days earlierfor PAO1 with 2 % (w/v) PE pyrolysis wax as carbon source.However, the level of PHA accumulated (% CDW) did notincrease compared to experiments when NH4Cl was suppliedas nitrogen source.

Finally, exogenous rhamnolipids produced and extractedfrom the cultures of P. aeruginosa GL-1 were added to theliquid media in order to further enhance growth and PHAaccumulation (Fig. 3c). The addition of 0.05 % w/vrhamnolipids to the culture media resulted in maximum bio-mass and PHA accumulation in strain PAO1 after 2 days ofincubation (Fig. 3c) which was an improvement, compared tocells grown in the absence of rhamnolipid where no PHAaccumulation occurred after 2 days of growth (Fig. 3a and b).

Fig. 3 Comparison of growth ofP. aeruginosaGL-1 and PAO1 on2 % w/v PE pyrolysis wax inshake flasks supplied withdifferent inorganic nitrogensources and in or without thepresence of 0.05 % w/vrhamnolipids (Rhl). Cell dryweight (CDW) (g l−1, filledtriangle), PHA content of cells (%of CDW, filled circle) weretracked over time. Conditions:a 0.25 g l−1 ammonium chlorideas nitrogen source (65 mgN l−1);b 0.19 g l−1 ammonium nitrate asnitrogen source (65 mgN l−1);c0.19 g l−1 ammonium nitrate (65mgN l−1) and 0.05 % Rhl. Note:Microorganisms were not able toaccumulate biomass fromRhl as asole carbon and energy source atthis concentration

Appl Microbiol Biotechnol

Monomer composition of PHAwas not drastically affected bythe change of nitrogen source or the presence of rhamnolipids(Table 4).

Given that increasing the PE pyrolysis wax concentra-tion to 2 % w/v, changing the inorganic nitrogen source to

ammonium nitrate and supplementing the cultures with0.05 % w/v rhamnolipids resulted in improved PHA ac-cumulation for both P. aeruginosa strains, we retested allmicroorganisms, which were able to grow on PE pyrolysiswax (0.05 % w/v, NH4Cl) but failed to produce PHA(Table 2), under these new conditions. Four strainsshowed not only improvement in biomass but were ableto accumulate PHA (Table 3). Acinetobacter calcoaceticusBD413 and P. aeruginosa NCIMB 14923, improved bio-mass (CDW) levels 1.2- and 2.7-fold, respectively, andboth accumulated low amounts of PHA (from 2.2 % to4.1 % CDW, respectively). P. aeruginosa RR1 improvedCDW levels 1.5-fold and accumulated 5.8 % of the celldry weight as PHA. P. aeruginosa NCIMB 14924achieved 3.1-fold higher biomass under the new growthconditions and accumulated 6.7 % CDW as PHA. None ofthe strains grew in control flasks where rhamnolipid, butno PE pyrolysis wax, was supplied to the growth medium.The composition of PHA was very similar to that isolatedfrom strains GL-1 and PAO-1 with (R)-3-hydroxydecanoicacid as the predominant monomer with both even anduneven carbon chain monomers appearing in the PHA(Table 4).

We have isolated PHA from cultures grown on PE pyrol-ysis wax in the presence of rhamnolipids and ammonium

Table 4 Monomer distribution of PHA accumulated under different conditions and by different strains using PE pyrolysis wax

Conditions Monomer composition (%)

3HHX 3HHP 3HO 3HN 3HD 3HUD 3HDD 3HTD

P. oleovoransB-14682

0.5 % PE wax, NH4Cl, 2 days nd nd 2±0 22±3 44±4 21±2 12±3 nd

P. aeruginosaGL-1

0.5 % PE wax, NH4Cl, 2 days 2±0 9±1 16±1 23±0 31±2 13±0 4±1 2±0

2.0 % PE wax, NH4Cl, 5 days nd 4±0 6±1 22±1 35±3 16±2 12±1 6±0

2.0 % PE wax, NH4NO3, 5 days 1±0 5±0 8±2 23±1 31±1 14±0 12±0 5±2

2.0 % PE wax, NH4NO3, Rhl, 2 days 1±0 6±0 13±1 22±0 26±1 13±1 12±2 8±1

P. aeruginosa PAO-1

0.5 % PE wax, NH4Cl, 2 days no PHA accumulation

2.0 % PE wax, NH4Cl, 5 days 2±0 8±2 6±1 29±3 36±1 15±0 5±1 nd

2.0 % PE wax, NH4NO3, 5 days 1±0 7±0 8±1 26±0 32±1 14±0 13±2 nd

2.0 % PE wax, NH4NO3, Rhl, 2 days nd 8±2 9±2 18±3 28±1 16±3 16±0 5±2

Other strains — 2.0 % PE wax, NH4NO3, Rhl, 2 days

A. calcoaceticusBD413 1±0 6 ±1 15 ±2 22±0 29±1 13±1 10±1 6±1

P. aeruginosaRR1 2±1 7±1 18±1 22±1 29±1 15±2 9±0 nd

P. aeruginosa 14923 nd 4±1 13±0 26±1 33±4 17±2 9±1 nd

P. aeruginosa 14924 2±1 7±0 16±1 24±0 27±0 11±1 12±0 3±2

PEwaxPE pyrolised wax, Rhl rhamnolipids 0.05%w/v, 3HHX3-hydroxyhexanoic acid, 3HHP3-hydroxyheptanoic acid, 3HO3-hydroxyoctanoic acid,3HN 3-hydroxynonanoic acid, 3HD 3-hydroxydecanoic acid, 3HUD 3-hydroxyundecanoic acid, 3HDD 3-hydroxydodecanoic acid, 3HTD 3-hydroxytridecanoic acid, nd not detected

Table 3 Growth and PHA production by bacterial strains using 2 % w/vPE pyrolysis wax as the sole source of carbon and energy, in the presenceof NH4NO3 and 0.05 % w/v rhamnolipids during 48 h

Strain CDW (g l−1) PHA (% CDW)

A. calcoaceticusBD413 0.24±0.10 4.1±0.4

P. aeruginosa 3924 ND ND

P. aeruginosaGL-1 0.39±0.04 18.9±0.7

P. aeruginosa PAO1 0.31±0.02 14.5±0.5

P. aeruginosaRR1 0.35±0.09 5.8±0.3

P. aeruginosa 14923 0.32±0.07 2.2±0.1

P. aeruginosa 14924 0.34±0.10 6.7±0.4

P. oleovoransB-14682 ND ND

Pseudomonas sp. GO20 ND ND

Microorganisms were not able to accumulate biomass from Rhl as a solecarbon and energy source at this concentration; all data is representationof at least three independent experiments

ND not detected, Rhl rhamnolipids at concentration of 500 mg l−1

Appl Microbiol Biotechnol

nitrate from two bacterial cultures— P. aeruginosaGL-1 andPAO-1 — and tested the physical properties of these twopolymers. Each of these polymers poses an onset of thermaldegradation occurring at 286 °C and 294 °C, for GL-1 andPAO1, respectively. Both are partially crystalline, as evi-denced by the presence of a melting peak (ΔH=10.1 J g−1). The melting temperatures of the PHA producedin our setup were found to be 35.1 °C for GL-1 and 36.0 °Cfor PAO-1. Thermal analysis showed a low glass transitiontemperature to be −41.0 °C for mcl-PHA produced by bothstrains. Molecular weights shown for these two strains(159.2 — GL-1; 160.8 kDa — PAO1) with correspondingpolydispersity index (1.8) of our polymers are comparableto those of typical elastomers (Kenny et al. 2008, Madisonand Huisman 1999, Preusting et al. 1990, van der Walleet al. 2001).

Discussion

The infrastructure to mechanically recycle post consumerplastic waste is in place in many countries globally.However, the percentage of plastic waste recycled is lowwith the best performing countries mechanical recyclingaround 25 % of plastic entering the market (EUPC andEuPR 2011; European Commission DG ENV 2011). Oneof the major reasons for low rates of mechanical recyclingare the low value products that arise from existingrecycling technologies (rx3 2011; Smith et al. 2011).New technologies that create value added products couldimprove recycling rates. The research described here in-vestigates the conversion of PE farm plastic to the biode-gradable polymer PHA with a view to expanding thepossibilities for PE recycling.

We have reported previously on the conversion of polysty-rene and polyethylene terephthalic acid to PHA using a two-step chemo-biotechnological process (Kenny et al. 2008;Ward et al. 2006). The conversion of PE to PHA has not beendescribed previously. The pyrolysis product of PE is a com-plex hydrocarbon mixture and while the production of PHAwas reported several times from single commercially availablehydrocarbons (Arino et al. 1998; de Smet et al. 1983; Sabirovaet al. 2006) the conversion of complex mixtures like a PEpyrolysis wax has not been described. We have observed thatmany known hydrocarbons degraders (Table 1) were not ableto utilise this complex substrate as a single carbon and energysource.

The strain Alkanivorax borkumensis SK2, which was re-ported to grow on a broad range of aliphatic hydrocarbons(C8–C32) (Schneiker et al. 2006; Yakimov et al. 1998), wasreported to produce both short chain length PHA (scl-PHA,grown on pyruvate) and mcl-PHA (grown on octadecane) asan extracellular network (Sabirova et al. 2006). When PE

pyrolysis wax was supplied as a carbon source to liquidcultures on either MSM or ONR7a medium (Sabirova et al.2006), A. borkumensis SK2 failed to grow, possibly due totoxicity of a broad range of alkanes and alkenes.

Pseudomonas putida GPo-1 has been shown to convertoctane to high levels of mcl-PHA (60 % of CDW) in fed batchand continuous processes (Hazenberg and Witholt 1997; Junget al. 2001; Kellerhals et al. 1999). However, previous reportsstated that the hydrocarbon substrate range for this strain islimited to C6 and C12 alkanes (Lageveen et al. 1988). Ourfindings support this observation as the strain failed to growand accumulate PHA when PE pyrolysis wax was the onlycarbon and energy source in a liquid culture.

Microorganisms have evolved systems to promote theuptake of water insoluble hydrocarbons through produc-tion of rhamnolipids which act as surfactants (Maier andSoberon-Chavez 2000; Noordman and Janssen 2002;Zhang and Miller 1995). Several studies have focusedon optimisation of rhamnolipid production in shakeflasks, containing synthetic media, by altering inorganicnitrogen sources (Arino et al. 1998; Zhang and Miller1992). Following a study carried out by Arino et al.(1998), which reports the ability of P. aeruginosa GL-1to produce rhamnolipids when grown on glycerol or n-hexadecane, we have tested ammonium nitrate as theinorganic nitrogen source with our strains (GL-1, PAO1and B-14682) in order to achieve better PE pyrolysis waxconversion to biomass and PHA. Indeed, this changetriggered earlier PHA accumulation with higher PE pyrol-ysis wax loading (2 % w/v). Following this, exogenouslyproduced rhamnolipid was introduced to flasks with PEpyrolysis wax (2 % w/v). This resulted in further im-provements in growth rate, biomass accumulation andearlier accumulation of PHA (Fig. 3a) compared to flaskswithout the rhamnolipid added (Fig. 3a and b). Thisobservation is in agreement with other authors reportingthat the addition of rhamnolipid enhances uptake of ali-phatic hydrocarbons (Zhang and Miller 1992). Moreover,the presence of biosurfactant allowed growth and PHAproduction in other tested strains previously not able togrow and produce PHA from PE pyrolysis wax (Table 3).To our knowledge, it has never been shown thatbiosurfactant addition increased PHA accumulation norallowed microorganisms previously incapable of accumu-lating PHA from hydrocarbons to start producing thisbiopolymer.

The work presented here demonstrates that a combinationof thermal and biological treatment resulted in the conversionof post consumer PE to a value-added product — PHA.Furthermore, the improvement of bioavailability resulted inimproved growth and PHA accumulation. Future investiga-tions will focus on bioprocess manipulation in the more con-trollable environment of a fermentor.

Appl Microbiol Biotechnol

Acknowledgements This project has been funded under a grant fromthe Environmental Protection Agency of Ireland (grant number 2008-ET-LS1). JNR is partially funded by Ministry of Education, Science andTechnological Development of Serbia Project No. 173048.

References

Alexander M (1975) Environmental and microbiological problems aris-ing from recalcitrant molecules. Microb Ecol 2(1):17–27

Al-Salem SM, Lettieri P, Baeyens J (2009) Recycling and recovery routesof plastic solid waste (PSW): a review. Waste Manag 29(10):2625–2643

Arino S, Marchal R, Vandecasteele JP (1998) Involvement of arhamnolipid-producing strain of Pseudomonas aeruginosa in thedegradation of polycyclic aromatic hydrocarbons by a bacterialcommunity. J Appl Microbiol 84(5):769–776

Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonasoleovorans as a source of poly(beta-hydroxyalkanoates) for poten-tial applications as biodegradable polyesters. Appl EnvironMicrobiol 54(8):1977–1982

Butler E, DevlinG,McDonnell K (2011)Waste polyolefins to liquid fuelsvia pyrolysis: review of commercial state-of-the-art and recent lab-oratory research. Waste Biomass Valor 2(3):227–255

de Smet MJ, Eggink G, Witholt B, Kingma J, Wynberg H (1983)Characterization of intracellular inclusions formed byPseudomonas oleovorans during growth on octane. J Bacteriol154(2):870–878

Doumenq P, Aries E, Asia L, Acquaviva M, Artaud J, GilewiczM, Mille G, Bertrand JC (2001) Influence of n-alkanes andpetroleum on fatty acid composition of a hydrocarbonoclasticbacterium: Marinobacter hydrocarbonoclasticus strain 617.Chemosphere 44(4):519–528

EUPC E, EuPR, PlasticsEurope (2011) Plastics — the facts 2011. Ananalysis of European plastics production, demand and recovery for2010. Brussels, pp 1–32

European Commission DGENV (2011) Plastic waste in the environment.BIO Inteligence Service (ed) Paris, pp 1–171.

Freitas F, Alves VD, Pais J, Carvalheira MN, Costa N, Oliveira R, ReisMAM (2010) Production of a new exopolysaccharide (EPS) byPseudomonas oleovorans NRRL B-14682 grown on glycerol.Process Biochem 45(3):297–305

Hazenberg W, Witholt B (1997) Effcient production of medium-chain-length poly(3-hydroxyalkanoates) from octane by Pseudomonasoleovorans: economic considerations. Appl Microbiol Biotechnol48(5):588–596

Hori K, Marsudi S, Unno H (2002) Simultaneous production ofpolyhydroxyalkanoates and rhamnolipids by Pseudomonasaeruginosa. Biotechnol Bioeng 78(6):699–707

Jung K, Hazenberg W, Prieto M,Witholt B (2001) Two-stage continuousprocess development for the production of medium-chain-lengthpoly(3-hydroxyalkanoates). Biotechnol Bioeng 72(1):19–24

Juni E, Janik A (1969) Transformation of Acinetobacter calcoaceticus(Bacterium anitratum). J Bacteriol 98(1):281–288

Kellerhals M, Hazenberg W, Witholt B (1999) High cell density fermen-tations of Pseudomonas oleovorans for the production of mcl-PHAsin two-liquid phase media. Enzyme Microb Technol 24(1–2):111–116

Kenny ST, Runic JN, Kaminsky W, Woods T, Babu RP, Keely CM, BlauW, O'Connor KE (2008) Up-cycling of PET (polyethylene tere-p h t h a l a t e ) t o t h e b i o d e g r a d a b l e p l a s t i c P HA(polyhydroxyalkanoate). Environ Sci Technol 42(20):7696–7701

Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, WitholtB (1988) Formation of polyesters by Pseudomonas oleovorans:

effect of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. ApplEnviron Microbiol 54(12):2924–2932

Madison L, Huisman G (1999) Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol BiolRev 63:21–53

Maier RM, Soberon-Chavez G (2000) Pseudomonas aeruginosarhamnolipids: biosynthesis and potential applications. ApplMicrobiol Biotechnol 54(5):625–633

Noordman WH, Janssen DB (2002) Rhamnolipid stimulates uptake ofhydrophobic compounds by Pseudomonas aeruginosa. ApplEnviron Microbiol 68(9):4502–4508

O'Connor KE, O'Leary NP, Marchesi JR, Dobson AD, Duetz W (2005)Isolation and characterization of a diverse group of phenylacetic aciddegrading microorganisms from pristine soil. Chemosphere 61(7):965–973

Preusting H, Nijenhuis A, Witholt B (1990) Physical characteristics ofpoly(3-hydroxyalkanoates) and poly(3-hydroxyalkenoates) pro-duced by Pseudomonas oleovorans grown on aliphatic hydrocar-bons. Macromolecules 23:4220–4224

rx3 (2011) The Irish recycled plastic waste arisings study. Dún Laoghaire,Ireland, pp 1–130

Sabirova JS, Ferrer M, Lünsdorf H, Wray V, Kalscheuer R,Steinbüchel A, Timmis KN, Golyshin PN (2006) Mutation ina “tesB-Like” hydroxyacyl-coenzyme A-specific thioesteraseg e n e c a u s e s h y p e r p r o d u c t i o n o f e x t r a c e l l u l a rpolyhydroxyalkanoates by Alcanivorax borkumensis SK2. JBacteriol 189(2):289–290

Scheiner D (1976) Determination of ammonia and Kjeldahl nitrogen byindophenol method. Water Res 10(1):31–36

Schlegel HG, Kaltwasser H, Gottschalk G (1961) A submersion methodfor culture of hydrogen-oxidizing bacteria: growth physiologicalstudies. Arch Microbiol 38(3):209–222

Schneiker S, dos Santos VAPM, Bartels D, Bekel T, Brecht M,Buhrmester J, Chernikova TN, Denaro R, Ferrer M, Gertler C,Goesmann A, Golyshina OV, Kaminski F, Khachane AN, Lang S,Linke B, McHardy AC, Meyer F, Nechitaylo T, Puhler A,Regenhardt D, Rupp O, Sabirova JS, Selbitschka W, YakimovMM, Timmis KN, Vorholter F-J, Weidner S, Kaiser O, GolyshinPN (2006) Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis. NatBiotech 24(8):997–1004

Schwartz RD, McCoy CJ (1973) Pseudomonas oleovorans hydroxyl-ation–epoxidation system: additional strain improvements. ApplEnviron Microbiol 26(2):217–218

Smith M, Wiesweg M, Koster R, Longworth K, Klemens C (2011)European plastics & polymers market report. CMAI EuropeGmbH, Düsseldorf, pp 1–14

Smits TH, Balada SB, Witholt B, van Beilen JB (2002) Functionalanalysis of alkane hydroxylases from gram-negative and gram-positive bacteria. J Bacteriol 184(6):1733–1742

Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, HickeyMJ, Brinkman FSL, Hufnagle WO, Kowalik DJ, Lagrou M, GarberRL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, BrodyLL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K,Spencer D, Wong GKS, Wu Z, Paulsen IT, Reizer J, Saier MH,Hancock REW, Lory S, Olson MV (2000) Complete genome se-quence of Pseudomonas aeruginosa PAO1, an opportunistic patho-gen. Nature 406(6799):959–964

Throne-Holst M, Wentzel A, Ellingsen TE, Kotlar H-K, Zotchev SB(2007) Identification of novel genes involved in long-chain n-alkanedegradation by Acinetobacter sp. strain DSM 17874. Appl EnvironMicrobiol 73(10):3327–3332

Timm A, Steinbüchel A (1992) Cloning and molecular analysis of thepoly(3-hydroxyalkanoic acid) gene locus of Pseudomonasaeruginosa PAO1. Eur J Biochem 209(1):15–30

Appl Microbiol Biotechnol

van Beilen JB, Smits TH, Whyte LG, Schorcht S, Rothlisberger M,Plaggemeier T, Engesser KH,Witholt B (2002) Alkane hydroxylasehomologues in Gram-positive strains. Environ Microbiol 4(11):676–682

van der Walle GAM, de Koning GJM, Weusthuis RA, Eggink G (2001)Properties, modifications and applications of biopolyesters. AdvBiochem Eng/Biotechnol 71:264–291

Ward PG, Goff M, Donner M, KaminskyW, O'Connor KE (2006) A twostep chemo-biotechnological conversion of polystyrene to a biode-gradable thermoplastic. Environ Sci Technol 40(7):2433–2437

Yakimov MM, Golyshin PN, Lang S, Moore ERB, Abraham W-R,Lünsdorf H, Timmis KN (1998) Alcanivorax borkumensis gen. nov.,sp. nov., a new, hydrocarbon-degrading and surfactant-producingmarine bacterium. Int J Syst Evol Microbiol 48(2):339–348

Yamamoto S, Harayama S (1996) Phylogenetic analysis of Acinetobacterstrains based on the nucleotide sequences of gyrB genes and on theamino acid sequences of their products. Int J Syst Bacteriol 46(2):506–511

Yuste L, MaE C, MaJ T, Karlson U, Puyet A, Rojo F (2000)Characterization of bacterial strains able to grow on high molecularmass residues from crude oil processing. FEMS Microbiol Ecol32(1):69–75

Zhang Y, Miller RM (1992) Enhanced octadecane dispersion and bio-degradation by a Pseudomonas rhamnolipid surfactant(biosurfactant). Appl Environ Microbiol 58(10):3276–3282

Zhang Y, Miller RM (1995) Effect of rhamnolipid (biosurfactant) struc-ture on solubilization and biodegradation of n-alkanes. Appl EnvironMicrobiol 61(6):2247–2251

Appl Microbiol Biotechnol