Biologia 64/2: 246—251, 2009Section Cellular and Molecular BiologyDOI: 10.2478/s11756-009-0050-6

Microbial degradation and physico-chemical alterationof polyhydroxyalkanoates by a thermophilic Streptomyces sp.

Chitwadee Phithakrotchanakoon1, Yosita Rudeekit2, Sutipa Tanapongpipat1,Thanawadee Leejakpai2, Sei-ishi Aiba3, Isao Noda4 & Verawat Champreda1*1Enzyme Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani12120, Thailand; e-mail: verawat@biotec.or.th2Biodegradation Plastic Laboratory, National Metal and Material Technology Center (MTEC), Pathumthani 12120, Thai-land3Research Institute on Innovative Sustainable Chemistry, AIST, Ikeda, Osaka, Japan4The Procter & Gamble Company, West Chester, OH 45069, USA

Abstract: A thermophilic Streptomyces sp. capable of degrading various aliphatic polyesters was isolated from a landfillsite. The isolate, Streptomyces sp. BCC23167, demonstrated rapid aerobic degradation of several polyesters, includingpolyhydroxyalkanoate copolymers, poly(ε-caprolactone) and polybutylene succinate at 50◦C and neutral pH. The degradingactivity was repressed by glucose and cellobiose, but tolerant to repression by other carbon substrates. Degradation of acommercial poly[(R)-3-hydroxybutyrate-co-3-hydroxyhexanoate] (PHBHx) by Streptomyces sp. BCC23167 progressed fromsurface to bulk as suggested by the slight decrease in polymer molecular weight. Differential scanning calorimetry analysisof PHBHx film degradation by Streptomyces sp. BCC23167 showed that relative crystallinity of the film increased slightlyin the early stage of degradation, followed by a marked decrease later on. The surface morphology of degraded films wasanalyzed by scanning electron microscopy, which showed altered surface structure consistent with the changes in crystallinity.The isolate is thus of potential for application in composting technology for bio-plastic degradation.

Key words: biodegradable plastic; polyhydroxyalkanoate; thermophile; catabolite repression; crystallinity.

Abbreviations: Mn, number average molecular weight; Mw, weight average molecular weight; PBS, polybutylene-succinate; PCL, poly(ε-caprolactone); PHA; polyhydroxyalkanoate; PHB, poly[(R)-3-hydroxybutyrate]; PHBHx, poly[(R)-3-hydroxybutyrate-co-3-hydroxyhexanoate]; PHBV, poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate]; PLLA, poly-L-lactic acid.

Introduction

Polyesters of bacterial origin have received much atten-tion as biodegradable alternatives to petroleum-basedplastics. Polyhydroxyalkanoates (PHAs) are biodegrad-able thermoplastics with similar material properties topolyolefins. Structurally diverse PHAs have been syn-thesized by microbial fermentation and chemical poly-merization (Reddy et al. 2003; Hazer & Steinbuchel2007). PHAs are readily degraded in different environ-ments by a variety of bacteria and fungi, either in iso-lation or as microbial consortia (Jendrossek et al. 1996;Jendrossek & Handrick, 2002). Practical application ofPHAs includes agricultural and biomedical products,and packaging.The mechanical properties of different PHAs are

strongly dependent on the type, content and dis-tribution of the copolymer units. NodaxTM is aseries of aliphatic PHA copolymers with improvedphysico-chemical properties. NodaxTM is composed of

3-hydroxybutyrate monomer and medium chain length3-hydroxyalkanoate branching co-monomers with sidegroups of at least three carbon units or more (Noda etal. 2005). Poly[(R)-hydroxybutyrate-co-hydroxyhexanoate](PHBHx) is a NodaxTM series commercial biodegrad-able plastic, for which large-scale industrial productionhas been reported (Chen et al. 2001). Studies of en-zymatic degradation of PHBHx have been conducted(Iwata et al. 1999; Kikkawa et al. 2002; Li et al. 2007),although to our knowledge, systematic studies on wholecell microbial degradation of this polymer under diverseconditions have been very limited.Composting is considered an efficient means of de-

composition of biodegradable plastics (Tokiwa & Cal-abia 2004). Composting is a high-temperature process;hence, thermophilic microbes capable of degrading var-ious kinds of polyesters are of interest (Takeda et al.1998; Romen et al. 2004, Takaku et al. 2006). Actino-mycetes capable of PHA degradation at high temper-ature, especially poly[(R)-3-hydroxybutyrate] (PHB)

* Corresponding author

c©2009 Institute of Molecular Biology, Slovak Academy of Sciences

Polyhydroxyalkanoate degradation by a Streptomyces sp. 247

and its copolymers have been isolated from differentthermophilic sites (Kleeberg et al. 1998; Calabia &Tokiwa 2004; Hoang et al. 2007; Tseng et al. 2007).Despite the potential importance of actinomycetes incomposting polyesters, no study of PHBHx degrada-tion by actinomycetes under high-temperature condi-tions simulating composting has been reported.In this work the actinomycete Streptomyces sp.

BCC23167 was isolated and characterized. The iso-late is capable of rapidly degrading different aliphaticpolyesters. Degradation of the commercial polyesterPHBHx by the isolate was further investigated, focus-ing on the change in PHBHx physico-chemical charac-teristics during decomposition. The study shows thatthe isolate has potential biotechnological application inbio-plastic composting systems.

Material and methods

MaterialsPHB powder (natural origin), poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (PHBV 8 mol% and 12 mol% HV),poly(ε-caprolactone) (PCL) and polybutylene-succinate(PBS) were purchased from Sigma-Aldrich (Germany).Poly-L-lactic acid (PLLA), M.W., i.v. 0.1–0.2 was obtainedfrom Polysciences Inc. (Warrington, PA, USA). NodaxTM:PHBHx (6.9 mol% HHx) was supplied by the Procter &Gamble Co. (OH, USA). PHB and PHB copolymer filmswere prepared by a solution-casting method (Kasuya et al.1996). The film (average thickness = 100 µm) was cut intopieces and sterilized by 70% ethanol.

Microorganism isolationA thermophilic actinomycete, Streptomyces sp. (hereafteridentified as BCC23167) was isolated from a landfill sitein Suphanburi province, Thailand, based on the ability onforming a clear zone on PHB emulsified agar according toCalabia & Tokiwa (2004). Temperature and pH toleranceof the isolate was studied based on clear zone formation onemulsified polyester agar plates under different conditions.The taxonomic identification of the isolate was determinedbased the analysis of the full-length 16S rDNA sequenceamplified using primers BSF8/20 and REVB as describedpreviously (Kanokratana et al. 2004). The sequence was de-posited in GenBank (Benson et al. 2009) with accessionnumber EU106048.

Microbial degradation of PHAsThe microbial degradation of various PHA films was car-ried out in triplicate. The primary culture was preparedby suspending the spores of the microorganisms grown onemulsified PHB agar plate in 0.8% Tween 20 to a densityof approximately 4.6 × 108 cfu/mL. A 500-mL Erlenmeyerflask containing 100 mL of basal salt medium was inocu-lated with the primary culture at 5% inoculum. Basal saltmedium (250 mg/L yeast extract, 10 mg/L FeSO4 · 7H2O,200 mg/L MgSO4 · 7H2O, 1000 mg/L (NH4)2SO4, 20 mg/LCaCl2 · 2H2O, 100 mg/L NaCl, 0.5 mg/L Na2MoO4.2H2O,0.5 mg/L Na2WO4, and 0.5 mg/L MnSO4 in 10.7 mMKH2PO4/K2HPO4, pH 7.1) was supplemented with 0.2%emulsified PHB as the sole carbon and energy source. Theculture was incubated at 50◦C with vigorous shaking at200 rpm for 2 days. The cells were harvested by centrifuga-tion at 5,000×g for 10 min and then re-suspended in basalsalt medium of the starting volume. The cell suspension

was used as a seed culture for aerobic PHA degradationstudy. The seed culture was inoculated at 5% in 5 mL basalmedium containing a surface-sterilized (70% ethanol) PHAfilm (25 mg), and incubated at 50◦C with shaking at 200rpm for 30 h. The residual film sample, cell biomass andculture supernatant were collected at time intervals for anal-ysis. After incubation, the residual films were washed withdistilled water and 70% ethanol, and then dried before resid-ual weight and physico-chemical property analysis. The as-says of repression of PHB degrading activity by simple car-bon substrates were performed in 5 mL basal salt mediumsupplemented with 1% (w/v) of the carbon substrate witha PHB film (25 mg) as described above. The cultures wereincubated at 50◦C with shaking at 200 rpm for 15 h and theresidual film samples were determined. The residual carbonsubstrates were analyzed by HPLC. Controls with no in-oculum were included in all experiments. The reported datawere analyzed from triplicate experiments.

Analytical techniquesCarbon substrate concentration analysis. Carbon substrateconcentrations were determined by HPLC on a WatersHPLC model 2360 equipped with a Waters 410 differen-tial refractometer and a SugarPak column (Waters, Milford,MA, USA). Water was used as a mobile phase at a flow rateof 1 mL/min.

Molecular weight determination. Polymer starting molec-ular weight and residual molecular weight were determinedby gel permeation chromatography on a Waters GPC model150-CV (Waters) equipped with a refractive index detec-tor and two PL gel 10 µm mix-B columns. Chloroform wasused as the eluent at a flow rate of 1 mL/min. A calibrationcurve was generated with polystyrene standards of molecu-lar weight 4.5 to 1,112 kDa.

Relative crystallinity and Tm analysis. Relative crys-tallinity and melting temperature were analyzed by differen-tial scanning calorimetry on a DSC822 differential scanningcalorimeter (Mettler Toledo, Columbus, OH, USA). Tem-perature scanning was from −30 to 200◦C. Relative crys-tallinity was calculated as the ratio of the heat of fusion(∆H) of the degraded film sample compared with intactfilm. Peak temperature was reported as melting tempera-ture (Tm).

Surface morphology analysis. Surface morphology ofthe PHA films was analyzed by scanning electron mi-croscopy using a JSM-6301F Scanning Electron Microscope(JEOL, Tokyo, Japan). The polymer films retrieved frommicrobial degradation and controls were washed with 70%ethanol, dried and coated with gold for analysis.

Results and discussion

Isolation and phylogenetic analysisEnvironmental samples were obtained from 33 differentlocations in Thailand from a variety of milieu, includingsoil, fresh water, waste water treatment systems, land-fill and compost. Several bacteria capable of degradingdifferent polyesters were isolated, based on clear zoneformation on emulsified polyester agar plates. The ma-jority of isolates were obtained from PHB agar, followedby PCL and PBS, while no PLLA degrading microor-ganism was obtained (data not shown). From more than50 bacterial isolates obtained from PHB agar, five werethermophilic isolates from landfill and compost sam-ples. Partial 16S rDNA sequence analysis putatively

248 C. Phithakrotchanakoon et al.

Table 1. Biodegradation activity of Streptomyces sp. BCC23167 on various polyesters.

Polyester Mw (kDa) Mw/Mn Clear zone formationa

PHB 555 3.1 +++PHBV 8% 210 2.3 +++PHBV 12% 358 1.9 +++PHBHx 6.9% (NodaxTM) 633 2.4 +++PCL 176 1.7 ++PBS 45 1.8 +PLLA 15 1.3 –

a +++ = forming clear zone in 1–2 d; ++ = forming clear zone in 2–3 d; + = forming clear zone within 4 d; – = no clear zoneformation.

Table 2. Effects of carbon substrates on catabolic repression of PHB degrading activity of Steptomyces sp. BCC23167. A PHB films(25 mg) was incubated with Streptomyces sp. BCC23167 in 5 ml basal salt medium supplemented with 1% carbon substrate (50 mg)as indicated. The culture was incubated at 50◦C with shaking at 200 rpm for 15 h.

Carbon substrate Degraded PHB film (mg) C substrate utilized (mg) CDWa (mg) Degraded PHB (mg)/CDW (mg)

Glucose 0 30 11.3 0Fructose 19.4 25 15.8 1.23Galactose 25 11 17.7 1.41Xylose 17.6 29 18.8 0.94Maltose 25 9.5 20.9 1.20Cellobiose 0 24 12.8 0Glycerol 24.8 12 20.8 1.19PHB controlb 16.2 n.d.d 10.3 1.57Film controlc 0 n.d. n.d. n.d.

a Cell dried weight. b PHB film inoculated with cells but not supplemented with carbon substrate. c PHB film with no cell inoculums.d n.d.: not determined.

identified the five thermophiles as Schlegelella thermo-depolymerans, Bacteroidetes bacterium S22-35, Pseu-domonas sp., Thermobifida fusca and Streptomyces sp.Bacteria from the same genera have been reported pre-viously for their ability to degrade PHAs (Kleeberg etal. 1998; Schober et al. 2000; Elbanna et al. 2003; Cal-abia & Tokiwa 2004). The Streptomyces sp. BCC23167was of particular interest due its high degrading ac-tivity on various polyesters. Based on its full-length16S rDNA sequence, the isolate is closely related tothermophilic streptomycetes, including S. thermovul-garis, S. thermonitrificans, S. thermoviolaceus and S.thermogriseus (99% identity), but distinct from thereported thermophilic Streptomyces strain MG (Cal-abia & Tokiwa 2004) and mesophilic Streptomyces,including Streptomyces sp. SNG9 (Mabrouk & Sabry2001).Streptomyces sp. BCC23167 formed wrinkled, dark

grayish colonies on PHB agar. It grew and degradedPHB effectively in the temperature and pH rangesof 25–55◦C and 6–9, respectively, with optimal condi-tions of 50◦C and pH 7–8. Clear zone formation as-says on different emulsified polyester agar showed thatStreptomyces sp. BCC23167 degrades polyesters selec-tively (Table 1). The preferred polyester substrate isPHB, in which clear zones were observed within 24 h.Clear zones were formed more slowly on PCL and PBSagar (48 and 72–96 h, respectively), and no clear zonewas observed on PLLA. Streptomyces sp. BCC23167is not expected to degrade PLLA, since most PLLA-degrading actinomycetes belong to the Pseudonocar-

diaceae family and related genera (Tokiwa & Jarerat2004).

Catabolite repression of PHB degrading activityBacterial PHA degrading activity is known to be regu-lated by simple carbon substrates (Manna et al. 1999,2000; Romen et al. 2004). Regulation of PHA degrad-ing activity generally acts through a catabolite repres-sion mechanism. To test for catabolite repression ofPHA degrading activity in Streptomyces sp. BCC23167,PHB degradation assays were performed by cultivatingthe bacteria in the presence of PHB film and variouscarbon substrate supplements (Table 2). Glucose wasfound to be a strong repressor of PHB degrading ac-tivity, since no PHB degradation was detected on glu-cose supplemented PHB, in accordance with previousreports on other streptomycetes (Manna et al. 1999,2000). Cellobiose showed similar repression effect to glu-cose, which could be explained by its partial hydrolysisto glucose (shown by HPLC profiling; data not shown).However, different from many bacteria including somestreptomycetes reported previously (Manna et al. 1999,2000; Mabrouk & Sabry, 2001), PHB degrading activityof our isolate showed marked tolerance to catabolite re-pression by other carbon substrates tested. As indicatedby the amount of PHB degraded/cell dried weight ratio,the PHB degrading activity was tolerant to cataboliterepression either by highly assimilated substrates (fruc-tose and xylose) or by the substrates assimilated at alower rate (galactose, maltose and glycerol).The different effect possessed by glucose and its

Polyhydroxyalkanoate degradation by a Streptomyces sp. 249

disaccharide maltose has been reported in gene regula-tion of maltose utilization pathway in S. coelicolor (vanWezel et al. 1997). This suggested that only glucoseis the key repressor on PHB depolymerase activity inStreptomyces sp. BCC23167, possibly due to its key rolein catabolite repression related to energy metabolism.Catabolic repression by glucose via a specific gene regu-lation mechanism was previously reported for control ofhydrolytic enzymes in S. lividans (Nguyen et al. 1997;Hodgson 2000).

Biodegradation of polyhydroxybutyrate copolymersThe time course of biodegradation of various PHBcopolymers is shown in Figure 1. During degradation ofall PHB copolymers, the cell biomass weight increased,along with a respective decrease in pH due to accumu-lation of the acidic hydrolysis products. Rapid degrada-tion of PHB was observed, as PHB film was completelydegraded to water-soluble products within 24 h at 50◦C.Under the experimental aerobic and thermophilic con-ditions, PHBV (8 mol% and 12 mol% of copolymer HV)was degraded markedly faster than PHB. It was ob-served that after 18 h of degradation, the 8 mol% and12 mol% PHBV films had 8 and 22% residue remaining,respectively. In contrast, 30% residue remained for thePHB films. PHBHx (6.9 mol% HHx) was degraded veryefficiently by Streptomyces sp. BCC23167 with com-plete degradation within 15–18 h. This marked pref-erence for PHBHx degradation over other copolymerscontrasts with previous reports on degradation of PHBcopolymers by isolated enzyme which showed degrada-tion rate in the order of PHBV > PHB > PHBHx bySCL-PHA depolymerase from Ralstonia pickettii dueto affinity of the enzyme to the substrates (Li et al.2007; Numata et al. 2007). The relative preference ofPHB copolymer degradation shown by Streptomycessp. BCC23167 is similar to previous reports on micro-bial degradation of PHAs in soil, water and activatedsludge under environmental conditions (Volova et al.1998; Wang et al. 2004). The variation in PHA copoly-mer degradation rates appear to be related to the de-gree of crystallinity of the polyesters, reported as PHB> PHBV > PHBHx (Wang et al, 2004; Li et al. 2007),and also differences in surface morphology, which makePHBHx more susceptible to microbial attack. On theother hand, mesophilic streptomycetes have been shownto degrade PHBV more slowly than PHB (Manna et al.1999), suggesting that substrate specificity of SCL-PHA

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Fig. 1 Time dependent biodegradation of PHA films. A PHA film(25 mg) was incubated with 5% inoculation of Streptomyces sp.BCC23167 in basal salt medium. The cultures were incubated at50◦C with shaking at 200 rpm. (A) Relative degradation rate onPHB, PHBV (8 and 12 mol% HV) and PHBHx (6.9 mol% HHx);(B) degradation profile of PHBHx (6.9 mol% HHx). Data werefrom triplicate experiments. Average SD is <10%.

depolymerases may differ among streptomycetes.

Physico-chemical alteration of PHBHx upon microbialdegradationChanges in the physico-chemical properties of degradedPHBHx (6.9 mol% HHx) were assessed by differentanalytical techniques (Table 3). Gel permeation chro-matography revealed that degradation of PHBHx ledto progressive decrease of the polyester’s weight average

Table 3. Alteration in physico-chemical properties of PHBHx films upon progressive biodegradation.

Time (h) Residual polymer wt (%) Mw (kDa) Relative crystallinity (%) Tm (◦C)

0 100 633 100 129.803 99 616 104.9 129.476 98 608 103.2 130.449 71 606 88.4 132.1812 14 600 71.5 128.9815 Not detectable n.d.a n.d. n.d.

a n.d.: not determined.

250 C. Phithakrotchanakoon et al.

molecular weight (Mw) from 633 kDa to 600 kDa in 12 hunder the experimental conditions, while no significantchange was observed for the control without cell inocu-lum (Mw = 632 kDa after 12 h incubation under thesame conditions). This slight decrease in Mw suggestedthat the microbial degradation of PHBHx films pro-gressed from surface to bulk. No change was observedin melting temperature (Tm) during degradation.Enzymatic hydrolysis of PHAs is believed to start

in the amorphous region, leading to increased surfacecrystallinity of the film (Abe & Doi 1999). So far, mostreports have been focused on the degradation using iso-lated enzymes, not by whole cells as occurring in nature.In this work, PHBHx films were degraded by Strepto-myces sp. BCC23167 whole cells and the change in rela-tive crystallinity was measured by differential scanningcalorimetry analysis (Table 3). An increase in relativecrystallinity was observed during the early phase of mi-crobial degradation on PHBHx, followed by marked de-cline in relative crystallinity compared with the control(97.5% relative crystallinity after 12 h). According tothe reported calibration curve of PHBHx (Noda et al.2005), this late-stage decline corresponds to a decreasein crystallinity from 42% to 30%. The early increasein relative crystallinity is probably due to preferentialdegradation of the amorphous region by extracellularPHB depolymerase (Hazer et al. 1994; Molitoris et al.1996; Abe & Doi 1999). The decrease in relative crys-tallinity was then due to the attack on the crystallineregion by the action of the colonized microorganisms,which attacked the film by both enzymatic action andphysical penetration, which finally resulted in completedegradation of the film structure.Scanning electron microscopy revealed the progres-

sive erosion of the PHBHx surface during microbialdegradation (Fig. 2). The degraded film’s surface ap-peared rough, with irregular holes observed through-out, which became more distinct during the degra-dation process. The alteration in film surface struc-ture was consistent with that reported by Nishida &Tokiwa (1993). The changes in surface morphology arethus consistent with the measured changes in physico-chemical properties of PHBHx films during biodegra-dation.In conclusion, this work reports the isolation of a

thermophilic streptomycete capable of degrading vari-ous aliphatic polyesters of biotechnological importanceat high temperature. This microorganism can rapidlydecompose various PHAs, including PHBHx, a com-mercial biodegradable plastic. The strain thus has po-tential for application in biodegradable plastic com-posting technology.

Acknowledgements

This work was supported by a research grant from the Na-tional Center for Metal and Material Technology (Grantnumber MT-3-49-POL-07-353-I). AIST fellowship for shortcourse training for V.C. was also appreciated. The authors

D

E F

A

Fig. 2. Surface morphology of PHBHx films during degradationby Streptomyces sp. BCC23167 analyzed by SEM at 10,000×.PHBHx films were incubated with Streptomyces sp. BCC23167in basal salt medium at 50◦C with shaking at 200 rpm. (A) Beforeincubation; (B) 3 h after incubation; (C) 6 h after incubation; (D)9 h after incubation; (E) 12 h after incubation; (F) control withno cell inoculated after 12 h.

would like to thank Dr. Philip Shaw for proofreading of themanuscript.

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Received June 10, 2008Accepted October 17, 2008