JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 80, No. 3, 265-269. 1995

Microbial Degradation of Poly(3-Hydroxybutyrate) and Polycaprolactone by Filamentous Fungi

YUJI ODA,‘* HIROYUKI ASARI,’ TEIZI URAKAMI,2 AND KENZO TONOMURA’

Department of Food Science and Technology, Fukuyama University, Fukuyama, Hiroshima 729-02’ and Mitsubishi Gas Chemical Company, Chiyoda-ku, Tokyo 1OO,2 Japan

Received 10 March 19951Accepted 7 June 1995

Five strains were isolated from soil as fungi able to degrade both poly(3-hydroxybutyrate) (PHB) and poly- caprolactone (PCL), and one of the strains, D218, identified as Puecilomyces lilacinus, was selected. In 10 d, D218 degraded PHB almost completely and 10% of the PCL, each at 0.1% in the culture media. Strain D218 excreted PHB and PCL depolymerases in media containing either PHB, PCL or PHB plus PCL. Both depolymerase activities were reduced when the medium was supplemented with either soluble starch, lactose or glucose. The optimum conditions for PHB depolymerase were pH 6.5 to 7.5 at 50°C, while those for PCL depolymerase were pH 3.5 to 4.5 at 3O’C. In the reaction mixtures used for the enzyme assays, the formation of 3-hydroxybutyrate from PHB and &-caprolactone from PCL was confirmed by high performance liquid chromatography.

[Key words: poly(3_hydroxybutyrate), polycaprolactone, Paecilomyces lilacinus, depolymerase]

Poly(3-hydroxybutyrate) (PHB) is receiving much at- tention as a raw material for biodegradable plastics (1, 2). This polymer is representative of poly(3-hydroxy- alkanoates) (PHAs), which are bacterial reserves of car- bon and energy (3). Environmental conditions for the growth of these bacteria, such as the type and feeding rate of the substrate, pH, temperature, aeration, and nitrogen sources often change the amount and composi- tion of PHAs (1). Since PHB homopolymer is hard and brittle, a copolymer of 3-hydroxybutyrate (HB) and 3- hydroxyvalerate (HV) with improved physical proper- ties has been developed, which is available commercially as Biopol (Zeneca Bio Products, Billingham, UK). In the copolymer, produced by Alcaligenes eutrophus using butyric and valeric acids as carbon sources, the impact strength and melting point have been shown to be regu- lated by the content of the HV unit (1). However, the growth conditions of this bacterium have to be strictly controlled for industrial production of the copolymer with a desired ratio of HB to HV units (4).

Polycaprolactone (PCL) is also a biodegradable plastic synthesized chemically by polymerization of E-caprolac- tone (dhexanolide). PCL is produced at lower cost than PHAs, but melts at 6O’C. Since PHB and PCL possess these different physical properties, it may be possible to change the physical properties of biodegradable plastics by simply blending PHB with PCL in certain ratios to meet the varied demand in the market (5).

Degradation of PHAs has been mainly shown with bacteria, but fungal degradation has recently been ob- served (1, 6, 7). Reports on the microbial degradation of PCL are limited compared with those on PHB. Tokiwa et al. (8) isolated a Penicillium sp. utilizing PCL for its growth and found PCL depolymerase activity in the ex- tracellular and intracellular fractions. Preliminary experi- ments by Dave et al. (5) indicated that crude enzyme from the culture fluid of Penicillium funiculosum ATCC 9644 hydrolyzed blends of PHAs and PCL.

To evaluate the degradation of such newly prepared

* Corresponding author.

blends, we isolated fungi which grow efficiently on both PHB and PCL as principal carbon sources, and investi- gated the production of enzymes depolymerizing these plastics by a selected fungus, Paecilomyces lilacinus D218.

MATERIALS AND METHODS

Chemicals PHB, of natural origin, was purchased from Aldrich Chemical Co. Inc. (Milwaukee, WI, USA). PCL was a commercial product (Placcel HIPTM) gifted from Daicel Chemical Industries, Ltd. (Hyogo). Both the PHB and PCL were in powder form and had average molecular weights of 469,000 and 10,000, respec- tively. Sodium 3-hydroxybutyrate and &-caprolactone (CL) were obtained from Nakarai Tesque (Kyoto). HB dimer was synthesized in the Niigata Research Labora- tory of Mitsubishi Gas Chemical Co.

Media A basal medium containing 0.5 g yeast ex- tract, 1 .O g (NH&Sod, 1.0 g NaN03, 1.0 g KH2P04, l.Og K2HP04, 0.5g MgS04.7H20, 0.1 g KCl, 0.01 g CaC12.2Hz0, 0.01 g FeS04.7Hz0, and 0.07 g ZnS04 was supplemented with either 1 g of PHB, PCL or sodium succinate, or 0.3 g of PHB plus 0.7 g of PCL, per liter of distilled water unless otherwise stated, and adjusted to pH 5.2. For a solid medium, 2.0% (w/v) agar was ad- ded.

Isolation of fungi Samples collected from soil and activated sludge were suspended in 5 ml of distilled water and left for several minutes. A 0.1 ml portion of the supernatant was transferred to 5 ml of the basal medium with both PHB and PCL, and incubated statically at 30°C for 7 d. This procedure was repeated three times to enrich the microorganisms degrading PHB and PCL. The culture broth was then diluted, spread on an agar plate of the basal medium with sodium succinate, and in- cubated at 30°C for 2 d. Five strains showing fungus-like colonies were picked up and maintained on agar slants of the basal medium with sodium succinate. P. funiculo- sum ATCC 9644 was used as a reference strain degrad- ing both PHB and PCL (5).

265

266 ODA ET AL. J. FERMENT. BIOENG.,

TABLE 1. Filamentous fungi degrading PHB and PCL

Polymer in the medium C32 Cl44

Strain

C213 D215 D218 ATCC 9644

PHB Weight of mycelium (mg/ml) Residual polymer (%) Protein in culture fluid @g/ml) PHB depolymerase (U/ml) PCL depolymerase (U/ml)

0.73 0.77 0.47 0.93 1.4 0.53 ND ND 27 ND ND 50

12 12.5 12.5 29.1 22 5 6.8 9.2 14.1 17.5 33.4 5.1 0.03 0.02 0.06 0.12 0.67 0.45

PCL Weight of mycelium (mg/ml) Residual polymer (%) Protein in culture fluid (mg/ml) PHB depolymerase (U/ml) PCL depolymerase (u/ml)

0.17 ND ND ND 0.37 0.13 95 100 100 100 89 91 ND ND ND ND 0.9 3

12.7 4.7 4.9 7.3 13.9 ND ND ND ND ND 0.52 1.15

ND: Not detectable. The fungi were grown in the medium containing 0.2% of either PHB or PCL for 10 d.

Culture Fungi were statically grown on 30ml of the basal medium supplemented with PHB, PCL or both in a Petri dish (diameter 9cm) at 30°C for 7 to lOd, and then passed through a paper filter. The supernatant obtained by centrifugation of the filtrate at 8,OOOXg for 10min was dialyzed against 20mM phosphate buffer (pH 6.0) and used for enzyme assays. The residue on the filter was transferred in chloroform/water (1 : 1) and shaken for 2min. After the two layers were separated, an equal volume of chloroform was further added to the water layer and this was shaken again. The polymer in the combined chloroform layer concentrated by evapora- tion and the fungal mycelium in the water layer were dried at 105°C for 2 h and their weights were deter- mined.

Enzyme assays Polymers were dropped in distilled water at 1.6 mg/ml, sonicated at 4°C for 10 min to form suspensions, and used for assays of PHB and PCL depolymerases. The commercial PCL product was previ- ously treated as follows for dispersal in water. PCL (1 g) was solubilized in 25 ml of boiling water in a 50-ml glass bottle and vortexed thoroughly after adding 1.5 ml of PlysurfrM A210G (Daiichi Kogyo Seiyaku Co., Kyoto) as a detergent. The bottle was left to cool at room tempera- ture. A precipitate that appeared was discarded and the supernatant was centrifuged at 4,000 x g for 5 min. The precipitated PCL was suspended in 25 ml of distilled water by sonication to remove the detergent, recovered by centrifugation at 4,000 x g for 5 min, and dried at 30°C overnight.

The standard assay mixtures, which contained 0.2ml of 1 M phosphate buffer (pH 6.0), a 0.25-ml suspension of either PHB (A650=5.2) or PCL (&s0=2.0), and the crude enzyme in a total volume of 2.0ml were incubated at 40°C. After 1 h for PHB depolymerase and 3 h for PCL depolymerase, the turbidity to insoluble plastics was measured at 650nm. The reaction for PHB depolymerase was stopped by heating in boiling water for 5 min. One unit of activity was defined as the amount of enzyme required to decrease the &so by 0.1 in 1 h. Protein was determined by the method of Brad- ford with bovine serum albumin as a standard (9).

High performance liquid chromatography (HPLC) Degradation products of the plastics were analyzed using a high performance liquid chromatograph (L-6000; Hitachi, Tokyo) equipped with a Shodex OHpak SB-804 column (Showa Denko Co. Ltd., Tokyo) and a UV- monitor (Hitachi). Phosphoric acid (0.1%) was used as

the mobile phase at a flow rate of 1 .O ml/min.

RESULTS

Isolation of fungi The isolated strains degrading both PHB and PCL are shown in Table 1. Most strains that were cultured in the medium containing PHB con- sumed PHB completely and excreted enzymes depoly- merizing not only PHB but also PCL after incubation at 30°C for 10d. PCL was only slightly hydrolyzed, and the mycelium weights and depolymerase activities were lower than when strains were grown on PHB. D218 was finally selected as the strain producing both PHB and PCL depolymerase more efficiently than the other four strains and P. funiculosum ATCC 9644, and this strain was used for the subsequent experiments. Strain D218 degraded Biopol, a commercially produced copolymer of HB and HV, as well as PHB, in the medium (data not shown).

Strain D218 formed conidia without sexual reproduc- tive organs and was identified as P. lilacinus by Union Bitech Co. (Osaka).

Production of PHB depolymerase and PCL depol- ymerase by P. lilacinus D218 Strain D218 was stati- cally grown on either PHB, PCL, or both and the con- sumption of these polymers and the depolymerase ac- tivities were followed (Fig. 1). In the medium containing PHB, both PHB and PCL depolymerase activities in- creased gradually with the decrease of PHB and reached a constant value after 6 d. Each activity, which is inter- preted as an endo-type depolymerase decreasing the turbidity of the polymer suspension, was found in the culture fluid but not in the mycelium. Strain D218 did not hydrolyze PCL as quickly as it did PHB, and about 90% of the polymer was still present in the medium 10 d after inoculation. As compared with PHB, PCL was a poor substrate for the fungal growth, resulting in lower productivity of PHB depolymerase. The time courses of PHB and PCL depolymerases in the medium containing PHB did not correspond to that in the medium contain- ing PCL, suggesting that these two enzymes are pro- duced independently. In the presence of both PHB and PCL, strain D218 seemed to hydrolyze PCL slowly after preferable consumption of PHB for 4 d, and both depolymerase activities were lower than those grown on PHB alone. Agitation of the culture media had little effect on the degradation of these polymers (data not shown).

VOL. 80, 1995 FUNGI DEGRADING PHB AND PCL 267

0 2 4 6 8 10

PCL

m

PHB+PcL

r\ l.4 1.2

1

0.8

0.6

0.4

0.2

0 0 2 4 6 8 10 0 2 4 6 8 IO

Culture period (d)

FIG. 1. Degradation of PHB and PCL by P. lilucinus D218. The fungus was grown in three media containing 1 g of PHB, 1 g of PCL, and 0.3 g of PHB plus 0.7 g of PCL per liter, respectively, and the PHB depolymerase ( l ), PCL depolymerase (A), residual polymer (0), and weight of mycelium (A) were determined as described in the text.

The effects of sugars on the production of depoly- merases were examined in P. lilacinus D218 grown in the medium containing PHB (Table 2). Addition of solu- ble starch, lactose or glucose had little effect on the pro- tein content in the culture fluid, but reduced the activ- ities of both depolymerases.

Some properties of PHB and PCL depolymerases from P. lihcinus D218 PHB and PCL depolymerase activities in the crude enzyme were measured under vari- ous conditions. Maximal activity of PHB depolymerase was found in a pH range from 6.5 to 7.5 and at 5O”C, while PCL depolymerase activity was the highest at pH 3.5 to 4.5 and 30°C. The optimum pH of PHB

Reaction period

60

PHB

1 HB monomer

,HB dimer

o----J-

I I I I 1 0 5 IO 15 20

Time (min)

depolymerase from strain D218 was similar to that ob- served in the enzyme from P. funiculosum ATCC 9644 (pH 6) (9) and far different from those of bacterial en- zymes (pH 7.5 to 8.5) (10-15).

The products hydrolyzed by the crude enzyme were analyzed by high performance liquid chromatography (Fig. 2). The retention times were lO.Omin for the HB monomer, 10.7 min for the HB dimer, and 15.3 min for CL. Before the reaction for PHB a single peak was observed, and the peaks derived from the HB monomer and HB dimer increased as the reaction proceeded. After 60min, the area derived from the HB monomer became larger than that from the HB dimer, indicating that the

PCL

I I I 1 5 10 15 20

Time (min)

FIG. 2. High performance liquid chromatograms of the hydrolyzed products from PHB and PCL. The hydrolyzed products in 10 ~1 of the reaction mixture were analyzed by HPLC as described in the text.

268 ODA ET AL. J. FERMENT. BIOENG.,

TABLE 2. Effects of carbon source on the production of PHB and PCL depolymerases by P. lilacinus D218

PHB PCL Protein in Weight of Addition depolymerase depolyrnerase culture fluid mycelium

(u/ml) (D/ml) @g/ml) (mglml)

None 42 0.46 21.0 1.4 Soluble starch 0.02 ND 21.1 3.1 Lactose 0.07 ND 33.5 0.8 Glucose 0.06 ND 15.5 2.2

ND: Not detectable. Strain D218 was grown in the medium containing 0.1% PHB sup-

plemented with the sugars each at 0.5% as shown above for 10 d, and the depolymerase activity, protein concentration in the culture fluid and weight of mycelium were determined.

dimer was further hydrolyzed to monomer by the crude enzyme. In the hydrolysis of PCL, only area of the peak derived from CL increased during the incubation period. Since no production of free acid was detected by titra- tion with 0.1 N NaOH (data not shown), 6-hydroxyhex- anoic acid hydrolyzed from PCL may be readily convert- ed to CL in the reaction mixture.

DISCUSSION

Though many microorganisms are concerned in the degradation of biodegradable plastics in natural environ- ments, fungi would seem to play an important role judg- ing from the fact that PHA-based plastics are generally degraded under environmental conditions in which fungi dominate, such as in compost, municipal landfills, and greenhouse peat (6, 7). Since the degradation of PCL has also been reported in Penicillium sp. (8), we focused on fungi in selecting a microorganism degrading both PHB and PCL. The selected strain, identified as P. lilaci- nus D218, degraded PHB and PCL more rapidly than the reference strain, P. funiculosum ATCC 9644, which has previously been used to examine the degradation of these blends (5), and secreted both PHB and PCL depolymerases efficiently in the medium containing PHB as the sole carbon source. PCL depolymerase was readi- ly assayed using a stable suspension that we developed in the present experiments, but its activity was much lower than that of PHB depolymerase. Tokiwa et al. (8) ob- served that Penicillium sp. strain 26-l degraded 0.1% of PCL in the medium completely in 12 d, consuming a part of the PHB for its growth; they did not, however, investigate the strain further.

The presence of either soluble starch, lactose or glu- cose in addition to PHB repressed the synthesis of PHB and PCL depolymerases by P. lilacinus D218. Matavulj and Molitoris (7) found that fungal degradation of PHA occurred in a mineral medium containing peptone and yeast extract more frequently than in media supplement- ed with glucose. High expression of depolymerases may thus depend on the presence of PHB, PCL, and related compounds, and also on starvation of the cells of car- bon sources as suggested with some bacteria (10-12).

Tokiwa and Suzuki (16) isolated Penicillium sp. strain 14-3 capable of utilizing polyethylene adipate, which is an aliphatic polyester like PCL, as the sole carbon source from soil. They found that the polyethylene adipate-degrading enzyme purified from strain 14-3 hydrolyzed various polyesters including PCL, but not PHB, and assumed this enzyme to be a lipase (17). Vari- ous polyesters, including PCL but excluding PHB and

aromatic polyesters, have been shown to be degraded by lipases from Rhizopus delemer and R. arrhizus (18, 19). In the present experiments, production of both enzymes seemed to be independently regulated in the media containing PHB, PCL, and PHB plus PCL (Fig. 1). The optimal pH and temperature of PHB depolymerase were differed from those of PCL depolymerase. The two enzymes seem to be different proteins judging from the differences in these properties in their presence in the crude enzyme, though the possibility that both activities are derived from the same protein is not excluded. Purification and characterization of the enzymes depolymerizing PHB and PCL are required to clarify further the mechanism of PHB and PCL degradation by P. lilacinus D2 18.

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