Plastic-Eating Fungi Found In The Amazon May Solve World’s Waste Problem

ORIGINAL: PSFK

A group of students and professors from Yale University have found a fungi in the Amazon rainforest that can degrade and utilize the common plastic polyurethane (PUR). As part of the university’s Rainforest Expedition and Laboratory educational program, designed to engage undergraduate students in discovery-based research, the group searched for plants and cultured the micro-organisms within their tissue.

Several active organisms were identified, including two distinct isolates of Pestalotiopsis microspora with the ability to efficiently degrade and utilize PUR as the sole carbon source when grown anaerobically, a unique observation among reported PUR biodegradation activities.

Polyurethane is a big part of our mounting waste problem and this is a new possible solution for managing it. The fungi can survive on polyurethane alone and is uniquely able to do so in an oxygen-free environment. 

The Yale University team has published their findings in the article ‘Biodegradation of Polyester Polyurethane by Endophytic Fungi’ for the Applied and Environmental Microbiology journal.

Biodegradation of Polyester Polyurethane by Endophytic Fungi

FIG. 1. Example of PUR-A plates initially used to screen for polyurethane-degrading activity after 2 weeks of fungal growth. (A) Negative control. (B) Pleosporales sp. strain E2705B.

ABSTRACT

Bioremediation is an important approach to waste reduction that relies on biological processes to break down a variety of pollutants. This is made possible by the vast metabolic diversity of the microbial world. To explore this diversity for the breakdown of plastic, we screened several dozen endophytic fungi for their ability to degrade the synthetic polymer polyester polyurethane (PUR). Several organisms demonstrated the ability to efficiently degrade PUR in both solid and liquid suspensions. Particularly robust activity was observed among several isolates in the genus Pestalotiopsis, although it was not a universal feature of this genus. Two Pestalotiopsis microspora isolates were uniquely able to grow on PUR as the sole carbon source under both aerobic and anaerobic conditions. Molecular characterization of this activity suggests that a serine hydrolase is responsible for degradation of PUR. The broad distribution of activity observed and the unprecedented case of anaerobic growth using PUR as the sole carbon source suggest that endophytes are a promising source of biodiversity from which to screen for metabolic properties useful for bioremediation.

INTRODUCTION

Tremendous increases in the manufacture and consumption of plastics over recent decades have led to numerous ecological and economic concerns. The persistence of synthetic polymers introduced into the environment by human industry poses a major threat to natural ecological systems. The low cost and ease of manufacture have increased global plastic demand more than 150-fold, with the production of 1.5 million tons in 1950 and 245 million tons as of 2006 (21). Despite recognition of the persistent pollution problems posed by plastic, global production is still increasing, with the largest increases expected in developing nations. The sheer volume of plastics produced each year presents a problem for waste disposal systems. The scale of this problem and the recalcitrance of some polymers to degradation necessitate investigation into effective methods for biodegradation of plastics. By gaining an understanding of the mechanisms of polymer degradation, a more efficient technique for the biodegradation of plastic waste can be achieved. To accomplish this goal, researchers need greater knowledge of how compounds are metabolized by existing organisms, an investigation of new organisms with bioremediation potential, and the characterization of novel metabolic capabilities. A basic understanding of the biological processes that lead to biochemical degradation will advance the development of new bioremediation techniques.

Polyester polyurethane (PUR) is a plastic widely used in industry and manufacturing that has been shown to be susceptible to biodegradation (6, 10). The polymer is generated by the condensation of a polyisocyanate and a polyol. This results in a carbon polymer composed of a series of urethane linkages. Variations in the spacing between urethane linkages, as well as the nature of the substitutions, can change the properties of the resulting polymer from linear and rigid to branched and flexible. In a liquid suspension PUR appears milky white and completely opaque. Like other polyurethanes, this product is synthesized commercially for the manufacture of textiles and textile coatings.

Enzymatic degradation of PUR by both fungi (4, 5, 6, 19) and bacteria (11, 12,14, 15, 17, 18, 23) has been demonstrated. Soil fungi comprise the majority of organisms screened for PUR degradation activity (4, 5). Fungi of the generaAlternaria, Aspergillus, Phoma, Pennicilium, Plectosphaerella, Geomyces, Nectria, and Neonectria were isolated with access to mixed nutrient sources from buried PUR samples (4). Geomyces pannorum was the most commonly isolated PUR-degrading organism with this method (4). Few organisms have been shown to degrade PUR as a sole carbon source. Aspergillus niger has some reported degradation activity; however, it was observed to be quite slow, with visible signs of degradation occurring only after 30 days (7).

Putative polyurethanases have been isolated and characterized from protein extracts of several organisms, including the bacteria Pseudomonas chlororaphis(25) and Comamonas acidovorans (1, 2), as well as the fungus Candida rugosa(8). The active enzymes have been classified as esterases (5, 13, 16), lipases (26), and proteases and ureases (19), suggesting degradation of the PUR substrate by cleavage of the ester bond.

In an effort to identify new organisms with novel metabolic capabilities for polymer degradation, we have embarked on an effort to explore the biological and chemical diversity of endophytes. This was achieved as part of an educational program to engage undergraduate students in discovery-based research (9, 28). Endophytes are hyperdiverse microorganisms, including bacteria and fungi, that live within the inner tissues of plants without causing overt disease symptoms (3). These organisms enter their hosts by penetrating exterior surfaces, and some play a key role in plant decomposition following host tissue death (3). Indeed, fungi such as these contribute to decomposition of lignocellulose polymers and are major contributors to the carbon cycle (3, 22, 24). The ability of these microorganisms to degrade a polymer as complex as lignocellulose would suggest that these organisms offer promise for their ability to degrade other complex polymers, such as those present in plastics. The unique biological niche of endophytes as endosymbionts of tissues rich in complex carbon polymers justifies the investigation of their wider metabolic capabilities. Each of the more than 300,000 land plant species on Earth potentially hosts multiple endophyte species. Only a small sampling of plants have been examined for their endophytic associations, yet many of these organisms can be readily cultured (3, 25, 28). Endophytes reach their greatest diversity in tropical forests. Individual trees can harbor hundreds of endophytic species, some of which are known but many of which are new to science (25).

In the current study, endophytes were isolated from plant stems collected in the Ecuadorian rainforest. A subset of these organisms was screened for their ability to degrade polyurethane. Several active organisms were identified, including two distinct isolates of Pestalotiopsis microspora with the ability to efficiently degrade and utilize PUR as the sole carbon source when grown anaerobically, a unique observation among reported PUR biodegradation activities.

MATERIALS AND METHODS

Plant sampling.Woody plants of various families were collected in the Yasuni National Forest in the Ecuadorian Amazonian rainforest. Some plants were targeted for their purported ethnobotanical uses, while others were sampled randomly. A 10-cm stem sample was collected and stored in an air-tight polyethylene bag at 4°C. Herbarium samples were prepared and deposited in the Peabody Herbarium at Yale University and the Herbario Nacional del Ecuador (QCNE).

Endophyte isolation.Plant stems were surface sterilized by immersion in ethanol for 10 s followed by brief flaming. Outer layers of tissue were removed from the stems, and three sections of the inner tissues of each sample were plated on potato dextrose agar (PDA) (Difco), a 1:10 dilution of potato dextrose medium in water agar (WA), a 1:10 dilution of glycerol arginine medium (GAM) in WA, and WA plates (25). All plates were sealed with Parafilm and monitored every 2 to 3 days for growth. As growth of microbes was detected, fungal organisms were isolated by transferring a hyphal tip to fresh PDA plates. The plates were again wrapped with Parafilm and stored in plastic containers. Permanent stocks of each organism were made by growing the organisms on triple-autoclaved barley seeds and storing them at -80°C.

Sequencing and phylogenetic analysis.Endophyte cultures were grown on PDA plates for 1 to 2 weeks at room temperature. DNA was extracted from approximately 100 mg of fungal material using the Qiagen DNeasy plant minikit. Approximately 10 ng of DNA was used as a template to amplify the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) by PCR as previously described (30). The primers used for ITS sequencing were ITS1 (5'-TCCGTAGGTGAACCTGCGGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), corresponding to the forward and reverse primers (30). The amplified fragments were verified for length using agarose gel electrophoresis and purified using a QIAquick PCR purification kit (Qiagen). Sequencing was performed at the Yale University W. M. Keck Facility on Applied Biosystems 3730cL DNA Analyzer machines. Forward and reverse sequences for each organism were aligned using the programs Pregap4 and Gap4 (25). Endophyte sequences were aligned to organisms present in the GenBank database on 1 August 2008 using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information 

(http://www.ncbi.nlm.nih.gov/GenBank/index.html).

Initial PUR clearance screen.Endophytes were first assayed for their ability to degrade PUR by growing them in the presence of Impranil DLF an anionic aliphatic aqueous PUR dispersion with 4% N-methyl pyrrolidone (NMP) (Bayer MaterialScience). Fifty-nine fungal endophytes were grown on solid PUR medium (PUR-A) containing 19 mM NaH2PO4, 33.5 mM K2HPO4, 7.6 mM (NH4)2SO4, 2.5 mM Na citrate, 250 µM MgSO4, 19 µM thiamine, 0.05% Casamino Acids, 147 µM FeCl3·6H2O, 14 µM ZnCl2·4H2O, 12 µM CoCl2·6H2O, 12 µM Na2MoO4·2H2O, 10 µM CaCl2·2H2O, 11 µM CuCl2, 12 µM MnCl2, 12 µM H3BO3, and 1.8 mM HCl. To 1 liter of this mixture was added 10 ml Impranil DLF and 15 g of agar. The polymer was added after autoclaving the medium to prevent deformation.

The solid medium screening assay followed the general method of Crabbe et al. (5). The PUR-A solid medium was added to sterile culture tubes in 10-ml aliquots. A 0.5-cm3 plug of fungus grown on PDA was added to each test tube using aseptic technique and allowed to grow undisturbed at 23°C. The bacteriumPseudomonas chlororaphis (ATCC 55729, from Gary Howard) and the fungusAspergillus niger (from Gary Strobel, Montana State University) were used as positive controls for polyurethane degradation (6, 7, 13). PUR degradation was evidenced by a change in medium appearance from opaque to translucent. After 2 weeks of incubation, the depth of polyurethane clearance was measured from the top of the medium to the lowest point of visible clearance.

The liquid medium assay followed the method described by Gautam et al. (8). PUR-L liquid medium was prepared using the same recipe as for the solid PUR-A medium without agar. PUR-L medium was added to sterile culture tubes in 10-ml aliquots and inoculated with a 0.5-cm3 plug of fungus grown on PDA. At the end of 2 weeks, the liquid cultures were homogenized by vigorous shaking. A 1.5-ml portion of each culture was transferred to an Eppendorf tube and centrifuged for 1 min at 4,200 × g in an Eppendorf MiniSpin centrifuge to selectively pellet the fungal matter. The absorbance of the supernatant liquid was measured on a Varian Cary 50 Bio UV-visible spectrophotometer at a wavelength of 600 nm, with sterile water as a blank. Dilutions of PUR-L medium with sterile water were measured to construct a standard curve for converting absorbance to percent clearance. Samples were measured each day for 2 weeks for optical absorbance to determine a relative rate of clearance.

Sole carbon source assay.Organisms identified as having PUR-degrading activity were tested for their ability to use PUR as the sole carbon source. For these studies, the substrate was Impranil DLN, which contains PUR suspended in only water (no N-methyl pyrrolidone is present). This isolates the Impranil as the sole source of carbon for metabolism and growth. The top five organisms from the initial activity screens were grown on Impranil DLN with no other carbon sources (PUR-Lmin). The fungal samples were washed prior to inoculation to remove all residual medium. These two considerations—the wash and the DLN substrate—ensure that the polymer is the sole source of carbon for fungal metabolism and growth. PUR-Lmin was prepared in a manner similar to that for the PUR-L medium but in the absence of sodium citrate, thiamine, Casamino Acids, or agar. The organism Aspergillus niger was tested as a basis for comparison.

Fungal cultures were grown for 1 week in potato dextrose broth (PDB). Stock cultures were homogenized by vigorous shaking, and 1 ml of each culture was centrifuged at 12,100 × g for 1 min. The supernatant was removed, and the fungal pellet was resuspended in 1 ml of the PUR-Lmin liquid medium. Samples were centrifuged and resuspended a second time to ensure removal of all residual PDB. The 1-ml sample of washed fungal material was added to sterile culture tubes to a final volume of 10 ml PUR-Lmin. The cultures were monitored for visual clearance of the opaque medium. Samples were measured every 2 days for 2 weeks for optical absorbance at 600 nm to determine an approximate rate of clearance. An increase in fungal mass correlating to PUR degradation was measured by lyophilizing mycelial mass from triplicate cultures containing minimal medium with and without PUR.

Anaerobic degradation of PUR.Culture tubes containing 9-ml aliquots of PUR-Lmin medium and 1 ml of washed fungal inoculums were incubated under anaerobic conditions. The anaerobic environment was generated using a BD GasPak anaerobic chamber. Liquid cultures of each isolate were inoculated in duplicate. One set was placed inside the anaerobic chamber, and the control set was placed outside the chamber in an aerobic environment. Both sets were incubated at 25°C for the duration of the study. After 1 and 2 weeks, samples were removed and clearance was determined by absorption at 600 nm.

IR analysis of PUR degradation.Infrared (IR) spectra of liquid PUR-Lminsuspensions were collected every 48 h using a Nicolet 6700 infrared spectrometer. A 50-µl portion of each sample was removed for each measurement and centrifuged for 60 s at 4,200 × g to settle out fungal material from the samples without significant sedimentation of the polymer. Liquid sample spectra were collected using deionized water as a background spectrum. Measurements were performed until complete degradation of PUR in the 10-ml culture was observed within 2 weeks.

Enzymatic characterization of putative polyurethanase.An extracellular enzyme fraction was prepared by growing a 1-liter liquid culture of the most active organism, Pestalotiopsis microspora E2712A, in PUR-Lmin and PDB media. After 10 days of incubation at 30°C, the culture was filtered through a 0.22-µm filter into a sterile container. Crude extracellular extract was stored at 4°C. To test for the activity of an extracellular enzyme, 4 ml of extract was added to 6 ml of PUR-Lmin medium. Samples were incubated for 2 h in a rotary incubator at 30°C.

Three mechanism-based inhibitors were used to characterize the activity. Phenylmethylsulfonyl fluoride (PMSF), a serine hydrolase inhibitor, was added to an aliquot of enzyme extract and added to PUR-Lmin medium to a final concentration of 1 mM. Iodoacetate, a cysteine hydrolase inhibitor, was added to a final concentration of 10 µM. EDTA at a concentration of 5 mM was used as a metallohydrolase inhibitor. Extract that was heat treated at 98°C for 20 min served as a negative control. Samples were incubated in a rotary incubator at 30°C, and clearance was observed macroscopically after 2 h.

Based upon initial observations that a serine hydrolase was implicated in PUR degradation, activity-based probes were used to test for a serine hydrolase protein in crude cell-free filtrates. The probe molecules contain a fluorophosphonate moiety that reacts specifically with the active-site serines of enzymes in the serine hydrolase family and a tetramethylrhodamine (TMR) tag (20). Reactions of the crude protein extract with the probe were carried out as previously described (20). The resulting SDS-polyacrylamide gel was visualized using a Fujifilm FLA-5100 fluorescent bed scanner with excitation at 532 nm and a 570-nm emission filter. The same gel was silver stained to visualize all proteins in the samples.

Crude protein from cell-free filtrate of Pestalotiopsis microspora E2712A grown in 1-liter cultures of PUR-Lmin minimal medium and PDB rich medium were concentrated and purified to approximately 90% purity by gel filtration chromatography (data not shown) using a Superdex 200 column (GE Healthcare) in buffer comprised of 10 mM MES (morpholineethanesulfonic acid) (pH 5.5) and 50 mM NaCl. The ability of the purified protein to degrade PUR was assayed as described above.

RESULTS

Initial PUR clearance screen.Impranil DLN, a polyester polyurethane (PUR), is an opaque milky suspension that becomes transparent upon degradation. Organisms capable of degrading this polymer display a zone of clearance around the growing culture (5). A collection of 59 fungal endophytic organisms isolated from plant samples in the Ecuadorian Amazon were screened for their ability to grow on and degrade polyester polyurethane using the PUR halo assay as the initial screen. Of the organisms screened, 18 organisms produced a halo of clearance such as that shown in Fig. 1. Two other organisms, identified by ITS sequencing as Guignardia mangiferae (E2702C) and Zopfiella karachiensis(E2719A), could grow on but not degrade PUR-A medium. These were used as negative controls in the subsequent studies. The host plants, isolation media, and identities of the 18 active fungi and two inactive control fungi are listed in Table 1.

TABLE 1.

Endophytes studied and the host plant species from which the endophytes were isolated

Endophytea	Highest-homology organism (maximum % identity)b	Host plant	Isolation mediumc
E2524A	Alternaria sp. (100)	Annonaceae, Annona muricata	PDA
E2914A	Alternaria dauci (100)	Malvaceae, Malva alcea	1:10 PDA
E2910B	Bionectria sp. (99)	Gesneriaceae, Drymonia semicordata	1:10 PDA
E2705G	Plectosphaerella sp. (94)	Piperaceae, Piper arboretum	1:10 PDA
E3432O	Edenia gomezpompae (99)	Fabaceae, Erythrina smithiana	1:10 GAM
E2702C	Guignardia mangiferae (99)	Melastomataceae,Axinaea sodiroi	1:10 PDA
E2611A	Lasiodiplodia sp. (100)	Moraceae, Naucleopsis oblongifolia	WA
E2711A	Pestalotiopsis microspora (100)	Monimiaceae, Siparuna aspera	1:10 PDA
E2712A	Pestalotiopsis microspora (100)	Myrtaceae, Psidium guajava	1:10 PDA
E2708A	Pestalotiopsis microspora (100)	Mimosaeae, Calliandra angustifolia	1:10 PDA
E2911H	Pestalotiopsis microspora (100)	Commelinaceae,Dicrorisandra ulei	PDA
E3317B	Pestalotiopsis microspora (100)	Annonaceae, Annona muricata	WA
E3412F	Pestalotiopsis microspora (99)	Fabaceae, Lonchocarpus glabrescens	1:10 PDA
E3314A	Pestalotiopsis sp. (100)	Sterculiaceae, Guazuma ulmifolia	1:10 PDA
E2520A	Pestalotiopsis microspora (99)	Myrtaceae, Psidium acutangulum	PDA
E3432K	Phaeosphaeria sp. (95)	Fabaceae, Erythrina smithiana	PDA
E2104E	Nectria sp. (97)	Onagraceae, Fuschia hybrida	1:10 PDA
E2705B	Pleosporales sp. (99)	Piperaceae, Piper arboretum	1:10 GAM
E2812A	Pleosporales sp. (99)	Sterculiaceae,Theobroma kakau	WA
E2719A	Zopfiella karachiensis (99)	Myrtaceae, Psidium guajava	WA

?a Each distinct fungal isolate was given a unique identification number.
?b DNA sequence identity for each endophyte was determined by ITS-5.8s rDNA sequencing and comparison to sequences in the GenBank database.
?c PDA, potato dextrose agar; 1:10 PDA, 1:10 dilution of potato dextrose medium in water agar; 1:10 GAM, 1:10 dilution of glycerol arginine medium in water agar; WA, water agar.