1

FUNGAL DEGRADATION OF POLYMERIC MATERIALS: MORPHOLOGICAL ASPECTS

Luiza Jecu1, Elena Grosu

2, Iulia Raut

1 , Marius Ghiurea

1, Mariana Constantin

1, Anicuta Stoica

3 ,

Marta Stroescu3, Gelu Vasilescu

1

1 National Research and Development Institute for Chemistry and Petrochemistry-ICECHIM, Spl.

Independentei 202, Bucharest, Romania, jecu.luiza@icechim.ro

2 SC INCERPLAST SA, Str. Ziduri Mosi 23, Bucharest, Romania

3 University Politehnica of Bucharest, Str. Polizu 1, 011061, Bucharest, Romania

Abstract

Plastics materials are ones of the most popular materials and indispensable in the present world because

their flexibility, toughness, excellent barrier and physical properties and ease of fabrication. But the

accumulation of plastics in the environment becomes a matter of great concern leading to long-term

environment, economic and waste management problems. In order to overcome these problems,

significant attention has been placed on biodegradable polymers, and also, on the identification of

microorganisms with degradative potential upon polymeric materials. In present paper, the microbial

degradation of polymeric materials was carried on by incubating with Aspergillus niger strain recognized

for the ability to grow and degrade a broad range of substrates. Polymeric films have been prepared by

solution casting of different proportion between components, alcohol polyvinylic (PVA), poly(3-

hydroxybutyrate) (PHB) with natural polymers (starch, biocellulose). The methods used to assess

biodegradation of polymeric materials are visual observation, optic and Scanning Electron microscopic

(SEM) observations. Significant changes in surface aspect were observed in connection with chemical

constituents of the polymeric films, conditions of biodegradative process as regarding the organism and

medium composition. Growth of fungus is observed on film surfaces.

INTRODUCTION

Over recent decades, developments in polymer science have resulted in polymeric materials that are

durable, long-lasting, and resistant to environmental factors. On the other hand, plastic wastes represent a

serious concern for the environment because of its recalcitrance to microbial attack. Degradation of waste

plastics through microorganism use represents one of the alternatives to deal with such problems.

Microorganisms such as bacteria and fungi are involved in the degradation of both natural and synthetic

plastics (Gu J. D., 2003). Polymers especially plastics are potential substrates for heterotrophic

microorganisms.

Poly(vinyl alcohol) (PVA) is recognized as one of the very few vinyl polymers soluble in water also

susceptible of ultimate biodegradation in the presence of suitably acclimated microorganisms.

2

Accordingly, increasing attention is devoted to the preparation of environmentally compatible PVA-based

materials for a wide range of applications. PVA presents an excellent compatibility with several natural

biopolymers and blends are expected to have potential for use in packaging applications. PVA can be

used by some bacteria, Pseudomonas, Brevibacterium, Bacillus megaterium, Alcaligenes, as a carbon

and energy source. Also works were focused on polymer degradation by fungi, Fusarium, Aspergillus,

Phanerochaete chrysoporium and it is of interest to use the potential of fungal strains, because they are

versatile organisms able to grow and degrade a variety of compounds, organic contaminants, polymeric

materials (Chiellini et al., 2003).

This study investigated the ability of Aspergillus niger to grow and degrade several blends of PVA. The

paper examines the effects of the fungal attack on the polymeric surface by qualitative assessment of

films biodegradation performed with optic and the scanning electron microscopy (SEM).

MATERIALS AND METHOD

Preparation of composites films. The experiments were carried out with the following blends types:

a) PHB-PVA blends (wt/wt): 95% PHB and 5% PVA.

b) PVA-Biocellulose (wt/wt): I – 4%, PVA, 1.5 %, wet BC; II – 4%, PVA, 3%, wet BC; III - 4%, PVA,

5%, wet BC.

c) PVA-starch blends (wt/wt): I – 50%, PVA; 15%, starch; 35%, glycerol; II – 40%, PVA, 15%,

starch;45%, glycerol; III – 30%, PVA; 20%, starch; 50% glycerol; IV - 30%, PVA, 25%, starch;

45%, glycerol.

The films were cut into pieces of 2 cm x 2 cm and sterilized at UV light for 10 minutes. Each film was then

aseptically transferred and individually placed into sterile medium.

Microorganism The microorganism used for PVA composites biodegradation was Aspergillus niger , from

Microbial Collection of INCDCP-ICECHIM (Romania). The fungal cultures were maintained at 40C, in a tub

test with dextrose-agar-potato medium.

Cultivation conditions. In the liquid cultivation, three types of media were used (g/L): A - basal mineral

medium with 2.0 yeast extract (pH 6.0); B - Sabouraud ¼ diluted medium without glucose (2.5 peptone;

mineral salts; pH 6.0) and C - Sabouraud ¼ diluted medium (2.5 peptone; 10.0 glucose; mineral salts; pH

6.0). The basic mineral medium contains (g/L): 1.0 NH4NO3; 1.0 K2HPO4; 0.5 MgSO4 x 7 H2O; 0.5 KCl; 2.0

yeast extract; pH 6. The culture was carried out, during a month, on a rotary shaker at 200 rpm and 28 0C,

in 300 mL Erlenmayer flasks containing 50 mL of the medium. Fermentation with fungus was performed

with two flasks in parallel.

Characterization of films biodegradation

Optic microscopy. The fungal cultures in agitated flasks were observed using optic microscope Olympus

BX 51.

3

Scanning electronic microscopy (SEM). The observation of the film surfaces and fracture were

performed with scanning electron microscope FEI-QUANTA 200. The film samples were dried and placed

on a metallic support, aluminum standard stub. The samples were processed at 10-15kV and 50-120 Pa

using a Large-Field detector. After incubation with fungal cultures, the pieces of polymer were taken out

from the culture and repeatedly rinsed with distilled water, fungal mycelium being removed carefully. The

films were dried at 35 0C and use for evaluation of biodegradation efficacy. Micrographs of the samples

were taken at different magnifications to identify holes and other changes on the surface during the

degradation process.

Results and Discussions

The biodegradation of poly(vinyl alcohol) composites films was tested using Aspergillus niger. Fungi are

widely used in biodegradation studies due to their robust nature and for their great source of diverse

enzymes (Lowe, 1992). The specific elements of a fungal growth are observed in the most relevant SEM

micrographs of Aspergillus niger cultures on PVA composites. SEM is a significant and reliable tool to

measure the morphological changes of degraded polymer (Labuzek et al., 2004; Guohua et al., 2006)).

Biodegradation of PVA-PHB materials

The Fig 1 presents the liquid culture of Aspergillus niger on polymeric substrates. It can be showed the

fungal mycelium grown on surface polymer.

a)Fungal culture in agitated flasks

4

b) Unwashed polymeric sample after 10 days of fungal cultivation

c) Washed polymeric sample after 10 days of fungal cultivation

d) Unwashed polymeric sample after 30 days of fungal cultivation

e) Washed polymeric sample after 30 days of fungal cultivation

f) Unwashed polymeric sample after 60 days of fungal cultivation

g) Washed polymeric sample after 60 days of fungal cultivation

Fig. 1. Cultivation of Aspergillus niger in liquid medium on PVA-PHB composites

After 60 days of contact with fungal biomass, the mycelium adherent to polymer becomes dark-brown.

Also, the colour of polymer changes from white-yellow to yellow –intense.

In Fig 2. are presented the optic observations, at 10, 21, 30 and 60 days of culture in liquid medium in

agitated flasks..

a)10 days of cultivations b) 21 days of cultivation

5

c)30 days of cultivation d) 60 days oif cultivation

Fig.2. Optic observations of fungal cultures on PVA-PHB films

In Fig 2d it can be seen an aspergillar head with spheric conidia brown colored. After 60 days, the fungal

culture is aging, the nutrients in medium are epuised.

a)PVA-PHB film b) hyphae grown on surface after 7 days

d) Conidia after 7 days e) Hyphae network after 14 days

6

f) Conidia after 21 days g) Aging culture after 60 days

Fig. 3. SEM micrographs of Aspergillus niger culture grown on PVA-PHB films

The micrographs from Fig 3. presents the specific growth elements of a fungal culture, such as conidia,

fialments and hyphae.

Biodegradation of PVA-biocellulose materials

Microbial cellulose has proven to be a remarkably versatile biomaterial and can be used in wide variety of

applied scientific endeavors, such as paper products, electronics, acoustics, and biomedical devices

(Czaja W. K. et al., 2007; Tsuchida, T., Yoshinaga, F., 1997; Wan et al., 2006). Bacterial cellulose fibers in

combination with other biocompatible material like PVA are useful to produce biocompatible

nanocomposites suitable for medical device applications (Wan and Millon, 2005). So it is of interest to

study the microbial degradation of such composites. PVA has OH groups and can hydrogen bond with

cellulose. Also, its structure and solubility parameter is much closer to that of cellulose, suggesting that

greater miscibility may be attained in the PVA/biocellulose. The experimental results for PVA-BC

degradation are presented in Fig. 4.

a) 10 zile

b) 30 zile

7

a) 10 zile

b) 30 de zile

a) 10 zile

b) 30 de zile

Fig. 4. Visual observations of Aspergillus niger cultures on PVA-BC films (m.mineral_ye0,2% = mineral medium with 0.2 % yeast extract; m. Sab1/4-Glc = Sabouraud medium, without glucose diluted ¼; m.Sab1/4 = Sabouraud medium, with glucose diluted ¼)

As it can be shown in Fig 4., the polymer type III was almost disrupted by the attack of fungal strain in

reach nutrients medium like Sabouraud medium with glucose. The mineral medium with 0.2% yeast

extract offered a lower level of nutrients, so the strain was not able to degrade the polymer.

Biodegradation of PVA-starch composites

PVA’s rheological properties, particularly its ability to produce highly resistant films and its hydrophilic

character, account for the improvements in the mechanical properties and performances of natural

polymers when mixed with PVA. Nowadays, particular attention is devoted to the utilization of materials

from renewable resources, such as agricultural over-productions and by-products as well as waste

materials. Starch-based materials originally attracted a great deal of interest because of their low cost,

real biodegradability, and renewable origins. The fungus colonized the polymer samples within days of

inoculation. Electron microscopic examination (Fig. 5) showed that the hyphae of Aspergillus niger had

adhered to polymer surface or even penetrated the polymer matrix. The material shows clear crack

initiation points, indicating that the polymer has become brittle.

8

a) 1000x; holes on polymer surface

b) 1000x; fungal hyphae on polymer surface

c) 10000x; conidia and filament

d) 1000x ; fungal network

Fig. 5. SEM micrographs of PVA-starch films

Also, the microbial propagation has been initiated from these cracks. Such colonization and adhesion by

microorganisms are a fundamental prerequisite for biodegradation of the polymer. Penetration and

cavities were higher in correlation with composition of polymer and nutrients level in culture medium.

Microorganisms that colonize the polymer surface can probably adhere by means of extracellular

polymeric substances

Conclusions

The fungal strain, Aspergillus niger, was able, in adequate conditions, to change not only the polymer

surface from smoother to rougher, but even to disrupt the polymer. The results of the degradation were

demonstrated by visual observations and scanning electron microscopy (SEM) analyses. The degree of

microbial degradation depends on the culture medium and on composition of polymeric materials. The

biodegradation process is facilitated by the presence of glucose in the culture medium, an easily available

carbon source.

References

Chiellini E et al.., 2003, Biodegradation of poly (vinyl alcohol) based materials, Prog. Polym. Sci., 28, 963–

1014.

9

Czaja W. K. et al., 2007, The future prospects of microbial cellulose in biomedical applications,

Biomacromolec.8(1):1-12.

P Gilan (Orr) et al., 2004, Colonization, biofilm formation and biodegradation of polyethylene by a strain of

Rhodococcus ruber, Appl. Microbial. Cell Physiol., 65, 97-104.

Gu J. D., 2003, Microbiological deterioration and degradation of synthetic polymeric materials: recent

research advances. Int Biodeterior. Biodegrad., 52, 69–91.

Guohua Z. et al., 2006, Water resistance, mechanical properties and biodegradability of methylated-

cornstarch/polyvinyl alcohol) blend film, Polym. Degrad. Stab., 91, 703-7011.

Łabużek S. et al., 2004, The susceptibility of polyethylene modified with Bionolle to biodegradation by

filamentous fungi, Polish J. Environ. Studies, Vol. 13, 59-68

Lowe, D. A., 1992, In Handbook of Applied Mycology, Fungal Biotechnology, Marcel Dekker: New York,

681− 706.

Shah, A. A., et al., 2008, Biological degradation of plastics: A comprehensive review, Biotechnol. Adv., 26,

246–265.

Tsuchida, T., Yoshinaga, F., 1997, Production of bacterial cellulose by agitation culture systems, Pure &

Appl. Chem., Vol. 69, 2453-2458.

Wan, W. K., and Millon, L. E., 2005, Poly(vinyl alcohol)-bacterial cellulose nanocomposite. U.S. Pat.Appl.,

Publ. US 2005037082 A1, 16.

Wan, W. K. et al., 2006, Bacterial cellulose and its nanocomposites for biomedical applications, ACS

Symposium Series, 938, 221-241

Aknowledgements: This research was supported by PNCDI II project 32-115/2008, financed by

UEFISCDI Romania.