Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Summary

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

Abstract

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Introduction

Degradation has historically been considered an undesirable attribute of plastic polymers, because breakdown shortens product life span and durability. Recently, awareness of the environmental problems presented by plastic waste in the natural environment^1,2,3 has made biodegradable plastics an attractive alternative to conventional plastic materials. Degradation (defined as structural changes, fragmentation, and reduction in molecular weight, integrity, and strength^4,5) occurs via a series of events, including both abiotic processes (thermal stress, photo-oxidation, hydrolysis, erosion and mechanical stress), and biological degradation⁶

Protocol

This procedure requires at least several months for incubation of BDM films in soil, and several more months for sequential bioassays both on agar plates and in agar-free, chemically defined broth to assess colonization and degradation. Individual methods are listed in the order they will be performed.

1. Incubation of BDM Films in Soil

Incorporate BDM films into soil under conditions mimicking those under which they will be expected to degrade. Acquire 400 g (dry weight equivalent) of resident soil and sandwich a 10 cm x 10 cm BDM with the soil inside a 13 x 13 cm nylon mesh (250 micron) bag. Close with nylon thre....

Results

In a recent study²⁴, four replicates each of three commercially-available BDMs labeled "biodegradable", plus an experimental film and a conventional plastic control, were placed over soil as mulch for tomato production in the spring of 2010 at Mount Vernon, WA, Knoxville, TN, and Lubbock, TX. In the fall of 2010, BDM film squares were cut from each weathered mulch in four replicate plots, and native soil was removed from directly beneath the area where the mulch sample had been excised. Each weathered BDM squa.......

Discussion

The procedure described herein represents a first-pass technique for isolating potential BDM degraders from soil, and was successfully used to isolate fungi from BDMs buried in soil for seven months. Fungi grew when reinoculated onto fresh BDM material of the same type, indicating that the isolated fungi were indeed colonizers, and that the films were not inhibitory to fungal growth. Isolation of plastic-degrading fungi and bacteria potentially could lead to their use, individually or in combinations, for amendments to s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Potato Dextrose Agar	Becton Dickinson	8X05491
Agar	Fisher	BP 1423-2
Chloramphenicol	Acros Organics	200-287-4
Glutaraldehyde	Electon Microscopy Sciences	16216-10	Toxic
Molecular sieve	Fisher	M-8892
Ethanol	Pharmco-Aaper	E200
Contrex	Decon Labs, Inc.	5204
Parafilm M	Pechiney Plastic Packaging	S37440
Mineral salts for buffers and media	Fisher	Various	Various vendors sell these reagents