Name: isolation of native soil microorganisms with potential for breaking down biodegradable plastic mulch films used in agriculture
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Description: Watch this Scientific Journal Video about Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture at JoVE.com

Dr. Stephen Alderman, Dr. David Leaf, and Erin Macri are gratefully acknowledged for help with microscopy. This research was funded through a grant from the NIFA Specialty Crops Research Initiative, USDA SCRI-SREP Grant Award No. 2009-02484. Briana Kinash, Kevin Kinloch, Megan Leonhard Joseph McCollum, Maria McSharry and Nicole Sallee provided excellent technical assistance and thoughtful discussions.

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 wei...

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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...

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 environment1,2,3 has made biodegradable plastics an attractive alternative to conventional plastic materials. Degradation (defined as structural changes, fragmentation, and reduction in molecular weight, integrity, and strength4,5) occurs via a series of events, including both abiotic processes (thermal stress, photo-oxidation, hydrolysis, erosion and mechanical stress), and biological degradation6. While abiotic processes can change the fragment size and characteristics of plastics, microorganisms are required for their ultimate mineralization to water and carbon dioxide (in aerobic conditions) and/or methane (under anaerobic conditions).
A substantial niche for biodegradable plastics exists in agriculture, where plastic mulches are used to prevent weed growth, to retain soil moisture and to increase soil temperatures7,8 . Hundreds of thousands of acres in the United States alone are covered with plastic mulches9, including mulches composed of biodegradable plastic. Following a crop growing season, the options for disposing of biodegradable mulches (BDMs) include disposal in a landfill, incineration for energy recovery10, degradation via composting, or degradation in the soil after tillage11. Of these, the least labor-intensive fate is plowing BDMs into the soil, but without efficient degradation and mineralization during non-crop months (generally in the winter), plastic fragments could remain and interfere with agricultural equipment during spring tillage and planting, and persist in the environment where they significantly impact wildlife, plant life, and microbiota1,2,3,10.
Although many plastic products, including agricultural mulch films, bear the label "biodegradable" or "compostable", in practice, degradation and mineralization may be too inefficient and/or too incomplete for in-soil decomposition to be a viable alternative for disposal of these products. For example, oxo-biodegradable polyethylenes achieved only 12.4% mineralization after one year of weathering and three subsequent months in a 58 &deg;C compost, and less than half that amount of mineralization occurred when the compost temperature was 25 &deg;C12. In the winter, soil temperatures at most locations would be lower than either of these temperatures, presumably resulting in even lower microbial activity and consequently, less mineralization. In addition to slow degradation rates, misuse of the term "biodegradable" has led to distrust of these products by consumers13,14, including those in the agricultural industry. Biodegradation is the conversion of polymers to carbon dioxide (and/or methane) and water14 by naturally-occurring microorganisms4. Therefore, biodegradation must be measured chemically; the physical association of microorganisms with a substrate does not imply microbial degradation of that material.
As part of an effort to examine sustainable use of BDMs in agriculture, this study focused on discovering microorganisms native to agricultural soils that colonize and degrade commercially-available BDMs. Standard test methods have been published for chemically measuring the breakdown of biodegradable plastics by abiotic and biological means15,16,17. However, these methods do not address degradation of plastics by individual microbial species, or provide methods for their isolation. The methodology herein more closely resembles standard methods designed to evaluate plastics for resistance to microbial breakdown after inoculating specimens with fungal spores18,19.
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 zones13. This method has been used previously to identify microorganisms that degrade polymers such as polyurethane20, poly-(butylene succinate-co-adipate)21, and poly(lactic acid)22. A similar method involves suspending pure powdered plastic in liquid medium where the plastic is the sole carbon source20,23. While these methods have the advantage of a defined system, they poorly mimic in situ degradation of BDMs. First, the surface area is distributed differently because BDMs are not dispersed in small particles throughout the soil matrix, but rather, sold and used as films. Second, the chemical makeup of BDMs is different from pure polymers. BDMs generally contain additives such as fillers, plasticizers, and colorants, and these additives may affect microbial growth and thereby, the rate of mineralization. For this reason, and because the composition of certain commercial films in this study were proprietary, plastic film in its field-ready form was utilized to isolate fungi and bacteria. For simplicity, the methods below are described only for fungi, with modifications noted where appropriate for bacterial isolations.
In a recent study24, three commercially-available BDMs and one experimental film were used at agricultural sites in three different regions of the United States for one growing season, and subsequently placed in mesh (250 micron) bags and buried for one winter in soil at the same sites. The 250 micron mesh openings allow fungal hyphae to penetrate while excluding roots and most soil fauna, and minimizing soil encroachment25,26. Nylon materials prevent bag degradation in soil. Following excavation, fungal isolates were recovered from BDM pieces and assessed for growth on minimal medium without a source of carbon except for the agar and a 5 cm x 5 cm surface-disinfested square of new, unused BDM film that was pre-disinfested. Most plastics used as films cannot be autoclaved without loss of integrity, so UV light was used to kill any microbial cells residing on the plastics. ISO 84619 recommends surface-disinfesting in 70% ethanol and subsequent drying, but if using this method, one must ensure that no component or additive of the film is adversely affected by the ethanol. Since BDMs presumably are manufactured to withstand sunlight, UV was chosen as a decontamination method.
Isolates that grew on BDM pieces better than on minimal medium alone were selected for further study. Agar, a polysaccharide produced by marine algae, is used to solidify microbial media because it is typically not utilized metabolically by agriculturally and medically notable microorganisms; however, agar-hydrolyzing enzymes have been isolated from marine bacteria27 and agar-hydrolyzing bacteria also have been isolated from soil28. BDM polymers and agar are both expected to be rare substrates for enzymes secreted by soil fungi, which have not evolved in environments that contain these polymers as potential nutrient sources, but both substrates are present in the plate bioassay described herein (Step 7). Fungi that use BDMs but not agar as a carbon source can be differentiated from fungi that use agar only, by comparing growth on agar-solidified medium containing i) no added carbon source except agar (negative control), ii) BDM films (experimental) and iii) glucose (positive control). Growth of all isolates is expected on minimal medium plus glucose; fungi not arising on glucose-containing plates may not be capable of growth on the particular minimal medium used in the experiment. Potential BDM degraders should grow on agar-solidified minimal medium + BDM film better than they grow on agar-solidified minimal medium alone. Fungi growing on minimal medium plates are agar-degraders or oligotrophs, and are also expected to grow on the agar associated with BDM films in bioassay plates, but not on the films themselves (unless they serendipitously also degrade BDM polymers).
To eliminate the possibility of seeing microbial growth due to utilization of agar and not of BDMs, we followed our initial assay for BDM colonization on agar plates with a bioassay in defined broth medium (Step 9). BDM pieces represented the only known carbon source in the bioassay tubes.
After the initial screening, and upon reviving glycerol stocks of the isolates, some formed scant but visible mycelia in liquid defined medium containing no known carbon source. These results suggested that some of the acquired isolates were oligotrophs - organisms that grow by scavenging very small amounts of carbon, nitrogen, and other nutrients dissolved either in the aqueous environment or existing as volatiles in the air29,30,31. Species identification via 18S ribosomal DNA analysis supported this view, as many of the isolates matched fungal genera previously reported to exhibit oligotrophy32. Oligotrophs, which are commonly saprophytes, require a broad range of metabolic capabilities for substrate utilization in a range of environments30. Thus, it is not surprising that the same fungi we isolated from BDMs (presumably requiring unusual enzymatic capabilities) demonstrated oligotrophic capacities, and were able to grow on trace contaminants such as skin oils from fingerprints, dust, or trace volatiles in the air. Due to the isolation of oligotrophs, we concluded that growth on a BDM surface alone could not be used to infer BDM breakdown. The methods described herein reflect our efforts to screen native BDM colonizers from agricultural soils for bona fide BDM breakdown.

<table border="1">
<tbody>
 <tr>
 <td>Reagent Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Potato Dextrose Agar</td>
 <td>Becton Dickinson</td>
 <td>8X05491</td>
 <td></td>
 </tr>
 <tr>
 <td >Agar</td>
 <td>Fisher</td>
 <td>BP 1423-2</td>
 <td></td>
 </tr>
 <tr>
 <td >Chloramphenicol</td>
 <td>Acros Organics</td>
 <td>200-287-4</td>
 <td></td>
 </tr>
 <tr>
 <td >Glutaraldehyde</td>
 <td>Electon Microscopy Sciences</td>
 <td>16216-10</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td >Molecular sieve</td>
 <td>Fisher </td>
 <td>M-8892</td>
 <td></td>
 </tr>
 <tr>
 <td >Ethanol</td>
 <td>Pharmco-Aaper</td>
 <td>E200</td>
 <td></td>
 </tr>
 <tr>
 <td >Contrex</td>
 <td>Decon Labs, Inc.</td>
 <td>5204</td>
 <td></td>
 </tr>
 <tr>
 <td >Parafilm M</td>
 <td>Pechiney Plastic Packaging</td>
 <td>S37440</td>
 <td></td>
 </tr>
 <tr>
 <td >Mineral salts for buffers and media</td>
 <td>Fisher</td>
 <td>Various</td>
 <td>Various vendors sell these reagents</td>
 </tr>
 </tbody>
</table>

isolation of native soil microorganisms with potential for breaking down biodegradable plastic mulch films used in agriculture

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.

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...

Watch this Scientific Journal Video about Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture at JoVE.com

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

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.

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

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