Full Paper
New Insight into Biodegradation of Polylactide (PLA)/Clay Nanocomposites Using Molecular Ecological Techniques Parveen Sangwan, Cameron Way, Dong-Yang Wu*
Novel molecular ecological techniques were used to study changes in microbial community structure and population during degradation of polylactide (PLA)/organically modified layered silicates (OMLS) nanocomposites. Cloned gene sequences belonging to members of the phyla Actinobacteria and Ascomycota comprized the most dominant groups of microorganisms during biodegradation of PLA/OMLS nanocomposites. Due to their numerical abundance, members of these microbial groups are likely to play an important role during biodegradation process. This paper presents new insights into the biodegradability of PLA/OMLS nanocomposites and highlights the importance of using novel molecular ecological techniques for in situ identification of new microorganisms involved in biodegradation of polymeric materials.
Polylactide (PLA) is an environmental friendly, economical and commercially available polymer that can be produced from renewable resources such as sugarcane, potatoes and corn.[1,2] It is widely used in various medical applications for controlled drug delivery, medical implants and absorbable sutures.[3] It is biocompatible and biodegradable, and offers great potential as disposable packaging material. However, further improvements in mechanical strength, thermal stability, gas permeability etc. are required to allow its use as a sustainable alternative to synthetic non-biodegradable plastics in a broader range of P. Sangwan, C. Way, D.-Y. Wu CSIRO Materials Science and Engineering, Gate 5, Normanby Rd, Clayton South, Victoria 3168, Australia Fax: (þ61) 3 9545 2829; E-mail:
[email protected] Macromol. Biosci. 2009, 9, 000–000 ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
applications in aerospace, automobiles and building construction. Development of polymer-layered silicate nanocomposites[4] has attracted significant research interests in recent years and it is considered as one of the effective means for improving properties of polymers including PLA. The concept was first explored by the Toyota group for preparation of nylon-layered silicates nanocomposites exhibiting significantly improved material properties.[5,6] Recent studies have reported that mechanical performance and the rate of biodegradation of PLA/organically modified layered silicates (OMLS) nanocomposites was significantly enhanced relative to neat PLA samples.[7–13] In these studies, assessment of polymer degradation was performed either by gravimetric analysis such as weight loss or respirometric measurement of total amount of CO2 evolved during the biodegradation process.[8,9,11] While these methods provide an indication of the biodegrad-
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DOI: 10.1002/mabi.200800276
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
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Introduction
P. Sangwan, C. Way, D.-Y. Wu
ability of polymers under specific set of conditions, they do not reveal any information about the diversity and population of microbial communities, which underpin the observed biodegradation performance. Microorganisms play vital roles in biodegradation and recycling of degradable polymers. Identification of the microbial communities involved in these processes and studying changes in their community composition and activity during biodegradation process is important for understanding the biodegradation mechanism. Although a few PLA-degrading microorganisms have been previously reported,[14,15] there is no information available relative to the PLA/OMLS nanocomposites. Considering the great potential of PLA nanocomposites in future commercial applications, identification of microbial species and/or the enzymes catalysing the biodegradation of these new materials could assist in selection of an efficient method for treatment or recycling at the end of their life cycle. Cultivation methods are traditionally used to identify and estimate the number of microorganisms in the environmental samples.[16–18] However, studies have shown that the majority of microorganisms have never been cultured under laboratory conditions,[19,20] which means that the results obtained from the traditional cultivation methods do not provide a true representation of the microbial communities in the environmental samples. In recent years, this limitation has been partially overcome by application of molecular ecological techniques to study microorganisms directly in their natural habitats. These novel techniques allow us to extract genetic information from microorganisms to confirm their identities without culturing them, and to relate the information on microbial diversity to ecosystem functions.[21,22] In one of our recent studies, the molecular techniques were successfully used to identify new PLAdegrading microorganisms belonging to the genera
Thermomonospora, Thermopolyspora and Paecilomyces.[23] The newly cultured species are currently being further investigated for their potential roles in degradation of other biodegradable polymers. This paper presents a first report on molecular investigation of biodegradation of three different PLA/ OMLS nanocomposites under aerobic composting conditions. Microbial community DNA was extracted from degraded neat PLA and three PLA/OMLS nanocomposite samples. Small-subunit rRNA gene sequences (16S rRNA and 18S rRNA) were amplified using polymerase chain reaction (PCR) and clone libraries were generated. The amplified cloned gene fragments were further sequenced and a comparative analysis was performed using basic local alignment search tool (BLAST) to compare these sequences to those available in the GenBank database. This yielded information on the identities of new gene sequences and provided an estimation of the genetic diversity in the examined samples. Parallel respirometric experiments were also set up to determine the rate of biodegradation (Australian Standard AS ISO 14855) for the neat PLA sample and the three PLA/OMLS nanocomposites.
Experimental Part Materials Polylactide 4032D (number–average molecular weight, Mn ¼ 1.1 105; weight–average molecular weight, Mw ¼ 1.7 105; polydispersity, Mw =Mn ¼ 1.6) from Natureworks was used in this study. It has a D content of 1.4 0.2% and a residual lactide content of less than 0.3%. Before compounding, the polylactic acid pellets were dried for 16 h at 70 8C in a hopper drier unit (SHD50A, CTS) connected to twin-bed dessicator (SD60, CTS). The three different kinds of OMLS used in this study were purchased from Sigma–Aldrich Co. The type of clays and organic modifier used for preparation of PLA/OMLS nanocomposites are summarized in Table 1. The OMLS powders
Table 1. Characteristics of the neat PLA and three PLA/OMLS nanocomposites analysed in this study.
Sample code
Layered silicate
Surfactant used for modification
Weight
CECa)
Mw T 10S3
M n T 10S3
g molS1
meq/100 g
g molS1
g molS1
Neat PLA PLA/ODA
Montmorillonite
Octadecyl-
Mw =M n
172
138
1.6
270
100
152
104
1.5
313
100
149
102
1.5
550
100
142
92
1.5
ammonium (ODA) PLA/DDA
Montmorillonite
Dimethyl dialkyl
PLA/TSA
Montmorillonite
Trimethyl stearyl
amine (DDA) ammonium (TSA) a)
Cation exchange capacity.
Macromol. Biosci. 2009, 9, 000–000 ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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DOI: 10.1002/mabi.200800276
Early View Publication; these are NOT the final page numbers, use DOI for citation !!
New Insight into Biodegradation of Polylactide (PLA)/Clay Nanocomposites . . .
were dried for 16 h at 105 8C in a convection oven with silica desiccant. The total organic carbon of the neat PLA and PLA/OMLS nanocomposites was determined using Leco CHN Analyser according to the Australian Standard AS1038.6.4 (HRL Technology Pty Ltd. Mulgrave, VIC, Australia).
Nanocomposites Preparation Polylactide pellets were dry mixed together with 4% w/w OMLS content and the mixture was melt extruded using a twin-screw extruder (Polylab 557-5052, Haake) operated at 180–200 8C (corotating high screw screws, L/D ratio ¼ 36, screw speed ¼ 200 rpm, material feed rate ¼ 45 g min1, vacuum port ¼ mid barrel). The extruded nanocomposites strands were air cooled, pelletized and then dried at 70 8C for 16 h in a convection oven with silica desiccant. Dried nanocomposites pellets were then pressed into 1 mm thick sheets with 1.5–1.8 MPa at 190 8C for 2 min. The moulded sheets were then press-cut into 1.2 1.2 0.1 cm3 film squares by pressing with 0.8–1.5 MPa at 120 8C for 40 s. Characteristics of the neat PLA and PLA/OMLS nanocomposites prepared in this study are summarized in Table 1.
Compost Sampling and Characterization Approximately, 2–3 months mature compost samples were collected from a commercial composting facility (Natural Recovery Systems, VIC, Australia) and transported to the lab within an hour of collection. The organic waste used in this facility consists of food-processing waste, supermarket produce wastes, sawdust and shavings, grass clippings, tree pruning, waste paper fibre and sewage sludge. In laboratory, compost was sieved through a sterile brass sieve with an 8-mm aperture size and any glass or stone pieces were manually removed. To determine dry weight of the compost, 25 g of fresh compost sample was weighed in an analytical balance and placed in a hot air oven at 105 8C for 3–5 d or until compost weight was consistent for two consecutive days. The conversion factor of fresh to dry weight for compost was calculated, and results were expressed per gram (dry weight) of compost. The pH of the compost was determined immediately after mixing compost in deionized water (ratio 1:5). Compost characteristics were: pH 8.2, dry weight 49% (Method 102, ANZECC), volatile solids 54% on dry basis (APHA 2540E), and C/N ratio 22 (APHA 5310B: total organic carbon and APHA 4500: total nitrogen).
before and after degradation. Narrow-polydispersity polystyrene standards were used for calibration and tetrahydrofuran (THF) was used as carrier solvent. All test samples were first dissolved in THF at 0.5% v/v and then filtered through 0.2 mm filters to remove clays particles. Solutions were then injected into GPC instrument at 1 ml min1 flow rate with columns and RI detector heated at 40 8C.
Transmission Electron Microscopy (TEM) A 70–90 nm sections of the samples were microtomed at 80– 100 8C using an Ultracut E microtome at a cutting speed of 0.05 mm s1. A Jeol 100S TEM was used at 100 keV to study dispersions of clay particles in the PLA matrix.
Assessment of Biodegradability by Respirometry (Measurement of Total CO2 Evolved During Aerobic Composting) The biodegradability of the neat PLA and the PLA/OMLS nanocomposites was determined according to Australian standard AS ISO 14855. The 3 L glass jars (bioreactors) were filled up with three different mixtures: blank (compost only), test (compost þ test polymer), and positive reference (compost þ cellulose), each in triplicate. These bioreactors were then placed inside an in-house built respirometer unit and temperature was maintained at 58 2 8C for 60 d. During this degradation period, compost moisture was maintained between 48 and 50% and pH 7.8–8.5 to ensure favourable conditions for compost microorganisms involved in biodegradation process. Aerobic conditions were maintained by continuous supply of sufficient airflow to the bioreactors and the contents of all bioreactor were shaken twice a week to ensure uniform distribution of air throughout the compost. An infrared CO2 analyser (Servomex) was used to measure the CO2 produced in each bioreactor and a pressure drop analyser (Alicate Scientific) was used to measure the flow rate of respired air. The CO2 and flow rate data were continually data-logged by computer for each respective bioreactor. The theoretical amount of CO2 produced by test and reference materials was assessed and the rate of biodegradation was calculated as described in the Australian Standard AS ISO 14855.
Molecular Studies Sampling
Weight Loss
Compost samples were collected from the surface (
