J Polym Environ (2012) 20:1019–1026 DOI 10.1007/s10924-012-0501-y
ORIGINAL PAPER
Methodology to Assess Silicone (Bio)Degradation and its Effects on Microbial Diversity Baptiste Laubie • Aure´lie Ohannessian Vale´rie Desjardin • Patrick Germain
•
Published online: 11 July 2012 Springer Science+Business Media, LLC 2012
Abstract The behavior of silicone elastomers in landfills has not been well studied. Their impact on the environment is not known and, consequently, it has not been possible to establish robust Life Cycle Assessments of these materials. In the first part of this study, a methodology for assessing silicone degradation pathways is described. The chemical and biological parameters were considered separately. Firstly, parameters such as pH, redox potential and cation concentration were monitored and then degradation in aerobic and anaerobic conditions was investigated. Any impacts on microbial diversity were also taken into account, using bio-molecular tools. In the second part, a case study on the degradation of Room Temperature Vulcanizable silicone elastomers was performed to validate this methodology. The results indicate that condensation catalysts play a key role at their end-of-life, in both chemical and biological degradation. Moreover, these compounds have a significant effect on microbial communities (similarities with blank samples \5 %). As a consequence, the choice of catalyst should be carefully considered to assess any environmental impacts. B. Laubie A. Ohannessian V. Desjardin P. Germain INSA of Lyon Laboratoire de Ge´nie Civil et d’Inge´nierie Environnementale, Lab. (LGCIE), University of Lyon, 20 av. A. Einstein, 69621 Villeurbanne cedex, France
Keywords Polymer end-of-life Polymer degradation Silicone elastomers Silicone waste
Introduction Life Cycle Assessment (LCA) is an impressive tool that allows us to quantify the environmental impacts of the production, use and disposal of polymers, and their associated processes. To develop robust LCAs, data quality is a key factor, especially as regards the disposal degradation pathway. Concerning solid polymers, and especially silicone elastomers, only a few studies have provided information on their behavior at their end-of-life. In this paper, a specific strategy was designed to monitor the physico-chemical and biological degradation of silicone in the environment. The two aspects were studied separately: chemical degradation was specifically related to siliconbased polymer hydrolysis and biodegradation was monitored in aerobic and anaerobic conditions. The evolution of bacterial biodiversity was also taken into account. Moreover, the impacts of vulcanization catalysts were evaluated to determine whether they should to be integrated into the LCAs.
A. Ohannessian e-mail:
[email protected]
Methodology Presentation
V. Desjardin e-mail:
[email protected]
This section is central to our study. It has been compiled so that it can be easily understood by the majority of researchers, whether they are chemists or biologists.
P. Germain e-mail:
[email protected] B. Laubie (&) INSA Lyon, LGCIE, 9 rue de la Physique, 69621 Villeurbanne, France e-mail:
[email protected]
End-of-Life of Silicone Elastomers The disposal of solid silicone elastomers varies from one country to another, but in most cases they are landfilled or
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incinerated. Recycling is very rare for silicone polymers because they are dispersed in the waste and they are only used in small quantities in consumer products [1]. When they are incinerated, they are oxidized into carbon dioxide, silica and water. The environmental impact may be assessed as for carbon-based polymers. When these silicone materials are present in landfills, they may be degraded by chemical and/or biological processes. It is important to understand, and to highlight different parameters affecting end-of-life polymers in these environmental conditions. Chemical parameters that need to be monitored during landfill disposal include pH, redox potential, cation concentration etc. Biological parameters concerning landfill disposal focus on the biodegradation potential, either due to aerobic or anaerobic microorganisms. These factors all seem to be interconnected. It is important to be aware that biotic and abiotic conditions have a strong impact on the chemical environment. In fact, microbial activity can influence waste structure modifications by inducing chemical degradation. For example, an increase in the acidity of the medium can occur due to organic acids produced by microorganisms [2]. On the other hand, there could be an increase in pH due to a specific step during waste composting [3]. To investigate the effects of these parameters, the labscale methodology was divided into 2 parts (Fig. 1). First, the chemical degradation of polymers in leachates was simulated by tests, under controlled conditions, in aqueous media. The second part relates to the biological experiments using macroscopic (to assess the polymer biodegradability) and microscopic (to assess polymer influence on microbial diversity) tests. Classical tests from OECD (Organization for Economic Co-operation and Development) and ASTM (American Society for Testing and Materials) recommendations were adjusted for assessing the aerobic and anaerobic potential biodegradation of Fig. 1 Scheme of the global methodology designed to monitor bio-chemical degradation during the end-of-life stage of polymers
various polymers. Each part of the investigation is complementary and contributes to an evaluation of the behavior of polymers during their disposal. Polymer Chemical Degradation Monitoring There is no standardized test for monitoring the chemical impact of polymer degradation at the end of their lives. Batch experiments were performed in aqueous phases to simulate the conditions of landfill leachates. In this way, synthetic conditions were established regarding the pH, redox potential and cation concentration. Leaching tests with real landfill leachates were more realistic, but many anomalies in the analysis can occur in a complex medium. Chemical tests consist of adding polymers, in aqueous solution, over different time periods. To accelerate the degradation rates, materials were cut into 3 mm length pieces (to increase the contact area) and samples were rotated. At different time points, samples were analyzed to monitor the release of degradation indicators into the aqueous phase. The crucial factor is the choice of indicators used to assess the level of polymer degradation. It depends on the nature of the polymer and its crosslinking composition. Concerning the most current polymers (with a carbon backbone), their degradation can be monitored by TOC (Total Organic Carbon) analysis of the solution. With this methodology, it is also possible to monitor the release of polymer fillers and polymerization catalysts. In the case of silicone elastomers, it was decided to monitor Silicon (Si) concentration in the aqueous phase by ICP-OES (Inductively Coupled Plasma—Optical Emission Spectroscopy). This indicator gives the rate of silicone network hydrolysis. It was also decided to monitor the release of catalysts because these chemical compounds could have very harmful impacts on the environment.
Polymer Chemical degradation pH, red/ox, landfill leachate …
Biological degradation Aerobic sludge
Leaching
Leaching Anaerobic sludge
Time [Degradation indicator]
Respiration
Release monitoring
Consortia activity Biogas production
[Filler or Catalyst]
Ecotoxicology
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Microbial diversity
DGGE
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Polymer Biodegradation Monitoring Aerobic Biodegradation When polymers are landfilled, the presence of O2 induces an initial aerobic degradation stage. It is, therefore, necessary to know the aerobic biodegradability potential of the materials studied during this step. Aerobic tests were conducted following the OECD 301F guidelines [4]. An OxiTop respirometry manometric system (WTW Ltd) was used to measure oxygen consumption. Microorganisms use oxygen from the atmosphere of the closed system to degrade organic substances and the carbon dioxide formed is absorbed by a NaOH trap. Due to the reduction in the amount of oxygen, the pressure in the bottle decreases and the pressure change is continuously recorded by a measuring head (a microprocessor vacuum manometer). This device is illustrated in Fig. 2a. A measure of biodegradability is provided by the ratio BOD (Biochemical Oxygen Demand) over COD (Chemical Oxygen Demand). BOD is directly correlated with oxygen consumption and represents the amount of oxygen needed by microorganisms to break down bioavailable organic matter. COD measurements give the total amount of organic compounds. Anaerobic Biodegradation At landfill sites, polymers are in a biotic environment mainly composed of anaerobic bacteria (when the oxygen concentration is decreasing). A whole sequence of various microorganisms is required to degrade the organic matter present into waste. Hydrolyzing microorganisms begin the reaction and then acidogenic bacteria convert the organic substances into lower molecular metabolites, such as alcohols and short-chain fatty acids. Acetogenic bacteria
Fig. 2 Experimental apparatus to assess aerobic (a) and anaerobic (b) biodegradation
degrade these substances into acetate, carbon dioxide, and molecular hydrogen. In the final phase, the action of methanogenic bacteria results in methane and carbon dioxide formation. This gas is called biogas. Volume production (manometric measurements) and the composition of biogas (gas chromatography analysis) can be used as indicators of the anaerobic biodegradation of polymers. The classical test used is called Biochemical Methane Potential (BMP) and it is performed according to USEAP, OPPTS 835.5154 [5]. This assay provides a measure of the anaerobic digestibility of a given substrate. This relatively inexpensive and reproducible method enables one to make comparisons of anaerobic degradability (by biogas production) between various polymers or other materials. The apparatus is described in Fig. 2b. At the beginning of the experiment, BMP bottles are flushed with nitrogen to ensure that anoxic conditions are established. Effect of Polymers on Microbial Diversity As has been previously stated, biotic conditions are very important in assessing the end-of-life stage. Even if silicones are not biodegraded, it is crucial to investigate whether these materials, and their fillers, have an impact on microbial diversity. A change in the microbial community could signify a disturbance in the biological equilibrium due to the presence of polymers. Understanding the diversity of microbial communities is hampered by the problems of easily identifying microorganisms. Any classification, based on physiological or biochemical parameters, is rarely possible because 99 % of microorganisms cannot be cultivated in the laboratory [6]. Nowadays, molecular tools enable microorganisms to be identified by comparing the sequence analysis of their Small SubUnit Ribosomal DNA (SSU rDNA) [7]. These techniques allow researchers to detect, and enumerate,
(b)
(a)
Manometer (pressure increase)
Mesuring head = microprocessor vacuum manometer (pressure decrease)
CO2 trap (NaOH) O2
Septum
Biogaz composition analysis
Polymer + inoculum + minimal medium CO2
N2
CO2 / CH 4
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microorganisms within their natural ecosytems (e.g. in compost, in sludge or in leachates). Denaturing Gradient Gel Electrophoresis (DGGE) is one of these techniques. It is a genetic fingerprinting method which provides a pattern of the microbial diversity in a sample, based on the physico-chemical separation of nucleic acid molecules. Copies of targeted genomic DNA sequences are separated in a polyacrylamide gel containing a linear gradient of DNA denaturants. Sequence variation, among the different DNA molecules, influences their melting behavior and, hence, molecules with different sequences stop migrating at different positions in the gel. Each microbial species corresponds to one particular band [8]. Figure 3 summarizes the whole experiment: the first step consists of DNA extraction from the biological matrix (aerobic or anaerobic sludge), followed by the amplification of the 16S rDNA portion by Polymerase Chain Reaction (PCR). The second step consists of denaturing gradient gel electrophoresis to analyse microbial diversity according to the addition of different polymers. The methodology presented here enables us to assess the behavior of silicone polymers during their disposal. It may lead to an explanation of how they are degraded and, also, how they affect microbial diversity. The following is a detailed example, using RTV (Room Temperature Vulcanizable) silicone elastomers, to illustrate this strategy and to evaluate its efficiency.
Aerobic or anaerobic conditions (activated or anaerobic sludges)
Biological tests with polymers (and blank sample without polymers)
DNA extraction
DNA amplification using PCR
PCR products separation by Denaturing Gradient Gel Electrophoresis (DGGE)
Case Study The aim of this section is to give examples of the results obtained using the methodology described, and also to present a comprehensive study report. Only the most relevant results are provided (for instance, anaerobic degradation is not detailed). RTV silicone elastomers were chosen to validate this strategy because: •
•
•
They are widely used (260 millions tons in 2008 [9]), and this amount is rapidly increasing, but no information is available, in the scientific literature, about their end-of-life. They are considered as inert material in landfill [1]. They are thought to cause problems, in biogas valorization in landfill, as a result of their degradation (presence of very abrasive silica deposits in powergeneration engines by oxidation of volatile degradation products). This reaction significantly increases the cost of this renewable energy source [10, 11]. A European directive requires that, as from 2014, RTV silicone products contain less than 0.1 % of standard catalysts (dibutyltin compounds). New families of catalysts are being developed, so this is a good opportunity to compare the environmental behavior of these new elastomers with the previous ones.
In the following section, examples of chemical degradation of the silicone matrix and the release of catalysts are shown, as well as aerobic biodegradation of elastomers. Finally, the effect of the catalysts on bacterial diversity is presented. Materials and Methods Elastomer Synthesis Three elastomers were synthesized with 3 different catalysts, but with the same silicone matrix: 86 % of a,xdihydroxypolydimethylsiloxane oil (20 Pa.s at 25 C), 3 % of vinyltrimethoxysilane (crosslinking agent), 10 % of fumed silica (filler) and around 1 % of catalyst [0.92 % of catalyst 1: dibutyltin dilaurate (Acima Speciality Chemicals), the original catalyst; 0.32 % of catalyst 2: a tetrasubstituted guanidine; and 1.13 % of catalyst 3: a bis (b-diketonate) zinc complex]. The mixtures were stirred for 60 s at 2,000 rpm (SpeedMixerTM DAC 150 FV) and the 3 elastomers, each 1 cm thick, were vulcanized at room temperature for 1 month. Chemical Degradation
Fig. 3 Experimental steps to study the influence of polymers on microbial diversity by Polymerase Chain Reaction—Denaturing Gradient Gel Electrophoresis (PCR-DGGE)
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Elastomer cubes, with 3 mm length sides (9 mm2 9 6), were placed in different aqueous media (50 g L-1) in
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100 mL high-density polyethylene bottles. Samples were rotated at 8 rpm, at room temperature, for 25 days. All the experiments were performed in triplicate. The aqueous media in contact with the elastomers were demineralized water (pH = 5.3; 18 MX cm-1, Veolia water STI), acidic solution (pH = 2 with HCl; Chimie-Plus Laboratories, 37 %), basic solution (pH = 12 with NaOH, Chimie-Plus laboratories, 98 %) and a landfill leachate (Rhoˆne-Alpes, France) filtered at 0.45 lm (cellulose acetate filter, Sartorius Stedim Biotech) (pH = 8.58, COD = 1.451 mgO2 L-1, TOC = 375 mg L-1). Si, Sn and other metal concentrations were analyzed in the aqueous phase by Inductively Coupled Plasma—Optical Emission Spectroscopy (ICP-OES) (Jobin–Yvon Horiba Ultima II) at the most sensitive emission line (Si: 251.611, Sn: 189.989). The level of measurement uncertainty was less than 5 %. Organic molecules were analyzed by UV absorption at their characteristic wavelength. Biological Degradation Aerobic tests were performed with OxiTop systems (WTW Ltd) at 20 C. Elastomers (1.5 g) or pure catalysts (112.0; 74.7; 37.3 mgCOD) were added to 88 mL of demineralized water, 2 mL of activated sludge (Rhoˆne-Alpes, France) and 10 mL of minimal medium (KH2PO4 = 2.83 g L-1, K2HPO4 = 14.61 g L-1, CaCl2, 2H2O = 0.27 g L-1, NH4Cl = 2.15 g L-1, MgSO4, 7H2O = 0.23 g L-1, FeSO4, 7H2O = 0.053 g L-1, ZnSO4, 7H2O = 0.03 g L-1). Two blank samples were included to assess the endogenous activity of the sludge (without any substrate) and the sludge activity (112 mgCOD of glucose). All experiments were performed in triplicate. To assess microbial sludge diversity, total DNA was extracted with a PowerSoil DNA Isolation kit (MoBio), according to the manufacturer’s protocol. The V3 region of
(a)
Catalyst 1
Chemical Degradation Results Leaching tests were performed in different aqueous solutions (3 different pHs: 2, 5.3 and 12) and in a landfill leachate. Degradation of 2 silicone elastomers (prepared with catalysts 1 and 2) was monitored by the release of Si into the aqueous phase (analyzed with ICP-OES). The release of catalysts was also studied by measuring metal concentrations in the medium (Sn for catalyst 1) and by UV detection for catalyst 2. The results are given in Fig. 4. The silicone elastomer prepared with catalyst 1 was affected much more by chemical degradation than the
(b)
Catalyst 2
300 250
[Catalyst] (mmol/L)
[Si] release (mg/L)
the 16S rDNA gene was amplified, by Polymerase Chain Reaction (PCR), with primers E334F-GC and E534R [12] using a JumpStartTM Taq Ready MixTM (Sigma-Aldrich) and a MJ MiniTM Personal Thermal Cycler (Bio-Rad Laboratories). The amplification program was 94 C for 5 min; 10 cycles of 94 C for 30 s, 65 C to 56 C for 30 s, 15 cycles of 92 C for 30 s, 55 C for 30 s, 72 C for 1 min and, finally, 72 C for 10 min. Then, PCR products were quantified using the Quanti-TM PicoGreen reagent (Invitrogen). DGGE was carried out using a Dcode Universal Mutation Detection System (Bio-Rad Laboratories). 450 ng of DNA PCR products migrated in a polyacrylamide gradient gel (8 % acrylamide–bisacrylamide 37.5:1, 60–30 % urea/formamide denaturant) for 16 h, at 75 V, in 19 TAE buffer (40 mM Tris–HCl, 20 mM acetic acid, 1 mM EDTA, pH 8.0) at 60 C. Visualization was carried out with a SybrGreen (Sigma-Aldrich) nucleic stain bath. DGGE gels were analyzed using QuantityOne gel analysis software (Bio-Rad Laboratories). Species identification was performed with an Applied Biosystems 3730xl DNA analyzer after band excision and DNA amplification. Dendrograms were created with the algorithm of Weighted Pair-Group Method, using Arithmetic averages (WPGMA).
200 150 100 50 0
Catalyst 1
Catalyst 2
0.4
0.3
0.2
0.1
0.0 Demineralized water (pH=6)
pH=2
pH=12
Landfill leachate
Fig. 4 Chemical degradation, after 25 days, of silicone elastomers synthesized with catalysts 1 and 2 in demineralized water, acidic, basic, and landfill leachate conditions: a silicone matrix degradation
Demineralized water (pH=6)
pH=2
pH=12
assessed by monitoring Si concentrations in aqueous phases; b release of catalysts in aqueous phases
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Glucose
Catalyst 1
Catalyst 2
(no chemical or physical bonds). Even if these two catalysts have the same properties regarding silicone elastomer polymerization, they have a completely distinct behavior during polymer end-of-life.
Catalyst 3
Oxygen consumption (mg)
80 70 60 50
Biological Degradation Results
40 30 20 10 0 0
5
10
15
20
25
30
Time (d)
Fig. 5 Oxygen consumption by aerobic microorganisms in the presence of 3 silicone elastomers, synthesized with catalysts 1, 2 and 3, with glucose but without any substrate (endogenous activity). The uncertainty of measurement is estimated from triplicates
elastomer prepared with catalyst 2 ([Si] [200 mg L-1 vs [Si] \15 mg L-1). Leaching tests with landfill leachates show the same trend. The choice of catalyst significantly modifies the network resistance and, consequently, influences the chemical degradation rate of the polymer. However, as regards catalyst concentrations, our findings are completely the opposite. In all conditions, the release of catalyst 2 was very high in the aqueous phase (80 % in acidic conditions, for example), indicating that this molecule is not strongly trapped by the silicone matrix
(a)
Catalyst 1
Catalyst 2
Aerobic biological degradation results from tests on silicone elastomers, synthesized using 3 different catalysts (catalysts 1, 2 and 3), are given in Fig. 5. Blank samples indicate that the starved sludge was active during the experiment (endogenous activity near to zero and glucose biodegradation at around 90 %). These results show that oxygen consumption was very low for elastomers, compared to the COD introduced (\1 %). The silicone matrix was, therefore, not directly biodegraded by microorganisms. This could be correlated with Watts’, Smith’s and Ohannessian’s experiments [11, 13, 14], which concluded that silicone polymeric chains are not used as carbon sources by aerobic consortia. However, Fig. 5 shows that the catalyst has an impact on aerobic degradation (oxygen consumption with elastomers 1 and 3 was higher than 10 mg O2, and with elastomer 2 it was similar to the endogenous activity), even if this compound is trapped within the solid matrix. This result was confirmed by degradation in the same conditions with pure catalysts: catalysts 1 and 3 are often used as the single carbon source (biodegradation rate higher than 40 %). It seems important,
Catalyst 3
(b) 0.05
Endogenous activity
Glucose
0.12 0.18
0.33 0.40
0.40
0.54 0.60
0.84 0.92
[catalyst 1]
[catalyst 2]
[catalyst 3]
3
4
Catalyst1
5
9
10
Catalyst3
11
6
7
Catalyst2
8
1
2
Blank samples
Fig. 6 Assessment of the impact of pure catalysts (at 3 different concentrations) on microbial diversity, in aerobic conditions, by DGGE: a DGGE patterns; b cluster analysis of the DGGE profile using the WPGMA algorithm
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Table 1 GenBank-EMBL accession number and identification for the 32 bands sequenced (marked with an asterisk on Fig. 6a) Lane
GenBank number
Identification
1
CP002542.1
Fluviicola taffensis
AB072406.1
Flavobacterium aquatile
AF430123.1
Pseudomonas sp.
JN033360.1
Pseudomonas sp.
JF813171.1
Pseudomonas sp.
HQ848376.1
Pseudomonas putida
HQ663922.1 AF430121.1
Pseudomonas anguilliseptica Pseudomonas sp.
FR682714.1
Lysobacter sp.
HQ848376.1
Pseudomonas putida
JF813171.1
Pseudomonas sp.
3
4
5 6
7
8 9
11
HM629401.1
Pseudomonas sp.
JF901149.1
Bacterium
HM629401.1
Pseudomonas sp.
GU255472.1
Acidovorax sp.
JF901149.1
Bacterium
CP002870.1
Pseudomonas putida
EU273858.1
Flexibacter sp.
GU255472.1
Acidovorax sp.
FJ950635.1
Pseudomonas putida
HM629401.1
Pseudomonas sp.
CP002870.1 HM629401.1
Pseudomonas putida Pseudomonas sp.
JF701966.1
Cupriavidus sp.
JF701966.1
Cupriavidus sp.
JF701966.1
Cupriavidus sp.
HM629401.1
Pseudomonas sp.
EF581816.1
Pseudomonas sp.
CP002870.1
Pseudomonas putida
CP002870.1
Pseudomonas putida
HM629401.1
Pseudomonas sp.
HM598288.1
Bacterium
therefore, to check whether these chemical compounds have an effect on microbial communities. Figure 6 shows the DGGE gel and dendrogram of aerobic sludge samples in contact with the three catalysts studied, at three different concentrations. Endogenous activity and the sludge sample, supplemented with glucose, are shown in lanes 2 and 1, respectively. The gel clearly demonstrates that catalysts have a strong impact on the sludge microbial community: different major bands from the endogenous activity are present, especially for catalyst 2 (lanes 6, 7, 8). For catalyst 3 (lanes 9, 10, 11), 7 species are predominant. These results are confirmed by the dendrogram: the original sludge (1) has only 5 % similarity with the catalyst samples (and 40 % with the glucose sample). For every catalyst, the lower concentration samples (5, 8,
11) are closer to the blank samples (1, 2) than the 2 higher concentrations. This seems consistent with the idea that microorganisms are less disturbed when the xenobiotic is less concentrated. However, for catalyst 2, a small increase results in a big change in diversity (there was only 54 % of similarity between the highest (6) and the intermediate (7) concentrations). Table 1 summarizes the bands sequencing results (names and Genbank accession numbers). The predominant species are from the Proteobacteria phylum (principally from the Pseudomonas genus), but species of the Bacteroidetes phylum are well represented in the catalyst 2 samples. Conclusions The methodology presented here is a good way to assess the degradation of silicone compounds at their end-of-life, on a chemical and biological scale. First, the extent of chemical resistance is shown by degradation batch tests, which simulate real landfill conditions. Secondly, BMP and OxiTop assays provide information on the biodegradability potential of the polymers studied. Finally, molecular biology is essential in assessing the material impact on microbial communities. Moreover, this approach has proved that catalysts play a significant role as regards the end-of-life behavior, affecting both the chemical and the biological degradation. As a consequence, these data should be taken into account in any LCA involving silicone elastomers. It would be interesting to apply more often the DGGE results (or other similar molecular fingerprints) to LCAs to evaluate the environmental impact of polymers on biodiversity. This methodology could be adapted to study the degradation of other polymers and biopolymers, and their fillers and catalysts, before carrying out LCAs. Acknowledgments The authors acknowledge the analytical department of the LGCIE for contributions to this study. The authors would also like to thank the Bluestar Silicones Company, which supplied the elastomers and Dr. Valerie James for proofreading this article.
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