Environ. Sci. Technol. 2010, 44, 4729–4734
Purification and Conformational Analysis of a Key Exopolysaccharide Component of Mixed Culture Aerobic Sludge Granules THOMAS SEVIOUR,† BOGDAN C. DONOSE,‡ MAITE PIJUAN,† AND ZHIGUO YUAN* Advanced Water Management Centre (AWMC) and Australian National Fabrication Facility (QLD Node), Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia QLD 4072, Australia
Received February 2, 2010. Revised manuscript received May 2, 2010. Accepted May 7, 2010.
The application of aerobic sludge granules in wastewater treatment could increase the intensity of wastewater treatment processes because of their greater density and size relative to conventional sludge flocs. It has been suggested that granules are distinguished from flocs by gel forming exopolysaccharides. In this study, evidence is presented linking a specific exopolysaccharide component with granule extracellular polymeric substance (EPS) gelation. Granular EPS comprised three components: high-molecular-weight (MW) exopolysaccharide, medium-MW proteins and glycosides, and low-MW proteins and glycosides. The high-MW fraction was separated by fractional precipitation and preparatory-scale gel permeation chromatography (GPC). The MW profile of this fraction appears to be exclusively attributable to high-MW polysaccharide. The exopolysaccharide exists as a gel at normal wastewater treatment operating pH (i.e., 6.0-8.5), whereas the low/mediumMW material does not. Conformational analysis by atomic force microscopy (AFM) of the dried material showed that the polysaccharide forms pearl-necklace-like, intramolecularly condensed structures when dissolved in Milli-Q water and partially relaxed helical aggregates when in alkali solution. Consequently, the gel-forming property of EPS in the aerobic sludge granules tested is probably associated with high-MW polysaccharide components.
Introduction The application of aerobic granular sludge as an alternative to floccular sludge in secondary wastewater treatment is attractive because their size and density allows the process to be operated with increased biomass concentrations and faster solid-liquid separation (1, 2). Despite these attractions, aerobic sludge granulation technology is yet to be widely applied, which is partly due to an inadequate understanding of how granules form. An importance for extracellular polymeric substances (EPS) in granule formation has been considered (3, 4). * Corresponding author. E-mail:
[email protected]. Tel.: +61-7-33654730. Fax: +61-7-33654726. † Advanced Water Management Centre (AWMC), The University of Queensland. ‡ Australian Institute for Bioengineering and Nanotechnology, The University of Queensland. 10.1021/es100362b
2010 American Chemical Society
Published on Web 05/17/2010
Lemaire et al. (5) observed an extensive matrix of EPS in aerobic granules, within which a low density of bacterial cells was embedded, suggesting EPS acted as carrier material. EPS are also considered to play a key role in activated sludge biomass flocculation (6). Differences in the physical properties and compositions of floc and granule EPS have been investigated in attempts to explain their contrasting influence on aggregate macrostructures. Tay et al. (7) noted that granules were distinguished from flocs by an overproduction of polysaccharides. Greater EPS hydrophobicity is commonly cited as important in stabilizing cell to cell interactions and initiating granulation (3, 8). McSwain et al. (4) postulated that the stability of granules depended on the presence of a protein core, whereas Adav et al. (9) suggested their mechanical stability arose from an outer EPS layer with a polysaccharide backbone. However, to the best of the authors’ knowledge, no one has reported the successful isolation of a class of macromolecules from the EPS of either granules or flocs and studied its physical properties in order to confirm a structural role. Seviour et al. (10, 11) characterized granules as hydrogels and demonstrated that granule EPS form a strong gel in the neutral pH region, whereas floc EPS do not. They also suggested (11) that polysaccharide material was the major gelling component. If this polysaccharide could be isolated and its gel-forming behavior shown to resemble those of the granules and EPS from which it was derived, such an outcome would suggest it has a structural role. Fractional precipitation together with preparatory-scale gel permeation chromatography (GPC) was used here to separate the gel-forming polysaccharide fraction from EPS. Fractional precipitation separates polymers on the basis on differing solubilities (12, 13), whereas GPC separates them on their molecular weights (14-16). To show that the isolated polysaccharide displays the same pH-dependent sol-gel transition as the granules and EPS, we used mechanical spectroscopy and atomic force microscopy (AFM). AFM enables individual polysaccharides to be visualized and gelation to be studied in terms of polymeric conformational changes (17). Cowman et al. (18, 19) have used AFM to demonstrate that hyaluronan assumes intramolecularly condensed forms, probably from counterion mediated electrostatic interactions, which indicates an adhesive material. Intermittent contact AFM in air is therefore appropriate for the study of polysaccharide conformational changes. Evidence of exopolysaccharide association would suggest an adhesive role for it in granule formation. The aim of this study was to isolate a key exopolysaccharide component from aerobic granule EPS and undertake conformational analyses on it to demonstrate a functional relationship with granule macrostructure. We believe this is the first time such a relationship has been established between an EPS and macrostructure of a wastewater aggregate.
Materials and Methods Granular Sludge Used. The aerobic sludge granules used in this study were sampled from a lab-scale sequencing batch reactor (SBR) treating abattoir wastewater. The influent wastewater had an average chemical oxygen demand (COD), total nitrogen (N), and total phosphorus (P) concentrations of approximately 600, 230, and 35 mg/L, respectively. The reactor was operated as an enhanced biological phosphorus removal (EBPR) process under alternating anaerobic-aerobic conditions, with a cycle time of 8 h. Granules were harvested at the end of the aerobic period and were recovered from the VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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supernatant after filtration through a 0.5 mm laboratory test sieve (Endecotts Ltd., London). For more details of SBR operation, refer to the Supporting Information. EPS Extraction. The method described by Seviour et al. (11) was used to extract EPS. Granules were freeze-dried for 24 h (FTS Thermal Systems, NY, USA). Lyophilized products were suspended in Milli-Q water (40 g DS/L) and solubilized by a variation of the formaldehyde/NaOH method reported by Liu and Fang (20). NaOH (1 M) was added to a final concentration of 0.1 M NaOH followed by incubation at 4 °C for 2 h. The reaction mixture was centrifuged at 12 000 g for 15 min at 4 °C, and the soluble fraction collected. Analytical GPC. Sample Preparation. The soluble fractions of EPS were recovered and dialyzed (Molecular Weight CutOff 3500) against Milli-Q water for 48 h at 4 °C. Dialyzed EPS was freeze-dried. Then 2 mg was dissolved in 2 mL of 0.01 M NaOH to a final concentration of 1 g/L. This protocol was repeated for the lyophilized exopolysaccharide precipitate and all sodium polystyrene sulfonate MW standards (16 600, 57 500, 127 000, 505 100, and 1 180 000 Da) (Scientific Polymer Products Inc., Ontario, NY). Aliquots (200 µL) of each solution were then transferred through 0.22 µm filters (Millipore, MA) into GPC sample vials (Waters, MA). EPS Molecular Weight Distribution. EPS MW distribution was determined with an analytical GPC system comprising Waters 515 High Performance Liquid Chromatography (HPLC) pump and Waters 717 Autosampler (Waters, MA, USA). The system was fitted with an Asahipak GS-320 HQ column with 7.5 mm diameter and 300 mm bed length (Shodex, Kawasaki, Japan). NaOH (0.025 M) was selected as the eluent because granule EPS was soluble under alkali conditions. The eluent flow rate was 0.2 mL/min and the sample injection volume 20 µL. RI detection was achieved with a Waters 410 Differential Refractometer and UV detection with a Waters 2487 Dual λ Absorbance Detector maintained at 25 °C. UV and RI detection for each sample run was conducted on consecutive sample runs. Exopolysaccharide Purification. Fractional Precipitation. Seviour et al. (11) demonstrated that fractional precipitation with cetyl pyridinium chloride (CPC) and methanol produced a material with the pH dependent sol-gel transition characteristic of granular EPS. CPC and methanol precipitation was therefore selected here for fractional precipitation of the polysaccharide from granular EPS. Lipids were removed by chloroform/methanol washing (2:1, 1:1, 1:2 (v/v)), the granules freeze-dried and EPS extracted as described above. Soluble EPS was diluted with Milli-Q water (1:9) and neutralized with 1.0 M HCl. Proteins were precipitated by adding 70% perchloric acid to a final concentration of 5% (v/v), and removed by centrifugation for 15 min (12 000 g at 4 °C). CPC was then added to the retentate (0.1% w/v). After 3 h at 4 °C, the precipitate was recovered by centrifugation (12,000 g at 4 °C for 15 min), washed with 0.1% CPC solution, redissolved in 2.5 M NaCl solution and the insoluble component discarded. Methanol (85% v/v) was added to the solution, and after 24 h at 4 °C, the precipitant was recovered by centrifugation, dialyzed as above, and freeze-dried. Preparatory GPC. A preparatory-scale GPC with an injection volume of 200 µL was set up to recover sufficient polysaccharide for subsequent rheological measurements. This consisted of a Waters 501 HPLC Pump, Prep Scale Manual Injection Valve (Rheodyne), 8 mm internal diameter column with 300 mm bed length (Pharmacia) and Varian RI-4 Refractive Index Detector. The Pharmacia column was packed with Toyopearl Size Exclusion Media HW-65F. EPS precipitate was dissolved in 0.025 M NaOH and filtered. Eluent was pumped at a flow rate of 1 mL/min. The high MW fraction was collected between t ) 12 and 16 min and the medium MW fraction between t ) 18 and 22 min. Samples were recovered in specimen bottles (70 mL, sterile plastic) 4730
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FIGURE 1. Molecular weight (MW) distribution of EPS derived from aerobic sludge granules. This indicates that the EPS contains high-, medium-, and low-MW fractions. All fractions effect a change in RI signal, whereas only the low and medium fractions absorb in the UV region. The RI signal is indicated by gray and the UV signal by black lines. from 1 mm plastic tubing exiting the RI detector. They were dialyzed against Milli-Q for 48 h and then freeze-dried. EPS Conformational Analysis. Rheology. Five mg of each of the freeze-dried high and medium MW fractions from the preparatory-scale GPC were dissolved separately in 1 mL of 0.025 M NaOH. The pH of soluble EPS preparations was reduced incrementally to pH 8.5 by adding 1.0 M HCl. Rheological testing was performed with a Rheometrics ARES strain-controlled rheometer (Rheometric Scientific Inc., NJ). All tests were conducted using 25.0 mm diameter parallelplate geometry at room temperature. EPS preparations were subjected to small and reversible periodic oscillations of frequency (ω) with stress (σ), measured from the torque exerted through the upper plate, enabling the frequency dependence of G′, G′′, and η* to be determined. AFM. One milligram of the high-MW EPS fraction was dissolved in Milli-Q water, filtered (0.22 µm, Millipore, MA) and two sets 2.5 µg/mL solutions were then prepared using Milli-Q and 1 mM NaOH, respectively, as diluent. Aliquots (2 µL) of each were then dispensed to freshly cleaved mica disks (ProScitec, QLD, AU). Prior to imaging, the samples were allowed to dry in a force fan Secador (Williston, VT) desiccator cabinet for two hours. AC mode height imaging of dried fractions was achieved with an Asylum Research MFP-3D BIO AFM placed on a Herzan antivibration table within a TCM acoustic isolation enclosure (Herzan, CA). All data was acquired in air at 21 ( 0.5 °C and 50% relative humidity. Samples were scanned using Arrow probes (Nanoworld, Switzerland) with a nominal spring constant of 2.8 N/m, at 0.5 to 1 Hz, and less than 200 mV set amplitude points. During scans, drive amplitude, scan rate amplitude, and set points were altered so that trace and retrace overlapped.
Results EPS Molecular Weight Distribution. On the basis of the relationship between elution time and MW of the sodium polystyrene sulfonate standards (see Figure S1 in the Supporting Information for calibration curve), the MW distribution of the ultraviolet (UV) and refractive index (RI) positive constituents of the EPS derived from granules is given in Figure 1. When coupled with UV or RI detection, GPC can
FIGURE 2. Photographic images of (A) freeze-dried crude granular EPS and (B) freeze-dried EPS following CPC and methanol precipitation. (C) Molecular weight profile of EPS following purification by fractional precipitation. The RI signal is indicated by gray and the UV signal by black lines. be used to distinguish between proteins and polysaccharides in wastewater aggregates because proteins absorb UV and polysaccharides give a strong RI signal (14, 16). The granular EPS was resolved into three main fractions; low- and medium-MW component both giving RI and UV signals and a high MW fraction giving only an RI signal. In their studies on EPS derived from floccular sludge treating wastewater, Go¨rner et al. (16) detected seven peaks ranging from 670 to 45 kDa. Ni et al. (14) also detected seven peaks for floccular EPS, although only three were considered important quantitatively with three peaks attributed to polysaccharides and four to proteins. It is therefore possible that either EPS from granular sludge is less polydisperse from floccular sludge or that the resolution is not as great as in other chromatographic studies into EPS from wastewater aggregates. Although the standard curve (Figure S1 in the Supporting Information) indicates a high correlation between elution time of the SPS standards and MW (R2 ) 0.9929), accurate MW determination would require EPS specific standards (14). This investigation, however, is directed more at characterizing their distribution or chemical nature than accurate MW determination. Figure 1 clearly shows that there is a high-MW fraction in the granular EPS which seems to contain exclusively polysaccharide. Polysaccharides are the materials of interest in this study and the high-MW fraction was therefore targeted in subsequent purification steps. The low- and medium-MW fractions, on the other hand, contain both polysaccharide and protein components. EPS Purification. Fractional Precipitation. The mediumMW peak clearly dominates the MW profile of the granule EPS and engulfs the high-MW peak. On the basis of Figure 1, it would be difficult to isolate the high MW polysaccharide fraction directly from granular EPS using GPC. For their separation, fractional precipitation using CPC and ethanol was performed. As illustrated by Figure 2, this reduced the brownish EPS tinge (Figure 2A, B), although the resulting MW profile of the precipitate indicates that all three fractions are still present following precipitation (Figure 2C). Fractional precipitation alone is therefore not sufficient to isolate the high-MW fraction. However, relative intensity of the peak corresponding to the high-MW fraction increased substantially, suggesting that this step led to its enrichment, and the sharper resolution of the two peaks meant it was now feasible to apply GPC to isolate the high-MW polysaccharide fraction directly from the precipitate.
Preparatory-Scale Gel Permeation Chromatography. Although examples of EPS studies using GPC have been cited, GPC was not used in any of these to purify and characterize any of the observed fractions, possibly because the HP-GPC systems were applied at analytical rather than preparatory scale. Thus, GPC was scaled up using Toyopearl Size Exclusion Media HW-65F. A 200 µL injection volume of precipitate in alkali solution at a concentration of 10 mg dry weight/mL was delivered per run. Figure 3A shows that even increasing the amount of material applied to the column by a factor of 100, a clear separation between the two dominant fractions was achieved. No UV detector could be connected to the preparatory-scale GPC setup. Consequently the low MW (i.e., high elution time) fraction, which has a stronger UV than RI signal, was not seen. In addition to the two major peaks with elution times of 14.0 and 19.8 min, respectively, a peak eluting after 12 min was also seen, which is probably the nonretained or void fraction. Given the profile presented in Figure 3A, the eluted material was collected from the RI detector between 12.0 to 16.0 min (fraction 1) and 18.0 to 22.0 min (fraction 2). These fractions were then dialyzed, freeze-dried and analyzed by HP-GPC, with the resulting MW profiles from fractions 1 and 2 presented in panels C and D in Figure 3, respectively. Figure 3C shows that the high-MW fraction produces a single, symmetrical peak with no UV signal, indicating that no peptide bonds are present in it. Therefore, fractional precipitation followed by molecular distillation using preparatory-scale GPC appears to purify the high-MW polysaccharide fraction from granular EPS. Figure 3D on the other hand shows that Fraction 2 contains predominantly the medium- and low-MW fractions, with possible traces of the high MW as indicated by broadening of the peak in the highMW region. EPS Conformational Analysis. Rheology. To investigate a role for the high-MW polysaccharide fraction in the structure and stability of aerobic sludge granules, the high -MW polysaccharide fraction was dissolved in weak alkali solution and the pH lowered to bring it to within the gel forming pH region for granules and granular EPS reported by Seviour et al. (11). Under alkali conditions, the EPS existed as a solution. When pH was lowered, the high-MW polysaccharide fraction formed a gel, as indicated by the frequency dependence of G′, G′′, and η* at pH 8.5 (Figure 4). G′ was greater than G′′ across all frequencies by approximately 1 VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. (A) Gel permeation chromatogram of EPS precipitate from preparatory scale GPC. (B) Photographic image of freeze-dried high MW polysaccharide component recovered from preparatory-scale GPC Molecular weight profile of. (C) high-MW polysaccharide component and (D) low-MW glycoprotein fraction from HP-GPC. The RI signal is indicated by gray and the UV signal by black lines.
FIGURE 4. Frequency dependence of G′ (•), G′′ (O) and η (×) of high MW exopolysaccharide fraction following dissolution in 0.01 M NaOH solution and subsequent 1.0 M HCl addition to reduce to pH to 8.5. Test undertaken at T ) 22 °C and strain 0.1. order of magnitude and η* decreased linearly. This is typical of polymeric gels (21), indicating that the isolated polysaccharide is gel forming. On the other hand, no pH-dependent sol-gel transition was observed for the low or medium MW fraction. The viscosity of this fraction was too low to be measured accurately and the results are not included. Atomic Force Microscopy. An AFM micrograph of the 2.5 µg/mL solution of the high MW exopolysaccharide fraction following deposition on mica substrate is presented in Figure 5A. It shows that following drying, the mica surface was covered with irregular exopolysaccharide aggregates of varying length. Although these compounds appear heterogeneous, closer inspection (Figure 5B) reveals that each compound on the frame consists of subunits tacked together end on end similar to the pearl necklace structures previously 4732
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FIGURE 5. (A) AC mode atomic force microscopy height profile of purified exopolysaccharide (2.5 µg/mL) from aerobic sludge granules in milli Q water. (B) Amplification of the square section from A. (C) Relative section height profile of purified exopolysaccharide along the line from B.
FIGURE 6. (A) AC mode AFM height profile of purified exopolysaccharide (2.5 µg/mL) from aerobic sludge granules in 1 mM NaOH. (B) Amplification of the square section from A. (C) Relative section height profile of purified exopolysaccharide along the white line from A. reported in the case of hyaluronan deposited on mica (19). The height of each subunit is approximately 4 nm, and a decrease in height between the subunits is consistent with fusion points (Figure 5C). To observe the effect of alkaline conditions on the conformation of the isolated high-MW exopolysaccharide fraction, we diluted a filtered solution to 2.5 using 1 mM NaOH and deposited it on a mica substrate. The AFM image of the exopolysaccharide fraction in 1 mM NaOH solution following drying is presented in Figure 6. It illustrates the dissociative effect of NaOH. The exopolysaccharide subunits in Figure 5 appear to have come apart and are in stages of becoming unravelled. Exopolysaccharide chains are drawn out and appear as strands either 0.8 µm high or 0.4 µm high. Cross-sectional analysis of these strands in basic conditions shows an average diameter of 600 pm, in agreement with the theoretical molecular dimensions for exopolysaccharides (22) considering the flattening effect induced by the tapping AFM probe.
Discussion The main objective of this study was to link a particular polysaccharide component with the aggregate macrostructure. This involved separating the high-MW polysaccharide
fraction from other low-MW materials, and then demonstrating a functional relationship. Granular EPS was analyzed by GPC with UV and RI detection (Figure 1). The granular EPS MW profile clearly shows the existence of a high MW polysaccharide component. This was then amplified by fractional precipitation and purified further by preparatory scale GPC to transform the EPS from brown (Figure 2A), to off-white (Figure 2B) to white (Figure 3B). The MW profile of the high MW fraction from preparatory scale GPC shows a single, symmetrical peak with no UV signal indicating that combining fractional precipitation with GPC produced a high MW polysaccharide fraction apparently free of proteins. Although the MW profile is symmetrical and the peak welldefined, the peak spans a broad MW range and it is therefore not possible to attribute it to a single polysaccharide or to suggest that a single polysaccharide dominates. The question was then how to relate this polysaccharide component with structural function. To the best of the authors’ knowledge, no such correlation has been made for flocs and floccular EPS, possibly because of the nature of flocs. These are relatively small and tend to entangle and coagulate, thus making mechanical testing of individual flocs problematic. Granules are larger and more regular in shape, and so can be prised apart readily, and studied in isolation. Seviour et al. (11) were therefore able to use mechanical spectroscopy to demonstrate that the granular EPS had a reversible sol-gel transition at pH 9, analogous to individual granules (10). Treating granules as gels allows for a functional relationship to be established between the aggregate and EPS. In this study, the high-MW polysaccharide isolate was shown to express a pH-dependent sol-gel transition and exist as a gel at neutral and acidic pH (Figure 4). The low and medium MW EPS fractions on the other hand had neither a viscosifying or gel-forming effect at neutral and acidic pH. Additionally, AFM demonstrated that at neutral pH the high MW exopolysaccharide forms intramolecularly condensed aggregates, similar to the gel-forming hyaluronan, indicating a high degree of intramolecular electrostatic attraction (19). Under alkali conditions these agglomerates come apart to reveal individual exopolysaccharide strands in a partially relaxed helical conformation. Gelation of many exopolysaccharides is characterized by such a helical transition, as for example with gellan where gelation results in either endto-end helical associations or side-by-side helical alignment (23). Comblike branched (1f3)-β-D-glucans in fact form triple helices that may in some cases fold in on themselves to form a cyclic structure (24). Like these glucans it is indeed possible that the subunits observed in Figure 5 also result from multihelical associations and chain bending. The AFM micrographs therefore demonstrate that unlike the low- and medium-MW component, the isolated high MW exopolysaccharide fraction displays gel-like associative behavior under neutral and acidic pH conditions. This is consistent with the reversible sol-gel transition observed for the granules (11), confirming that the high-MW EPS fraction is derived from the structural matrix of the granules. It can therefore be concluded that the gel-forming constituent is high MW polysaccharide. In contrast, the low- and medium-MW fraction may have a nonstructural function, fixed to the gel as an energy or nutrition source (25) or to facilitate cell-cell communications (26). It may also be hydrophilic “filler”, residing in the polysaccharide gel to assimilate water, swell the network, and open up pores for nutrient diffusion in and out of the granules (27). As well as providing insight into the likely mechanisms of gelation, these AFM images inform the purification process, in particular the size fractionation and performance of prepscale GPC. From Figure 5A, only a single type of aggregate is present with no nonaggregating material, whereas in VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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soluble form, the exopolysaccharide appears to exist in different stages of association. This might explain the broad peak observed in the MW profile of the purified polysaccharide component (Figure 3C). GPC separates compounds on the principle of size exclusion. Eluent and gel selection are therefore obviously important for good separation. Dilute NaOH solution was chosen as eluent as the dissociative effect of alkali on aerobic sludge granules and floccular sludges has been reported (21). Molecules in different stages of association will interact differently with the gel, which will affect their retention times. As previously noted, it is not possible to attribute the high MW fraction to a single polysaccharide. However, even if it were a single exopolysaccharide, the topologies observed in Figure 6 indicate that the broad peak seen in the chromatographic profiles would still be expected under these conditions. Structural characterization of the exopolysaccharide will be the subject of future research and is necessary to propose and confirm an associative mechanism. This will determine if it is a single or multiple polysaccharide and may help identify which microbial population(s) is responsible for its production and understanding the metabolism and molecular biology of the synthesizing population(s). A high-MW polysaccharide component of mixed culture aerobic sludge granules was separated from the low and medium MW EPS fractions and shown to display gel-like associative behavior under neutral and acidic pH conditions, whereas the low- and medium-EPS fractions did not. The gel forming property of EPS in the aerobic sludge granules tested is therefore likely associated with high-MW polysaccharide compounds. Granules are characterized by the existence of a gel-forming EPS at neutral and acidic pH. The high-MW exopolysaccharide component is therefore postulated to be crucial to granule formation and structure.
Acknowledgments This work was funded by the Environmental Biotechnology Cooperative Research Centre (EBCRC) Pty Ltd, Australia. AFM work was completed at the Queensland Node of the Australian National Fabrication Facility. Thomas Seviour is an Australian Postgraduate Award and EBCRC Scholarship recipient. We recognize the contributions of Dr. Les Edye from the Sugar Research Institute, Queensland University of Technology, and Dr. Jennifer Waanders and Mr. Graham Kerven from the School of Land, Crop and Food Sciences, The University of Queensland, for their generosity in making available analytical and preparatory-scale chromatographic facilities used in this study, and their expertise. We also thank Prof Robert Seviour, La Trobe University, for helpful discussions and valuable input.
Supporting Information Available Calibration curve for the analytical GPC showing elution time of polymer MW standards.This material is available free of charge via the Internet at http://pubs.acs.org.
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ES100362B
