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Application of Polyhydroxyalkanoate Granules for Sizing of Paper Robert Bourbonnais and Robert H. Marchessault* Department of Chemistry, McGill University, 3420 University Street, Montreal, QC, H3A2A7 Canada Received December 23, 2009; Revised Manuscript Received February 3, 2010
Polyhydroxyalkanoates (PHAs) are characterized by the chemistry of the biodegradable inclusions inside the microbial membrane. They are produced by a wide variety of bacteria, where they function as energy and carbon storage materials. This intracellular Bioplastic forms a stable latex suitable for surface treatments of paper such as sizing and coating. In this work, we compare native granules and artificial granules made from market poly(3hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-hydroxyvalerate), P(3HB-co-3HV), for their ability as sizing agent. Paper sizing was assayed by measuring the resistance of sized paper to penetration by aqueous fluids. Our results indicate that the sizing effect of PHAs is dependent on several factors, such as, paper drying temperature, drying time, pressure, and polymer composition, that is, homopolymer, random copolymer, and texture of granules. The sizing efficiency of the copolymer is generally poor compared to the PHB homopolymer. In addition to water permeability, the tensile strength of sized paper was measured and physical properties of granule suspensions were recorded using SEM microscopy, X-ray diffraction, and dynamic light scattering.
Introduction Most grades of paper products designed for use in printing, writing, and packaging benefit from the use of sizing agents. The usual objective of sizing is to impart aqueous liquid resistance to the cellulose product end-use. In addition to the most commonly used sizing agents, rosin and starch, a variety of water-dispersible synthetic resin polymers are also used as sizing agents. A biodegradable polyester, polyhydroxyalkanoates (PHAs), has been proposed to produce self -supporting films and coated papers.1,2 PHAs are a class of hydrophobic stereoregular biopolyesters produced by many bacteria in the form of submicrometer inclusions that serve as energy and carbon storage materials. They fit the aspiration of environmentalists for bioplastics that return to nature through composting. This renewable Bioplastic was shown to occur as a stable aqueous suspension, latex, suitable for surface treatments of paper such as sizing and coating.3 Poly(3-hydroxybutyrate), PHB, was the first of the PHAs to be discovered and is the most common in nature.4,5 Many other PHAs were produced by bacterial fermentation including a random copolyester of R-3-hydroxybutyrate and R-3-hydroxyvalerate, P(3HB-co-3HV).6 They are produced industrially by batch fermentation techniques that first involve exponential cell multiplication, and in a second stage, the exhaustion of a limiting nutrient other than carbon causes accumulation of PHA in bacterial cells, typically up to 75% of the bacterial dry weight.7 Following chemical and enzymatic treatments, they can be isolated from bacterial cells as a stable latex suspension.8 Native PHA granules in vivo are amorphous and stabilized by a protein/ phospholipid coating that prevents granules from crystallization and coalescence.9 Horowitz and Sanders10 reported a procedure to prepare artificial PHB granules, from PHB solution, with similar properties as native granules. In the artificial granules, a surfactant was used as a substitute for native protein/lipid coating. They are amorphous, very stable in suspension, and * To whom correspondence should be addressed. E-mail: robert.
[email protected].
crystallize on drying, which makes them potentially useful as coating agent. In this work we compare the efficiency of various artificial and native granule preparations made from PHB and P(3HBco-3HV) for sizing of Whatman paper.
Experimental Section Production of PHB Native Granules. PHB native granules were supplied by Polyferm Canada Inc. The bacteria Ralstonia eutropha was grown on glucose as a sole carbon source. PHB synthesis was induced by the limitation of phosphorus and nitrogen during fermentation. PHBloaded bacterial cells were harvested and washed by centrifugation. PHB native granules were then released from the cells by sequential treatments with 0.2 M NaOH, sodium dodecyl sulfate (SDS, 0.5%) in 0.1 M NaOH, and 0.1 g/L lysozyme. Following cleaning by washing and centrifugation, PHB native granules were stored in concentrated suspension in distilled water. PHA Artificial Granules Preparation. PHA artificial granules were prepared as described by Horowitz and Sanders.10 Four different spraydried PHAs were obtained from Imperial Chemical Industries: PHB homopolymer and three copolymers P(3HB-co-3HV) composed of 7, 21, and 30% of HV. PHA (1 g) was dissolved in 20 mL of chloroform and emulsified in 400 mL of an aqueous surfactant (5 mM cetyltrimethylammonium bromide, CTAB) using an ultrasonic disruptor for two periods of 2 min at 700 W. The chloroform was evaporated by heating at 70-75 °C for 1.5 h. The granules were then concentrated by centrifugation for 1 h at 25000 × g and kept in 5 mM CTAB. Dynamic Light Scattering. Dynamic light scattering measurements were made using a vertically polarized 50 mW He-Ne laser manufactured by Spectra Physics. The scattering plane was perpendicular to the incident light polarization. The incident wavelength was 632.8 nm. A commercial goniometer (Brookhaven Instruments BI-2030) was used with its original integrated optics to measure the scattered light at 90°. Wide Angle X-ray Diffraction. Wide angle X-ray diffraction patterns were recorded using a Bruker AXS diffractometer (Siemens Kristalloflex 780 generator), operated at 40 kV and 40 mA. The X-ray beam was collimated with a 0.5 mm pinhole and a graphite mono-
10.1021/bm9014667 2010 American Chemical Society Published on Web 02/15/2010
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Figure 1. Dynamic light scattering results of PHB artificial and native granules.
chromator to obtain Cu KR (0.1542 nm) radiation. The diffraction powder patterns or equatorial scans were recorded by a HI-STAR area detector. Field Emission Scanning Electron Microscopy (FE-SEM). Scanning electron microscopy was performed at FPInnovations, PointeClaire, QC, with a Hitachi Ultra-High-Resolution Analytical FE-SEM SU-70 and at the Centre for Biorecognition and Biosensors at McGill University with a Hitachi FE-SEM S-4700. Examination of surface and cross-section of paper sheets were performed on bevel-edge razor blade cuts of paper samples. ATR-IR Spectroscopy of Paper Surface. Attenuated total reflection infrared (ATR-IR) spectroscopy was performed on a PerkinElmer Spectrum BX FTRI spectrophotometer equipped with a MIRacleTM ATR accessory from PIKE Technologies. At 45° with a Diamond crystal plate the penetration depth is about 2 µm into the sample. Treatment of Paper with PHA Suspension. A total of 1 mL of the suspension of PHA was spread over a sheet of Whatman filter paper 1. Polymer spreading was performed with a 10 cm wide BYK-Gardner Film Casting Knife adjustable clearance applicator. The treated paper was dried under tension with a temperature controlled stainless steel paper dryer (Noram). Unless specified, papers were dried for 10 min at 110 °C. Pressing and heating of paper was performed with a Carver laboratory press (Model B) equipped with steel-heated platens (up to 260 °C). Water Penetration Measurement of Sized Paper: Drop Test. This method derived from the PAPTAC Standard F.1H was developed for absorbent type papers. The principle consists in determining the rate of absorption of a fixed volume of water to pass through the surface of the paper sheet. For Whatman papers, 15 µL of water was used. Tensile Properties of Sized Paper. Machine direction of Whatman paper was determined by examining the shape of the wet spot following water drop absorption. Tensile stress and strain measurements were recorded using a constant rate elongation apparatus, Instron Model 4400. Paper strips (15 mm wide per 8 cm long) were cut along the machine direction and fixed between two clamping jaws separated by 5 cm. Load versus elongation of paper was plotted using a constant rate of elongation of 5 mm/min. Maximum load, load at break, and tensile strain (% of elongation) at break were measured.
Results and Discussion Characterization of PHA Granules. All preparations of PHA artificial granules formed stable colloidal suspensions suitable for paper application. Small amounts of surfactant (CTAB 5 mM) prevent the coalescence and crystallization of PHA in suspension by forming surfactant-coated particles. The dynamic light-scattering study (Figure 1) showed that the particle size of all artificial granules range from 100 to 400 nm, with a mean diameter of around 200 nm. However, PHB native granules, isolated from fermentation by Ralstonia eutropha, were more polydisperse (500-1300 nm) with an effective mean
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diameter of 960 nm, which is about 5 times larger than the artificial granules. Scanning electron microscopy (Figure 2a) also shows that the PHB artificial granules are submicrometer size with a soft and sticky texture and have a tendency to coalesce to form large aggregates during drying. On the other hand, the size of the native granules are more heterogeneous, with some particles as low as 200 nm and others as high as 1.5 µm (Figure 2b), but they appear more rigid and unsticky and do not coalesce during drying. The difference in texture between artificial and native granules may be attributed to their molecular weight difference. Using NMR studies, Shaw et al.11 reported that the molecular weight of PHB artificial and native granules are ∼200000 and 500000 g/mol, respectively. The X-ray equatorial scans derived from the powder patterns of dried artificial granules of PHB show that the crystallinity of PHB (Figure 3a,b) was increased by raising the drying temperature from 80 to 110 °C, as shown by the narrowing of the scattering shoulders between 20 and 25°. For P(3HB-co3HV21%) (Figure 3c), the two sharp diffraction peaks were leftshifted due to changes in the PHB lattice as HV content of the copolymer increases due to isodimorphism.12 X-ray scans of sized Whatman paper (results not shown) also indicate crystallinity of PHA in paper following drying, but the scattering shoulders between 20 and 25° were not distinguishable from cellulose peaks. Paper Sizing with PHA Granule Suspensions. In this work, the paper sizing efficiency was assayed by the “drop test”, which measures the resistance of paper to penetration of aqueous liquids. Penetration of liquid in paper is a complex process involving changes in paper porosity, fiber swelling, hydrophobicity, and so on. Whatman filter paper was used as a model for sizing papers made from chemical pulps. Artificial granule suspensions of PHB and three different P(3HB-co-3HV) samples were compared for their sizing efficiencies of Whatman paper (Figure 4). For either 10 or 20 mg of polymer applied on the paper (i.e., ∼1.5 and 3%, based on paper weight), the sizing efficiency of PHB homopolymer was shown to be about 20 times greater than any of the three copolymers tested. With unsized Whatman paper, a 15 µL water droplet takes less than 2 s for complete absorption into the paper whereas it takes over 7 min for PHB sized paper (3 g/m2). To test higher concentrations of polymers, we have compared the sizing effect of artificial granules of PHB and P(3HB-co3HV21%), with increased charges up to 60 mg (9%; Figure 5). Sizing with PHB at concentrations lower than 8 mg (1.2%) is relatively poor, whereas at higher concentration (g8 mg) the homopolymer is highly effective and seems to level off around 20 to 30 mg. On the other hand, the sizing efficiency of the copolymer containing 21% valerate increased linearly up to 60 mg, but the overall efficiency is much lower than the PHB homopolymer. PHB loaded bacterial cells and isolated native granules obtained from lysed cells were assayed and compared to artificial granules. As shown in Figure 6, both bacterial cells and isolated PHB native granules were poor sizing agents under optimal conditions for drying of paper sized with artificial granules (i.e., 110 °C). Thus, when 20 mg of cells or polymer suspensions were applied on paper, the efficiency of artificial granules was shown to be around 30-60 times higher than native preparations. The difference in sizing efficiency between PHB artificial and native granules might be attributed to various factors including particle size and surface properties of the granules. As shown above (Figures 1 and 2), artificial granules are smaller, softer, and more sticky in comparison to native granules. Higher
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Figure 2. Scanning electron microscopy of (a) PHB artificial granules at 30000× and (b) PHB native granules at 20000×. Granule suspensions were dried at room temperature on glass and coated with platinum/gold.
Figure 3. Wide-angle X-ray powder equatorial scans of (a) PHB art. gr. dried at 110 °C; (b) PHB art. gr. dried at 80 °C; and (c) P(HB-coHV21%) art. gr. dried at 80 °C.
Figure 4. Sizing of Whatman paper with artificial granules of PHB and P(3HB-co-3HV). Papers were dried 10 min at 110 °C.
softness and coalescence of artificial granules, as compared to native granules, will likely facilitate the formation of a hydrophobic layer on paper fibers during paper drying at 110 °C. Effect of Paper/Polymer Drying Temperature on Sizing. The temperature and the time used to dry the PHA-sized papers was shown to be important to optimize sizing effects. As shown in Figure 7, the sizing effect of PHB homopolymer increases with drying temperature from 80 to 110 °C and then decreases at higher temperatures following 10 min of drying. However, by shortening the drying time down to 2 min, the sizing effect of PHB did not decrease at temperatures over 110 °C. One explanation of the sizing effect of PHB might involve the granule’s response to annealing. Lauzier et al.13,14 showed that annealed PHB native granules develop a core/shell morphology and that upon annealing, the noncrystalline chains of
Figure 5. Effect of increased charge of PHB and P(3HB-co-3HV21%) artificial granules on Whatman paper sizing. Papers were dried 10 min at 110 °C.
Figure 6. Sizing of Whatman paper with PHB native vs artificial granules. Papers were dried 10 min at 110 °C. Drop test of cells and native granules are shown on left Y axis, whereas artificial granules are shown on right axis.
the core are folded into the shell, which at higher temperature leads to formation of holes in the core. Thus, during drying of sized paper for a short time (i.e., 2 min), the increase of temperature results in enhanced polymer crystallinity and lower water permeability. Eventually, drying at a higher temperature for enough time (10 min), formation of holes and cracks in the granules might be responsible for greater water permeability. Statton15 has made extensive studies of this phenomenon on polyethylene single crystals. For P(3HB-co-3HV21%) and PHB native granules, the degree of paper sizing remains plus or minus constant all over the drying temperature range up to 140 °C for either 2 or 10 min of drying. Knowing that melting temperatures of PHB and P(3HB-co-3HV21%) are 180 and 125 °C, respectively,12 only the valerate copolymer is expected to melt during paper drying at 140 °C, where no change in sizing efficiency was observed.
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Figure 7. Sizing efficiency of artificial granules (PHB a.g. and PHBHV a.g) and PHB native granules (PHB n.g.) following 2 or 10 min of drying at temperatures from 80 to 140 °C. All granule suspensions were used at 20 mg/mL (i.e ∼3% on paper). Figure 10. ATR-IR of Whatman paper sized with increasing amount of PHB. The graph of the absorbance at 1720 cm-1 vs the amount of PHB added on the paper is also shown.
Figure 8. Sizing efficiency following drying and pressing for 2 min at 160 °C and 14 MPa. PHB artificial granules (PHB a.g.) and PHB native granules (PHB n.g.) are shown on right Y-axis and the 21% valerate copolymer (PHB-HV a.g.) is shown on the left axis. All granule suspensions were used at 20 mg/mL (i.e ∼3% on paper).
Figure 11. SEM of a cross section of Whatman paper sized with PHB artificial granules dried at 110 °C. Magnification of the major figure is 2000× and the smaller picture at the top right shows a 10× magnification of the region pointed by the arrow.
Figure 9. Strength properties of Whatman sized paper with PHB (3% on paper) under different conditions: PHB native granules dried at 110 °C (PHB n.g. Dr110 °C) or pressed at 160 °C (PHB n.g. Pr160 °C) and PHB artificial granules dried at 110 °C (PHB a.g. Dr110 °C) or pressed (14 MPa) at 160 °C (PHB a.g. Pr160 °C). A control of unsized Whatman paper pressed at 160 °C was also added.
Effect of Pressure and Temperature on Sizing. As shown in Figure 8, a substantial improvement of the sizing effect was obtained following the application of pressure to paper sized with either native or artificial granules of PHB, but only when the temperature reaches about 160 °C. For native granules, the sizing effect is maximum (drop test over 1.5 h). Under these conditions, the native granules are soft and spread into the pores and over the fiber (see SEM of PHA-Sized Paper). At higher temperature (i.e., 175-180 °C), PHB begins to decompose as shown by yellowing and the crotonic acid odor. In contrast, a small decrease of sizing effect was observed with the copolymer P(3HB-co-3HV21%) following pressing at 160 °C. Tensile Strength of PHB-Sized Paper. Figure 9 shows tensile strength properties of Whatman paper sized with PHB (3% on paper) under different conditions: PHB native granules dried at 110 °C or pressed at 160 °C and PHB artificial granules dried at 110 °C or pressed at 160 °C. A control of unsized Whatman paper pressed at 160 °C was also added. Compared
to the control sample, a gain of approximately 20% in tensile stress (measured as maximum load applied to the paper strip before weakening and break appear) of sized paper was obtained with PHB native granules, only when dried and pressed at 160 °C. For all other samples, that is, PHB native granules dried at 110 °C and PHB artificial granules dried at 110 °C or pressed at 160 °C, no significant improvements were noticed when compared to unsized paper. For tensile strain (measured as % of paper strip elongation before breaking), there were no obvious changes between all sized samples and the control paper. Measurement of PHA on the Surface of Sized Paper by ATR-IR Spectroscopy. In attenuated total reflection infrared (ATR-IR) spectroscopy, the penetration depth of the IR wave in the sample is in the order of 1-2 µm. Thus, only the polymer in a surface slice of the paper sample is detected by this technique. The carbonyl group of PHA has a strong absorption peak at 1720 cm-1 and measures the amount of PHA on the surface of paper samples (Figure 10). A straight line can be drawn through the absorption readings at 1720 cm-1 and thus provides a method to estimate the amount of the polymer in the surface of paper slice. SEM of PHA-Sized Paper. Scanning electron microscopy of the surface and cross-section of sized papers revealed that both artificial and native granules spread on the paper surface, penetrate into the paper, and are found inside and onto both sides of the paper. As shown in Figure 11, PHB artificial
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were used for paper sizing. Good sized paper was obtained following impregnation of paper with artificial granules of PHB when dried at moderate temperature (∼110 °C), whereas native granules of PHB were shown to be a poor sizing agent in this temperature range. This difference in sizing effect can be attributed to several factors including particle size of native granules versus artificial granules (1 vs 0.2 µm), and the physical state and morphology of the granules after drying. At moderate temperature, native granules stay granular and disperse in paper, while artificial granules coalesce and form films and bundles at fiber surfaces. However, highly sized papers were obtained using either native or artificial granules following pressing and heating at higher temperature (∼160 °C) for a short period. Under these conditions, granules melt and form a thin hydrophobic film covering the fiber surface. Interestingly, all artificial granule preparations, made from valerate copolymers, were about 20 times less efficient than PHB homopolymer even though they have similar particle size, stability, and morphology. The SEM images of sized paper have demonstrated how to transform printing paper into composite material that could open new applications for paper. The nanosize granules dispersed throughout the fiber matrix and post-treated with temperature, pressure, and cold rolling are potentially fiber reinforced films. The fiber orientation could be both in plane and in the machine direction, creating composites other than by compression or extrusion molding. Acknowledgment. Financial support was received from National Resource Canada/FPInnovations. We thank M. Paice, L-P. Scheffer, B. Ramsay (Polyferm Canada), J. Petlicki, R. Allem, P. Furasek, S. Essiembre, and L. Mongeon for advice, assistance, and materials.
References and Notes Figure 12. SEM of Whatman paper sized with PHB native granules (a) dried at 110 °C and (b) pressed at 160 °C. The smaller picture at the top right of each figure shows a 10× magnification of the region pointed by the arrow.
granules have a tendency to coalesce and form somewhat flat bundles in paper pores and on the surface of fibers. A magnified view of a bundle of coalesced granules is shown in the picture at the top right of Figure 11. Formation of hydrophobic clusters on the fiber surface will likely influence the penetration of liquid into paper. However, native granules do not seem to coalesce, they stay dispersed inside and on the surface of paper, and upon drying and pressing at temperatures under 150 °C, they keep their granular morphology. Figure 12a shows a SEM picture of paper sized with native granules and dried at 110 °C. The low sizing efficiency of native granules can be explained by the fact that drying as a powder leaves the surface of fibers uncoated and free to absorb water. However, pressing and heating at 160 °C, the granules melt and form a fine film on the fiber surface as shown by the disappearance of granules (Figure 12b), resulting in a huge gain of sizing efficiency.
Conclusions Thermoplastic biodegradable latex made from either native PHB granules or artificially reconstituted from PHA solutions,
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