23rd European Biomass Conference and Exhibition, 1-4 June 2015, Vienna, Austria PRODUCTION OF BIO-DEGRADABLE POLYURETHANE FOAM FROM SUGARCANE BAGASSE AND BIODIESEL-DERIVED CRUDE GLYCERINE Sanette Marxa, Wilmar Odendaala, Anné Williamsa, Urban Vermeulenb, Anne Groblerb, LC Mullera aFocus Area: Energy Systems, School of Chemical and Minerals Engineering, North-West-University (Potchefstroom Campus), Potchefstroom, South Africa, Tel: +27 18 299 1995, Fax: +27 18 299 1535, Email:sanette.marx@nwu.ac.za bDST/NWU Preclinical Drug Development, Faculty of Health Sciences, North-West-University (Potchefstroom Campus), Potchefstroom, South Africa) ABSTRACT: As the threat of the depletion of fossil based fuels becomes more of a reality, there is a need to find alternative carbon sources for the production of a wide range of chemicals, polymers and plastics. One plastic that is used in almost every aspect of everyday life is polyurethane foam. Currently it is produced through polyols and diisocyanates that are all obtained from fossil based resources such as crude oil and coal. Lignin is a by-product from the second generation bioethanol process and crude glycerol is a by-product of biodiesel production through transesterification. These two by-products can be combined to produce polyurethane foam of high quality. In this study, the influence of reaction parameters such as temperature, pH of the crude glycerol, and reaction time on the hydroxyl numbers and the properties of the produced foam was investigated. Foam properties such as density, cell size and compression strength was studied. Results showed that the hydroxyl numbers and thus the properties of the lignin and crude glycerin used as reagents have a significant influence on the foam density and rigidity. The foams were tested for biodegradability using a standard ASTM method and it was shown that the produced foams degraded faster than commercial rigid polyurethane foam. Polyurethane foams were also applied as ssDNA immobilization scaffolds by the DST/NWU Preclinical Drug Development Platform, on campus, to determine the potential to be used as part of a diagnostic system. Results showed that polyurethane foam is a viable starting material for immobilization scaffolds in the development of an affordable and practical point-of-care tuberculosis diagnostic system. Keywords: biodegradable, polyurethane, glycerin, bagasse, lignin. 1 INTRODUCTION used after chemical modification is used to produce polyols for polyurethane production [6]. Fossil fuels are used not only for the production of In some studies where lignin was chemically electricity or as sources of fuel, but also for the modified for the production of polyurethane, such as that production of a large variety of products, especially of Borges da Silva et al. [6], polyurethanes with similar plastic products. Unfortunately, fossil fuels are not characteristics to those produced with petroleum based renewable and because of this, a point will eventually be components were obtained. reached where it is no longer economically viable to If polyurethanes can be produced with the use of extract fossil resources. Even at this point in time, as a waste biomass materials such as lignin and crude result of the ever increasing demand for energy globally, glycerin, this will provide alternative options for the the costs of fossil fuels, and by extension products industries from whence these waste materials are produced from them, are increasing [1]. There is also an obtained. Biodiesel producers will therefore have an ever increasing level of concern about the impact that additional source of income, which will make the fossil fuel products have on the environment [1]. New production of biodiesel more economically viable and carbon sources are thus necessary to produce plastics for which will accelerate the growth of that industry. It everyday use and as alternative sources of energy. would also reduce the dependence of the polyurethane Biomass is known to be renewable and can be used as an industry on fossil based raw materials, which would alternative carbon source. reduce their carbon footprint and potentially result in One biomass based product that has been receiving more biodegradable polymers. increasing attention as an alternative fuel to fossil based It is, however, important to note that varying fuels, is biodiesel. However, the growth of the biodiesel production conditions for the chemical modification of industry has been hampered due to the relatively high lignin, would result is varying polyol, and by extension cost of its production [2]. The most preferred industrial polyurethane, characteristics. As such it is important to method for biodiesel production is transesterification [3] determine how the production conditions influence the which produces large amounts of crude glycerin [4] that polyol and polyurethane characteristics. One possible has little commercial value and is currently a liability to application of biodegradable bio-based polyurethane is biodiesel producers [1]. the medical industry. According to the World Health Another biomass waste material that has been Organization [9], tuberculosis (TB) is a serious health assessed for its potential as a raw material for production problem with nearly 9 million new infections each year, of commonly fossil based products, such as plastics, is and 1.5 million reported deaths in 2013. The diagnosis of lignin [5]. Lignin is most commonly produced by the TB is thus crucial in reducing the number of deaths due paper and pulp industry through the use of the Kraft to TB infection [10]. Current testing methods are pulping process [6]. However, the current market for expensive and the commonly used tests are not time lignin is limited and the vast majority of the produced efficient [11]. Problems commonly experienced with the lignin gets burned to recover energy and pulping testing of TB are the state of the sample to be analyzed chemicals [7]. [12] and inhibitors present in the sample [13]. Low These sources of waste biomass have been used in numbers of bacteria could also lead to false-negative the production of polyurethane in the past. In general, results. The time taken to analyze samples influences the glycerin is used as a solvent [8] while lignin, preferably effectiveness of a method. Many methods cannot meet 1075 23rd European Biomass Conference and Exhibition, 1-4 June 2015, Vienna, Austria the requirements for clinical diagnosis [12]. contained 49.3% glycerol with a pH of 10.8 and was used The DST/NWU Preclinical Drug Development without any further purification. Platform (PCDDP) has developed a new testing method Commercial Kraft lignin was obtained from Sigma by means of rapid cell lysis in a lysis micro reactor Aldrich chemical company and used as received from the (LMR) whereby clinical samples can be processed supplier. A crude glycerin to lignin weight ratio of 15:1 quickly and effectively [13]. was used for all experiments. Liquefaction was done in a Cell walls are weakened during cell lysis causing the closed glass flask using a hot oil bath to heat the mixture cell to rupture, releasing the genetic material [14]. High to the desired temperature and a magnetic stirrer (250 temperatures, rapid mixing and the buffer solution aid in rpm) was used to ensure uniform mixing of the reaction the cell lysis [13]. The scaffold may be used to mixture. The influence of reaction temperature (120- concentrate the ssDNA that is present in the biological 180°C), pH of the crude glycerin (7.5-8.5) and reaction sample by means of interaction of the hydrophobic bases time (1 to 2 hours) were investigated using a traditional of ssDNA with the hydrophobic surface of the linear experimental design. The pH of the crude glycerin material[13]. The scaffold also acts as the transport was adjusted prior to liquefaction using a standard 0.1 M mechanism between the different testing stages. sulfuric acid solution. In this study the effectiveness of polyurethane After completion of the reaction, the reaction was synthesized from lignin and crude glycerin as scaffolds quenched by placing the reaction flask in cold water. The for TB DNA analysis is compared to that of scaffold reaction product was removed from the flask and prepared from poly-ethylene glycol (PEG) with a weighed to determine the polyol yield. The polyol was molecular weight of 200 g.mol-1 (hydroxyl number of used as recovered from the reaction flasks without further 563). The influence of different synthesis parameters on washing or purification. the hydroxyl number and some properties of the foam were investigated as well as the biodegradability of the 2.3.2 Determination of hydroxyl number lignin-based foams compared to commercial The experimental procedure followed for polyurethane foam and starch. determination of the hydroxyl numbers of the polyols used is presented in Figure 1. All reactions were done in 250 ml GL-45 laboratory glass bottles with blue pp screw 2 MATERIALS AND METHODS caps and pouring rings. 2.1 Materials PEG200 (Average molecular weight of 190 – 210 g.mol-1), purchased from Sigma Aldrich was used as synthetic polyol in this study. Dabco DC5357 (Air Products & Chemicals) was used as a silicone surfactant and N,N-dimethylcyclohexylamine was used as gelling catalyst. Methylene diphenyl diisocyanate (MDI, Bayer) with a NCO group content of 30% was used for all foam syntheses. All materials were used as received from the supplier without further purification. 2.2 Preparation of poly-ethylene glycerol scaffolds Polyurethane foams were prepared according to the methods described by Hu et al. [15]. Polyol, surfactant and catalyst were added to a mixing cup and vigorously mixed using an electric stirrer for 10-15 s. Diisocyanate was then added to the polyol mixture, and vigorously mixed for a further 10-15 s. After the foaming process was complete, the foam was allowed to cure for 24 hours at room temperature. A band saw was used to cut the Figure 1: Experimental procedure for determination of foam into disks with a width of approximately 1 cm. The hydroxyl number scaffolds were cut from the disk using a laser cutter (Speedy 300, Trotec) and a scalpel. The dimensions of The hydroxyl numbers of the polyols were the scaffolds were 60 mm x 2 mm x 0.7 mm. The determined with the use of method D (Imidazole- influence of isocyanate index (100 and 200) on the catalyzed phthalic anhydride pressure bottle), as suitability of the polyurethane foam to be used as described in the ASTM standard [16] for hydroxyl scaffolds were investigated using a catalyst loading of 2.5 number determination of polyurethane raw materials. and 5 g with a fixed gelling agent amount of 0.84 g. Foams were prepared as described for poly-ethylene glycol-based foams. 2.3 Preparation of lignin-based scaffolds 2.4 Characterization of foams 2.3.1 Liquefaction of lignin in crude glycerin Crude glycerin was obtained as by-product from the 2.4.1 Microscopy analysis transesterification of virgin sunflower oil. A methanol to Sample blocks, with a width and length of as close as oil ratio of 6:1 was used with a reaction temperature and possible to 1 cm and a thickness of approximately 0.3 time of 60°C and 60 minutes respectively. The glycerin cm, were cut from the produced polyurethane foams with was allowed to settle from the reaction mixture by gravity the greater surface area being a side view of the foam. and collected by decantation. The glycerin thus obtained 1076 23rd European Biomass Conference and Exhibition, 1-4 June 2015, Vienna, Austria The blocks were placed under a stereoscopic light The desiccators were carefully shaken periodically to microscope set to 10 times magnification and the focus of break up the barium carbonate layer that forms on the the microscope as well as the amount of light shone on barium hydroxide solution as it absorbs CO2 to ensure the sample was set to obtain the optimal picture. A Nikon effecting CO2 absorption at all times. camera attachment along with a computer program was The desiccators were removed and opened used to capture pictures of the samples. periodically for a period of approximately 20 minutes to These samples were used to determine the cell sizes refresh the air within the desiccators and during this time of the produced foams by simply measuring the phenolphthalein was added to the barium hydroxide dimensions of numerous cells and determining its solution and the barium hydroxide solution was titrated average dimension. with a 0.05 N hydrochloric acid solution to a clear end point. The amount of hydrochloric acid needed for the 2.4.2 Density analysis titration of each sample was used to calculate the amount The produced foam was cut into blocks with of CO2 generated by each sample and, by extension, the dimensions as close to 4cm by 4 cm by 2.5 cm as percentage of carbon lost by each of the test materials. possible. The final dimensions of the blocks were A fresh batch of barium hydroxide solution was carefully measured in order to determine the volume of added to each desiccator before it was resealed again. the blocks, after which the blocks were weighed with the use of a mass balance. The weight of the blocks was then 2.5 DNA capturing divided by the volume to calculate the density of the The suitability of polyurethane as ssDNA foam in kg·m-3. immobilization scaffold was tested through the novel TB diagnosis method set out by the DST/NWU Preclinical 2.4.3 Compressibility analysis Drug Development Platform [13]. Scaffolds were placed A 100kN MTS Landmark servo hydraulic test system in the lysis microreactor, with the reference H37 Rv was used to run compression tests on the same foam mycobacterium cells and lysis buffer solution. The lysis sample blocks previously cut for density determination. conditions include temperatures of 95°C and mechanical The data obtained from these tests were subsequently stirring at 3600 rpm for 7 minutes. The scaffold was used to determine both the compression strength and removed from the lysis microreactor and transferred to a elasticity modulus of compression for the foams. PCR (polymerase chain reaction) cuvette. PCR mastermix and DNA primers for selected gene sequences 2.4.4 Biodegradability analysis of mycobacteria were added to the cuvette, which was A soil mixture containing one third Culterra potting placed in a Philisa rapid thermocycler (Streck Inc., soil, one third a reddish sandy soil obtained Omaha, NE). 4 µl of Novel Juice was added to the approximately 15 meters from a stream (26°41’00.1”S samples and loaded into the wells of a agarose gel 27°05’54.5”E) and one third soil obtained from a consisting of 2% agarose, 33 ml of 3x Gel-red dye, 10 ml residential garden (26°42’00.8”S 27°04’54.4”E) was of 10x Bionic Buffer and 47 ml ddH2O. Gel used for testing the biodegradability of the produced electrophoresis on an agarose gel at 70V for 90 minutes polyurethane foams. was used to visualize the PCR products. A UV The moisture holding capacity of the soil was transilluminator was used to obtain digital images of the determined by saturating a portion of the dry soil mixture 2%, 33 ml Gel red, agarose gel. with water and allowing it to drain over a period of 48 hours, after which the amount of water that was retained within the soil was determined. 3 RESULTS AND DISCUSSION The biodegradability test was set up in accordance with ASTM standard (D5988-12) [17]. 125 g of soil was 3.1 Effect of liquefaction variables on hydroxyl numbers added to 250 mL desiccators along with sufficient samples of the materials to be tested in powdered form or 3.1.1 Effect of reaction time crushed to less than 1 mm in diameter, to ensure that 250 The influence of reaction time on the hydroxyl values mg of carbon was added to all but the blank and technical of polyols prepared from lignin at a constant reaction control tests. Elemental analysis of the foams were used temperature of 150°C, a feed glycerin pH of 8 and a to determine the carbon content. Starch was used as the solvent to lignin ratio of 15:1 is presented in Figure 2. positive reference material and a commercial polyurethane foam (Rigifoam) was used for comparison. 450 A 4.72 g.L-1 solution of ammonium phosphate was 400 added to the soil for each test specimen to ensure a C:N 350 ratio of 12.5:1. The same volume? of ammonium 300 phosphate solution was also added to the blank tests. 250 Distilled water was added to the soil of all the test 200 samples and blank tests to the extent of the soil reaching 150 80% of its water holding capacity. 100 A 150 mL beaker containing 100 mL of a 0.025 N 50 barium hydroxide solution was placed within each of the 0 desiccators in order to capture the CO2 generated in each 0.8 1 1.2 1.4 1.6 1.8 2 2.2 along with a 100ml beaker containing 50 mL of distilled Reaction time (hr) water to ensure minimal moisture loss from the soil during the tests. The desiccators were sealed using high vacuum Figure 2: Effect of reaction time on hydroxyl numbers of grease and stored in a cool, dry and dark place. Kraft-lignin based polyols 1077 OH number (mg KOH/g) 23rd European Biomass Conference and Exhibition, 1-4 June 2015, Vienna, Austria From Figure 2 it can be seen that the hydroxyl value 400 decreases as reaction time increases. During liquefaction 360 of lignin, ether and ester bonds between lignin units are 320 cleaved [18] resulting in relatively high hydroxyl values 280 at short reaction times. As liquefaction reaction time is 240 increased, more of the liquefied hydrocarbons and 200 glycerol within the solvent is thermally degraded or 160 oxidized reducing the hydroxyl value [19]. Lim et al. [20] 120 also determined that polyols with the greatest hydroxyl 80 values had the lowest molecular weights. 40 0 7.4 7.6 7.8 8 8.2 8.4 8.6 3.1.2 Effect of reaction temperature Glycerol pH The influence of reaction temperature on the hydroxyl values of polyols prepared from lignin at a constant reaction time of 1.5 hours, a feed glycerin pH of Figure 4: Effect of crude glycerol pH on hydroxyl 8 and a solvent to lignin ratio of 15:1 is given in Figure 3. numbers of Kraft-lignin based polyols 450 Mahmood et al. [21] determined that, at a relatively 400 low pH, liquefaction intermediates will repolymerize, 350 resulting in polyols with higher molecular weights. This 300 implies that an increase in pH would result in more 250 alkaline polyols that have lower molecular weights. 200 Mahmood et al. [21] however determined that these changes would have no impact on the overall conversion. 150 The observed decrease in molecular weight with an 100 increase in pH was deemed to be as a result of ether 50 linkages being attacked and broken by water or hydroxyl 0 110 130 150 170 190 ions. This means that a point would be reached where Reaction temperature ( C) there would be no more linkages to break, resulting therein that a further increase in pH would have no further impact on the polyol molecular weights. The low Figure 3: Effect of reaction temperature on hydroxyl hydroxyl numbers of polyols produced with Kraft lignin numbers of Kraft-lignin based polyols at low pH values is likely due to the repolymerization of liquefaction intermediates [21]. As the pH increases the According to Hu et al. [8] the decrease in hydroxyl hydroxyl value of the produced polyols also increases value as observed during liquefaction is a result of due to lower degrees of repolymerization. oxidation, degradation and condensation reactions taking place during liquefaction, and that reduces the number of 3.2 Foam characterization hydroxyl groups within the reaction mixture. These reactions occur preferentially at low temperatures, which 3.2.1 Microscopy analysis explains why polyols prepared at lower temperatures The of liquefaction conditions on the cell size of PUF have higher hydroxyl values. As the reaction temperature foams are shown in Figure 5. increases however, the hydroxyl values of the produced polyols decreases, flattening out at approximately 165°C. pH = 8, T = 150°C pH = 8,  = 1.5 hr  = 1.5 hr, T = 150°C The latter may be due to the fact that as the oxidation, degradation and condensation reactions continue, there are less material readily capable of undergoing these reactions, resulting in the observed decrease in hydroxyl value with an increase in reaction temperature becoming less significant. 3.1.3 Effect of crude glycerin pH The influence of crude glycerin pH on the hydroxyl values of polyols prepared from lignin at a constant reaction time of 1.5 hours, a reaction temperature of 150°C and a solvent to lignin ratio of 15:1 is given in Figure 4. Figure 5: Microscope images at 10 times magnification of different Kraft-lignin based PUF prepared from polyols that were prepared at different conditions 1078 OH number (mg KOH/g) Increasing reaction time OH number (mg KOH/g) Increasing temperature Increasing pH 23rd European Biomass Conference and Exhibition, 1-4 June 2015, Vienna, Austria Thirumal et al. [22] found that lower density foams increased further, the aromaticity of the produced foam is were generally found to have greater cell sizes. Hakim et also increased, leading to low foam mobility and larger al. [23] suggested that polyols with higher hydroxyl expansion, resulting in foams with reduced density. values would produce foams with smaller cells sizes. The average cell size of foams produced from Kraft 3.2.3 Compressibility analysis lignin polyols was found to be comparatively small. This The influence of hydroxyl number on the is likely due to the higher hydroxyl values of the Kraft compressibility of the prepared foams per unit of density lignin polyols, which would lead to a higher crosslink is given in Figure 8. density within the foam, making cell growth more difficult and thereby reducing the average cell size of 90 these foams. 80 It was also observed that, contrary to results from 70 literature [24], no correlation between the average cell 60 sizes of the produced foams or their cell size distributions 50 and the hydroxyl values of the polyols used to produce 40 them could be drawn. The results obtained in relation to 30 the comparison between average cell size and hydroxyl 20 value can be seen in Figure 6. 10 0 0 50 100 150 200 250 300 350 400 1400 OH number (mg KOH/g) 1200 1000 Figure 8: Effect of hydroxyl number on Young’s 800 elasticity modulus for the foams prepared from Kraft- 600 lignin based polyols 400 Young’s elasticity modulus is dependent on the 200 density of the foam as well as the thickness of the cells’ 0 walls. An increase in density with an increase in 175 205 235 265 295 325 355 385 415 OH number (mg KOH/g) hydroxyl number does not influence the elasticity of the foam, because the cell walls of the foam is still relatively thin, which results in a low elasticity. As the hydroxyl Figure 6: Effect of hydroxyl number of average cell size number increases further, the density of the foam of foams prepared from Kraft-lignin based polyols decreases as a result of larger air bubbles in the foam. As can be seen from the microscopy analysis, the foams with 3.2.2 Density analysis a low density has thick cell walls, which results in higher The effect of hydroxyl number on the density of elasticity moduli. This result correlated with the density foams prepared from Kraft-based lignin is presented in and microscopy analysis. Figure 7. 3.2.4 Biodegradability analysis 310 The carbon loss over time of the foams prepared from Kraft-lignin based polyols is compared to the carbon loss 260 of starch as reference materials and commercial polyurethane foam in Figure 9. 210 8 160 7 6 110 5 60 4 0 50 100 150 200 250 300 350 400 OH number (mg KOH/g) 3 2 Figure 7: Effect of hydroxyl number on density of foams 1 prepared from Kraft-lignin based polyols 0 0 5 10 15 20 25 Time (days) Although there were some outliers, the general trend is an initial increase in density up to a hydroxyl number of approximately 300, after which the density decreases. Figure 9: Carbon loss (wt %) of prepared foams (), According to Aroúja et al. [25] the aromaticity of the commercial foam () and starch () polyols play an important role in the density of the produced foams. Initially, the increase in hydroxyl After a 121 day soil incubation of the samples, the groups with an increase in hydroxyl number leads to final carbon loss % for Kraft lignin based foams was greater crosslinking of the polyols, resulting in an 5.77±0.4%. The final carbon loss of the commercially increase in the density of the produced foams, as was also obtained Rigifoam and the starch were 1.48±0.13% and observed by Hakim et al. [23]. As crosslinking is 6.64±0.92% respectively. 1079 Density (kg/m³) Cell size (μm) % Carbon loss Young's elasticity modulus (kPa) 23rd European Biomass Conference and Exhibition, 1-4 June 2015, Vienna, Austria From these results it can clearly be seen that the The ssDNA fragments immobilized by the scaffolds foams produced from the polyols produced during this were 123 base pairs in size. Effective ssDNA study are indeed more degradable than their commercial immobilization was characterized qualitatively with a counterpart, but not as much as the starch reference bright band at the desired DNA amplicon value. PEG200 material. The greater level of biodegradability for foams with excess MDI followed by PEG 200 showed potential produced during this study is likely a result of the in immobilizing ssDNA although repeatability needs to glycerol and lignin containing polyols produced foams be improved. No bands were observed in the with structures more similar to the natural food of the amplification of the Kraft-lignin based foams. The microbes within the soil, enabling them to degrade the potential of PEG 200 with excess MDI could be foam more easily. explained possibly by the unreacted diisocyante present that supplied excess NCO groups. The presence of NCO 3.2.5 ssDNA capturing groups were indicated by FTIR. NCO is an electrophilic The properties of the prepared PEG200 foams used as group that can interact with the nucleophilic sites of reference material for ssDNA capturing showed that the ssDNA[30]. The negative result obtained for the Kraft- foams were similar in structure to rigid foams and had a based lignin is mostly due to the high porosity and density of 210 kg.m-3. hydrophilicity of the scaffolds that resulted in the FTIR spectra for the PEF200 foams showed urethane scaffolds absorbing all the liquid in the PCR vial. linkages at approximately 3300 cm-1 (NH bonds) [25], 2870 cm-1 (CH2 and CH -13 bonds) [26] and 1720 cm (CO bonds) [27]. The presence of a peak at 1600 cm-1 4 CONCLUSIONS indicated the presence of aromatic ring structures, which contributed to the aromaticity of the foams [28]. The The results from this study showed that polyurethane presence of isocyanurate rings were observed at scaffolds can be used to capture TB ssDNA by means of approximately 1415 cm-1 [29]. The presence of an NCO a combination of hydrophobic-hydrophobic and group at 2270 cm-1 [25] indicated that the isocyanate was electrophilic-nucleophilic interactions. Although Kraft- not completely consumed in the reaction [28]. lignin based scaffolds were not successful in capturing The results of the ssDNA immobilization tests for the ssDNA, it was proven that the direct relationship between PEG200 foams and the Kraft-lignin based foams are the foams characteristics and the hydroxyl number of the shown in Figures 10 and 11 respectively. Three scaffolds Kraft-lignin based polyols makes it possible to tailor were tested for each foam. A positive and negative make the foams for ssDNA capture. The hydroxyl control were used to ensure that the process progressed numbers are directly related to the polyol preparation correctly. A DNA ladder was used to size the amplicons. conditions and thus it is possible to predict the properties of the foam from the reaction conditions. L - + 1a 1b 1c 2a 2b 2c Biodegradability tests showed that the lignin based foams were more degradable than commercial rigid polyurethane over a period of 121 days. 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