Bioresource Technology 183 (2015) 259–261
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Structural characterisation of pretreated solids from flow-through liquid hot water treatment of sugarcane bagasse in a fixed-bed reactor Prashant Reddy a,b,⇑, Prabashni Lekha c, Wienke Reynolds d, Christian Kirsch d a
Sugar Milling Research Institute NPC, c/o University of KwaZulu-Natal, Durban 4041, South Africa Department of Chemistry, Durban University of Technology, P.O. Box 1334, Durban 4000, South Africa c CSIR/UKZN, Forestry and Forest Product Research Centre, Durban 4041, South Africa d Institute of Thermal Separation Processes, Hamburg University of Technology, Eißendorfer Strasse 38, D-21073 Hamburg, Germany b
h i g h l i g h t s FEG-SEM and TEM on sugarcane bagasse pretreated with flow-through liquid hot water. Deposition of lignin droplets on pretreated bagasse clearly observed from FEG-SEM. Density of droplets and average droplet size increased with temperature increase. Average droplet sizes were between 67 and 238 nm. Lignin migration across cell wall observed from TEM.
a r t i c l e
i n f o
Article history: Received 17 January 2015 Received in revised form 8 February 2015 Accepted 9 February 2015 Available online 18 February 2015 Keywords: Sugarcane bagasse Liquid hot water Flow-through FEG-SEM TEM
a b s t r a c t Untreated sugarcane bagasse and sugarcane bagasse pretreated with flow-through liquid hot water (LHW) treatment (170–207 °C and 204–250 ml/min) in a fixed-bed reactor have been structurally characterised. Field emission gun scanning electron microscopy (FEG-SEM) and transmission electron microscopy (TEM) were used to investigate changes in the residues, in particular due to the fate of lignin. FEGSEM results show that the LHW treatment modified the surface morphology of the pretreated bagasse with lignin droplets being observed on the fibre surface. TEM showed an increase in the plant cell wall porosity and lignin migration across the plant cell wall. Increases in pretreatment temperature were observed to increase the average size and density of lignin droplets on the fibre surface. The results provide evidence that for LHW flow-through treatment, just as for batch treatment, lignin repolymerisation and deposition on the surface of pretreated sugarcane bagasse is an important consideration. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Biomass pretreatment with liquid hot water (LHW) is a physicochemical process where pressurised water (typically between 160 and 230 °C) is employed. The influence of the reactor configuration in LHW treatment has been debated as flow-through methods have been considered as being superior to batch methods for the solubilisation of lignin (Mosier, 2013). Structural characterisation of pretreated solids from biomass pretreatment processes is important for understanding biomass deconstruction processes, including the fate of lignin.
⇑ Corresponding author at: Sugar Milling Research Institute NPC, c/o University of KwaZulu-Natal, Durban 4041, South Africa. Tel.: +27 31 273 1306; fax: +27 31 273 1302. E-mail address: [email protected] (P. Reddy). http://dx.doi.org/10.1016/j.biortech.2015.02.057 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Lignin has been noted to undergo depolymerisation followed by repolymerisation during LHW pretreatment (Donohoe et al., 2008) and this has important repercussions for pretreatment strategies. Deposition of lignin droplets on pretreated biomass solids has been noted in several studies (Donohoe et al., 2008; Kristensen et al., 2008; Xiao et al., 2011). Structural studies on residues from various pretreatments includes LHW on corn stover (Donohoe et al., 2008), wheat straw (Kristensen et al., 2008); sugarcane bagasse (Wang et al., 2012; Yu et al., 2013a,b; Zhang and Wu, 2014), salt cedar (Xiao et al., 2011); LHW and ammonia on sugarcane bagasse (Yu et al., 2013b); and dilute acid pretreatment on corn stover (Donohoe et al., 2008). There have been few structural studies on LHW-pretreated sugarcane bagasse reported and, to our knowledge, no SEM and TEM work has been reported to date for bagasse residues from flow-through LHW treatment. A key objective for this work was to investigate the extent and nature of the
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P. Reddy et al. / Bioresource Technology 183 (2015) 259–261
deposition of lignin droplets in the flow-through LHW treatment of sugarcane bagasse at temperatures between 170 and 207 °C with field emission gun scanning electron microscopy (FEG-SEM) and transmission electron microscopy (TEM).
Table 2 Reaction conditions and lignin droplet sizes for pretreated bagasse.
2. Methods 2.1. Materials Washed sugarcane bagasse from a local sugar mill in KwaZuluNatal, South Africa was oven-dried and shipped in vacuum-sealed bags to the Hamburg University of Technology (TUHH) in Germany. The chemical composition of the bagasse, shown in Table 1, was determined with a National Renewable Energy Laboratory (NREL) analytical procedure (Sluiter et al., 2011). The bagasse was not reduced in size or treated further prior to the LHW experiments. Unmilled bagasse has an extremely wide range of particle shapes and sizes. The results from the sieve analysis of the bagasse are shown in Table S1 (Supplementary data).
2.2. Pretreatment The apparatus used in this work, a 3 L high-pressure fixed-bed reactor, was developed at the Institute of Thermal Separation Processes at TUHH. Pressurised (5 MPa) hot water was passed through 0.6 kg of dry bagasse in the reactor for a predetermined amount of time at a specified flow rate. The pretreated solids were retrieved from the reactor once the equipment cooled down sufficiently. Six experiments were performed at temperatures between 170 and 207 °C and flow rates between 200 and 250 ml/min; five of these experiments were performed at the same flow rate (250 ml/min) with one experiment at a lower flow rate of 204 ml/min. The reaction conditions are shown in Table 2. The porosity of the fixed bed (Lewandowski and Beyenal, 2013) was calculated from the void volume and the reactor volume (2.98 L) as follows:
Porosity ¼ ðVoid volume=Reactor volumeÞ
ð1Þ
The average void volume and porosity values for the fixed bed in this work are 2.3 L and 0.77, respectively.
2.3. Analyses 2.3.1. Field emission gun-scanning electron microscopy (FEG-SEM) The oven-dried pretreated bagasse samples were mounted on aluminium stubs using double-sided carbon tape. The samples were coated with gold using a Polaron SC500 sputter coater and viewed with a Carl Zeiss Ultra Plus FEG-SEM at an accelerating voltage of 5 kV. The FEG-SEM images were obtained at magnifications ranging from 200 to 100,000. Images captured at the highest magnification were analysed using Image Pro Plus V7.0 to obtain diameters of the lignin droplets.
Table 1 Chemical composition of untreated sugarcane bagasse on a dry weight basis. Carbohydrates (%)
a b c d
Lignin (%)
Glucan
Xylan
Ara + man
42.5
24.4
2.9
a
Arabinan and mannan. Acid-soluble lignin. Acid-insoluble lignin. Water and ethanol extractives.
ASL 4.6
b
AIL
Extractives (%)d
Ash (%)
5.6
1.2
c
18.8
a
Temperature (°C)
Flow rate (ml/min)
Reaction time (min)
Droplet sizea (nm)
170 178 192 195 195 207
250 250 250 250 204 250
35 35 35 35 44 35
66.5 93.7 104.3 100.3 100.4 238.3
Average droplet size.
2.3.2. Transmission electron microscopy (TEM) The oven dried bagasse samples were placed in fresh 100% Spurr’s epoxy resin (Spurr, 1969). The resin moulds were incubated for 72 h in a desiccator with activated silica gel and phosphorous pentoxide. Resin changes were performed daily and after 3 days, the resin embedded samples were polymerised in an oven (70 °C for 8 h). The resin blocks were sectioned using a Leica Ultracut EM UC7 ultramicrotome. Ultra-thin sections, approximately 100 nm thick, were collected on copper grids. Thereafter, the grids were stained for 30 min with a 1% potassium permanganate solution prepared in 0.1% sodium citrate for lignin localisation (Donaldson, 1992). The samples were viewed at 100 kV with a JEOL 1010 TEM instrument.
3. Results and discussion 3.1. FEG-SEM FEG-SEM micrographs of the untreated sample and the pretreated samples are shown in Fig. S1 (Supplementary data). The zoomed inlays provide a closer view of the surface characteristics of the fibres. The untreated bagasse fibres (Fig. S1a) showed a smooth surface with no significant deposits. The pretreated bagasse fibres (Fig. S1b–g) have a rougher and corrugated appearance with the presence of deposits. Deposits were also observed by Donohoe et al. (2008) on dilute acid- and LHW-pretreated corn stover and were studied using FTIR, NMR, antibody labelling and cytochemical staining, where it was shown that the deposits contained lignin. In our study, cytochemical staining was also used, and dense staining which has previously been shown to represent lignin was obtained (Donaldson, 1992; Donohoe et al., 2008), as discussed later in the TEM results. The micrographs show that the density of lignin droplets generally increased with an increase in pretreatment temperature (Fig. S1b–g), as in the work of Xiao et al. (2011) on salt cedar. With the melting point of lignin between 90 and 190 °C (Mosier, 2013), it is conceivable that higher temperatures favour increased lignin depolymerisation and associated repolymerisation. In this work, characterisation of the droplets revealed differences in shape and size. As in the work of Donohoe et al. (2008), both spherical and flattened droplets were observed (Fig. S1f and g). There was an overall increase in the average droplet size with increasing pretreatment temperature with droplet sizes in the range of 67– 238 nm, as shown in Table 2. This trend was also reported in the work of Yu et al. (2013b). This could have major repercussions for the enzymatic hydrolysis of the residues as large lignin droplets can reduce access of the enzymes to the cellulose surface (Yu et al., 2013b). Interestingly, for the LHW treatments in this work at the same temperature (195 °C) and different flow rates of 204 ml/ min and 250 ml/min, the average droplet size was the same. Donohoe et al. (2008) observed lignin droplets in the range of 5 nm to 10 lm on both dilute acid- and LHW-pretreated samples from both batch and flow-through type reactors.
P. Reddy et al. / Bioresource Technology 183 (2015) 259–261
In comparison with previous SEM work on batch LHW pretreatment of sugarcane bagasse, the SEM results from the study of Wang et al. (2012) at 180 °C (for 20 min) with a 5% (w/v) solids to liquid ratio do not provide evidence of droplets of lignin on the surface of the fibres. In the work by Zhang and Wu (2014), at temperatures between 160 and 200 °C (for 20 min) with a solids to liquid ratio of 5% (w/v), the authors mention ‘‘particle-sized debris’’ on the surface of pretreated bagasse from the SEM images and from the FT-IR results mention the probability that ‘‘released lignin re-deposited on the material’’. However, globular lignin droplets, as observed in this work, could not be clearly observed from the SEM images presented in the work by Zhang and Wu (2014). In the study by Yu et al. (2013b) with a solids to liquid ratio of 20% (w/v) at 160 °C (for 30 min), droplets were clearly observed in the SEM images. From the above, it seems likely that in addition to temperature and reaction time, the solids to liquid ratio could play an important part in lignin redeposition on pretreated biomass. However, this requires extensive investigation to account for the influence of other variables such as particle size on the occurrence of lignin deposition. 3.2. Transmission electron microscopy The use of TEM with the well-established cytochemical stain i.e. potassium permanganate (Donaldson, 1992; Donohoe et al., 2008), was used to localise lignin across the untreated and pretreated bagasse fibre walls. Fig. S2 (Supplementary data) shows cross-sectional images of untreated and pretreated bagasse fibres. The untreated sample (Fig. S2a) showed a rigid and compact cell wall structure with the middle lamella (ML), primary cell wall (PCW) and S1 and S2 layers of the secondary cell wall (SCW) being well preserved. The pretreated fibres (Fig. S2b–g) displayed a porous ultrastructure due to the removal of hemicellulose and lignin from within the fibre wall. In terms of lignin distribution, from the TEM image of the stained untreated sugarcane bagasse (Fig. S2a), lignin appears relatively evenly distributed with no intensely stained regions. Migration of lignin across the cell wall was observed after staining with potassium permanganate. Lignin molecules appeared to move from the inner SCW toward the outer ML (shown by arrows in Fig. S2c–g). The density of lignin staining increased with temperature, with the highest temperature of 207 °C showing the most intense staining (Fig. S2g). The shapes of the lignin droplets were prominent where disk and globular-shaped lignin droplets were visible in Fig. S2d and g, respectively. For the two experiments at 195 °C with the different flow rates, the slower flow rate (Fig. S2f) appears to have resulted in more coalescence and slower migration of lignin through the fibre wall compared to the higher flow rate (Fig. S2e). From the above results, it is observed that lignin deposition is significant under the conditions investigated. The presence of lignin droplets has an adverse effect on the further processing of pretreated solids, especially for enzymatic hydrolysis. This has prompted investigation of alternative routes such as using LHW as a post-treatment for an organosolv process (Cybulska et al., 2012) or using aqueous supercritical hydrolysis of the pretreated biomass to produce cellulosic sugars (Renmatix, 2015). Also, the role of reactor configuration (batch versus flow-though) requires greater investigation of parameters such as solids to liquid ratios (batch) and flow rates (flow-through) to be better understood for minimising lignin redeposition on pretreated biomass.
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4. Conclusions It has been demonstrated that flow-through LHW treatment of sugarcane bagasse, as with batch treatment, is clearly not exempt from the occurrence of lignin in the hydrolysate repolymerising and depositing on the surface of pretreated fibres. This has been confirmed in this work through the use of FEG-SEM and TEM. The increase in the deposition of lignin droplets (density and size) with increasing temperature observed here requires careful consideration of optimal parameters when designing experiments for further processing of the solid residues such as enzymatic hydrolysis. Acknowledgement Prashant Reddy acknowledges the financial support from the Sugar Milling Research Institute NPC for a research visit to the Hamburg University of Technology. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.02. 057. References Cybulska, I., Brudecki, G., Lei, H., Julson, J., 2012. Optimization of combined clean fractionation and hydrothermal treatment of prairie cord grass. Energy Fuels 26 (4), 2303–2309. Donaldson, L.A., 1992. Lignin distribution during latewood formation in Pinus radiata D. Don. IAWA Bull. 13 (4), 381–387. Donohoe, B.S., Decker, S.R., Tucker, M.P., Himmel, M.E., Vinzant, T.B., 2008. Visualizing lignin coalescence and migration through maize cell walls following thermochemical pretreatment. Biotechnol. Bioeng. 101 (5), 913–925. Kristensen, J.B., Thygesen, L.G., Felby, C., Jørgensen, H., Elder, T., 2008. Cell-wall structural changes in wheat straw pretreated for bioethanol production. Biotechnol. Biofuels 1, 5. Mosier, N.S., 2013. Fundamentals of aqueous pretreatment of biomass. In: Wyman, C.E. (Ed.), Aqueous Pretreatment of Plant Biomass for Biological and Chemical Conversion to Fuels and Chemicals. John Wiley & Sons, Chichester, pp. 129–143. Lewandowski, Z., Beyenal, H., 2013. Fundamentals of Biofilm Research, second ed. CRC Press, Boca Raton, p. 593. Renmatix, 2015. (accessed on 7.01.15). Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2011. Determination of Structural Carbohydrates and Lignin in Biomass, Laboratory Analytical Procedure (LAP). Technical Report NREL/TP-510-42618, Version 0708-2011. National Renewable Energy Laboratory (NREL), Golden, CO, USA. Spurr, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26 (1), 31–43. Wang, W., Zhuang, X., Yuan, Z., Yu, Q., Qi, W., Wang, Q., Tan, X., 2012. Effect of structural changes on enzymatic hydrolysis of eucalyptus, sweet sorghum bagasse, and sugarcane bagasse after liquid hot water pretreatment. Bioresources 7 (2), 2469–2482. Xiao, L., Sun, Z., Shi, Z., Xu, F., Sun, R., 2011. Impact of hot compressed water pretreatment on the structural changes of woody biomass for bioethanol production. Bioresources 6 (2), 1576–1598. Yu, Q., Zhuang, X., Lv, S., He, M., Zhang, Y., Yuan, Z., Qi, W., Wang, Q., Wang, W., Tan, X., 2013a. Liquid hot water pretreatment of sugarcane bagasse and its comparison with chemical pretreatment methods for the sugar recovery and structural changes. Bioresour. Technol. 129, 592–598. Yu, Q., Zhuang, X., Yuan, Z., Qi, W., Wang, W., Wang, Q., Tan, X., 2013b. Pretreatment of sugarcane bagasse with liquid hot water and aqueous ammonia. Bioresour. Technol. 144, 210–215. Zhang, H., Wu, S., 2014. Impact of liquid hot water pretreatment on the structural changes of sugarcane bagasse biomass for sugar production. Appl. Mech. Mater. 472, 774–779.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Structural characterisation of pretreated solids from flow-through liquid hot water treatment of sugarcane bagasse in a fixed-bed reactor Prashant Reddy a,b,⇑, Prabashni Lekha c, Wienke Reynolds d, Christian Kirsch d a
Sugar Milling Research Institute NPC, c/o University of KwaZulu-Natal, Durban 4041, South Africa Department of Chemistry, Durban University of Technology, P.O. Box 1334, Durban 4000, South Africa c CSIR/UKZN, Forestry and Forest Product Research Centre, Durban 4041, South Africa d Institute of Thermal Separation Processes, Hamburg University of Technology, Eißendorfer Strasse 38, D-21073 Hamburg, Germany b
h i g h l i g h t s FEG-SEM and TEM on sugarcane bagasse pretreated with flow-through liquid hot water. Deposition of lignin droplets on pretreated bagasse clearly observed from FEG-SEM. Density of droplets and average droplet size increased with temperature increase. Average droplet sizes were between 67 and 238 nm. Lignin migration across cell wall observed from TEM.
a r t i c l e
i n f o
Article history: Received 17 January 2015 Received in revised form 8 February 2015 Accepted 9 February 2015 Available online 18 February 2015 Keywords: Sugarcane bagasse Liquid hot water Flow-through FEG-SEM TEM
a b s t r a c t Untreated sugarcane bagasse and sugarcane bagasse pretreated with flow-through liquid hot water (LHW) treatment (170–207 °C and 204–250 ml/min) in a fixed-bed reactor have been structurally characterised. Field emission gun scanning electron microscopy (FEG-SEM) and transmission electron microscopy (TEM) were used to investigate changes in the residues, in particular due to the fate of lignin. FEGSEM results show that the LHW treatment modified the surface morphology of the pretreated bagasse with lignin droplets being observed on the fibre surface. TEM showed an increase in the plant cell wall porosity and lignin migration across the plant cell wall. Increases in pretreatment temperature were observed to increase the average size and density of lignin droplets on the fibre surface. The results provide evidence that for LHW flow-through treatment, just as for batch treatment, lignin repolymerisation and deposition on the surface of pretreated sugarcane bagasse is an important consideration. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Biomass pretreatment with liquid hot water (LHW) is a physicochemical process where pressurised water (typically between 160 and 230 °C) is employed. The influence of the reactor configuration in LHW treatment has been debated as flow-through methods have been considered as being superior to batch methods for the solubilisation of lignin (Mosier, 2013). Structural characterisation of pretreated solids from biomass pretreatment processes is important for understanding biomass deconstruction processes, including the fate of lignin.
⇑ Corresponding author at: Sugar Milling Research Institute NPC, c/o University of KwaZulu-Natal, Durban 4041, South Africa. Tel.: +27 31 273 1306; fax: +27 31 273 1302. E-mail address: [email protected] (P. Reddy). http://dx.doi.org/10.1016/j.biortech.2015.02.057 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Lignin has been noted to undergo depolymerisation followed by repolymerisation during LHW pretreatment (Donohoe et al., 2008) and this has important repercussions for pretreatment strategies. Deposition of lignin droplets on pretreated biomass solids has been noted in several studies (Donohoe et al., 2008; Kristensen et al., 2008; Xiao et al., 2011). Structural studies on residues from various pretreatments includes LHW on corn stover (Donohoe et al., 2008), wheat straw (Kristensen et al., 2008); sugarcane bagasse (Wang et al., 2012; Yu et al., 2013a,b; Zhang and Wu, 2014), salt cedar (Xiao et al., 2011); LHW and ammonia on sugarcane bagasse (Yu et al., 2013b); and dilute acid pretreatment on corn stover (Donohoe et al., 2008). There have been few structural studies on LHW-pretreated sugarcane bagasse reported and, to our knowledge, no SEM and TEM work has been reported to date for bagasse residues from flow-through LHW treatment. A key objective for this work was to investigate the extent and nature of the
260
P. Reddy et al. / Bioresource Technology 183 (2015) 259–261
deposition of lignin droplets in the flow-through LHW treatment of sugarcane bagasse at temperatures between 170 and 207 °C with field emission gun scanning electron microscopy (FEG-SEM) and transmission electron microscopy (TEM).
Table 2 Reaction conditions and lignin droplet sizes for pretreated bagasse.
2. Methods 2.1. Materials Washed sugarcane bagasse from a local sugar mill in KwaZuluNatal, South Africa was oven-dried and shipped in vacuum-sealed bags to the Hamburg University of Technology (TUHH) in Germany. The chemical composition of the bagasse, shown in Table 1, was determined with a National Renewable Energy Laboratory (NREL) analytical procedure (Sluiter et al., 2011). The bagasse was not reduced in size or treated further prior to the LHW experiments. Unmilled bagasse has an extremely wide range of particle shapes and sizes. The results from the sieve analysis of the bagasse are shown in Table S1 (Supplementary data).
2.2. Pretreatment The apparatus used in this work, a 3 L high-pressure fixed-bed reactor, was developed at the Institute of Thermal Separation Processes at TUHH. Pressurised (5 MPa) hot water was passed through 0.6 kg of dry bagasse in the reactor for a predetermined amount of time at a specified flow rate. The pretreated solids were retrieved from the reactor once the equipment cooled down sufficiently. Six experiments were performed at temperatures between 170 and 207 °C and flow rates between 200 and 250 ml/min; five of these experiments were performed at the same flow rate (250 ml/min) with one experiment at a lower flow rate of 204 ml/min. The reaction conditions are shown in Table 2. The porosity of the fixed bed (Lewandowski and Beyenal, 2013) was calculated from the void volume and the reactor volume (2.98 L) as follows:
Porosity ¼ ðVoid volume=Reactor volumeÞ
ð1Þ
The average void volume and porosity values for the fixed bed in this work are 2.3 L and 0.77, respectively.
2.3. Analyses 2.3.1. Field emission gun-scanning electron microscopy (FEG-SEM) The oven-dried pretreated bagasse samples were mounted on aluminium stubs using double-sided carbon tape. The samples were coated with gold using a Polaron SC500 sputter coater and viewed with a Carl Zeiss Ultra Plus FEG-SEM at an accelerating voltage of 5 kV. The FEG-SEM images were obtained at magnifications ranging from 200 to 100,000. Images captured at the highest magnification were analysed using Image Pro Plus V7.0 to obtain diameters of the lignin droplets.
Table 1 Chemical composition of untreated sugarcane bagasse on a dry weight basis. Carbohydrates (%)
a b c d
Lignin (%)
Glucan
Xylan
Ara + man
42.5
24.4
2.9
a
Arabinan and mannan. Acid-soluble lignin. Acid-insoluble lignin. Water and ethanol extractives.
ASL 4.6
b
AIL
Extractives (%)d
Ash (%)
5.6
1.2
c
18.8
a
Temperature (°C)
Flow rate (ml/min)
Reaction time (min)
Droplet sizea (nm)
170 178 192 195 195 207
250 250 250 250 204 250
35 35 35 35 44 35
66.5 93.7 104.3 100.3 100.4 238.3
Average droplet size.
2.3.2. Transmission electron microscopy (TEM) The oven dried bagasse samples were placed in fresh 100% Spurr’s epoxy resin (Spurr, 1969). The resin moulds were incubated for 72 h in a desiccator with activated silica gel and phosphorous pentoxide. Resin changes were performed daily and after 3 days, the resin embedded samples were polymerised in an oven (70 °C for 8 h). The resin blocks were sectioned using a Leica Ultracut EM UC7 ultramicrotome. Ultra-thin sections, approximately 100 nm thick, were collected on copper grids. Thereafter, the grids were stained for 30 min with a 1% potassium permanganate solution prepared in 0.1% sodium citrate for lignin localisation (Donaldson, 1992). The samples were viewed at 100 kV with a JEOL 1010 TEM instrument.
3. Results and discussion 3.1. FEG-SEM FEG-SEM micrographs of the untreated sample and the pretreated samples are shown in Fig. S1 (Supplementary data). The zoomed inlays provide a closer view of the surface characteristics of the fibres. The untreated bagasse fibres (Fig. S1a) showed a smooth surface with no significant deposits. The pretreated bagasse fibres (Fig. S1b–g) have a rougher and corrugated appearance with the presence of deposits. Deposits were also observed by Donohoe et al. (2008) on dilute acid- and LHW-pretreated corn stover and were studied using FTIR, NMR, antibody labelling and cytochemical staining, where it was shown that the deposits contained lignin. In our study, cytochemical staining was also used, and dense staining which has previously been shown to represent lignin was obtained (Donaldson, 1992; Donohoe et al., 2008), as discussed later in the TEM results. The micrographs show that the density of lignin droplets generally increased with an increase in pretreatment temperature (Fig. S1b–g), as in the work of Xiao et al. (2011) on salt cedar. With the melting point of lignin between 90 and 190 °C (Mosier, 2013), it is conceivable that higher temperatures favour increased lignin depolymerisation and associated repolymerisation. In this work, characterisation of the droplets revealed differences in shape and size. As in the work of Donohoe et al. (2008), both spherical and flattened droplets were observed (Fig. S1f and g). There was an overall increase in the average droplet size with increasing pretreatment temperature with droplet sizes in the range of 67– 238 nm, as shown in Table 2. This trend was also reported in the work of Yu et al. (2013b). This could have major repercussions for the enzymatic hydrolysis of the residues as large lignin droplets can reduce access of the enzymes to the cellulose surface (Yu et al., 2013b). Interestingly, for the LHW treatments in this work at the same temperature (195 °C) and different flow rates of 204 ml/ min and 250 ml/min, the average droplet size was the same. Donohoe et al. (2008) observed lignin droplets in the range of 5 nm to 10 lm on both dilute acid- and LHW-pretreated samples from both batch and flow-through type reactors.
P. Reddy et al. / Bioresource Technology 183 (2015) 259–261
In comparison with previous SEM work on batch LHW pretreatment of sugarcane bagasse, the SEM results from the study of Wang et al. (2012) at 180 °C (for 20 min) with a 5% (w/v) solids to liquid ratio do not provide evidence of droplets of lignin on the surface of the fibres. In the work by Zhang and Wu (2014), at temperatures between 160 and 200 °C (for 20 min) with a solids to liquid ratio of 5% (w/v), the authors mention ‘‘particle-sized debris’’ on the surface of pretreated bagasse from the SEM images and from the FT-IR results mention the probability that ‘‘released lignin re-deposited on the material’’. However, globular lignin droplets, as observed in this work, could not be clearly observed from the SEM images presented in the work by Zhang and Wu (2014). In the study by Yu et al. (2013b) with a solids to liquid ratio of 20% (w/v) at 160 °C (for 30 min), droplets were clearly observed in the SEM images. From the above, it seems likely that in addition to temperature and reaction time, the solids to liquid ratio could play an important part in lignin redeposition on pretreated biomass. However, this requires extensive investigation to account for the influence of other variables such as particle size on the occurrence of lignin deposition. 3.2. Transmission electron microscopy The use of TEM with the well-established cytochemical stain i.e. potassium permanganate (Donaldson, 1992; Donohoe et al., 2008), was used to localise lignin across the untreated and pretreated bagasse fibre walls. Fig. S2 (Supplementary data) shows cross-sectional images of untreated and pretreated bagasse fibres. The untreated sample (Fig. S2a) showed a rigid and compact cell wall structure with the middle lamella (ML), primary cell wall (PCW) and S1 and S2 layers of the secondary cell wall (SCW) being well preserved. The pretreated fibres (Fig. S2b–g) displayed a porous ultrastructure due to the removal of hemicellulose and lignin from within the fibre wall. In terms of lignin distribution, from the TEM image of the stained untreated sugarcane bagasse (Fig. S2a), lignin appears relatively evenly distributed with no intensely stained regions. Migration of lignin across the cell wall was observed after staining with potassium permanganate. Lignin molecules appeared to move from the inner SCW toward the outer ML (shown by arrows in Fig. S2c–g). The density of lignin staining increased with temperature, with the highest temperature of 207 °C showing the most intense staining (Fig. S2g). The shapes of the lignin droplets were prominent where disk and globular-shaped lignin droplets were visible in Fig. S2d and g, respectively. For the two experiments at 195 °C with the different flow rates, the slower flow rate (Fig. S2f) appears to have resulted in more coalescence and slower migration of lignin through the fibre wall compared to the higher flow rate (Fig. S2e). From the above results, it is observed that lignin deposition is significant under the conditions investigated. The presence of lignin droplets has an adverse effect on the further processing of pretreated solids, especially for enzymatic hydrolysis. This has prompted investigation of alternative routes such as using LHW as a post-treatment for an organosolv process (Cybulska et al., 2012) or using aqueous supercritical hydrolysis of the pretreated biomass to produce cellulosic sugars (Renmatix, 2015). Also, the role of reactor configuration (batch versus flow-though) requires greater investigation of parameters such as solids to liquid ratios (batch) and flow rates (flow-through) to be better understood for minimising lignin redeposition on pretreated biomass.
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4. Conclusions It has been demonstrated that flow-through LHW treatment of sugarcane bagasse, as with batch treatment, is clearly not exempt from the occurrence of lignin in the hydrolysate repolymerising and depositing on the surface of pretreated fibres. This has been confirmed in this work through the use of FEG-SEM and TEM. The increase in the deposition of lignin droplets (density and size) with increasing temperature observed here requires careful consideration of optimal parameters when designing experiments for further processing of the solid residues such as enzymatic hydrolysis. Acknowledgement Prashant Reddy acknowledges the financial support from the Sugar Milling Research Institute NPC for a research visit to the Hamburg University of Technology. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.02. 057. References Cybulska, I., Brudecki, G., Lei, H., Julson, J., 2012. Optimization of combined clean fractionation and hydrothermal treatment of prairie cord grass. Energy Fuels 26 (4), 2303–2309. Donaldson, L.A., 1992. Lignin distribution during latewood formation in Pinus radiata D. Don. IAWA Bull. 13 (4), 381–387. Donohoe, B.S., Decker, S.R., Tucker, M.P., Himmel, M.E., Vinzant, T.B., 2008. Visualizing lignin coalescence and migration through maize cell walls following thermochemical pretreatment. Biotechnol. Bioeng. 101 (5), 913–925. Kristensen, J.B., Thygesen, L.G., Felby, C., Jørgensen, H., Elder, T., 2008. Cell-wall structural changes in wheat straw pretreated for bioethanol production. Biotechnol. Biofuels 1, 5. Mosier, N.S., 2013. Fundamentals of aqueous pretreatment of biomass. In: Wyman, C.E. (Ed.), Aqueous Pretreatment of Plant Biomass for Biological and Chemical Conversion to Fuels and Chemicals. John Wiley & Sons, Chichester, pp. 129–143. Lewandowski, Z., Beyenal, H., 2013. Fundamentals of Biofilm Research, second ed. CRC Press, Boca Raton, p. 593. Renmatix, 2015. (accessed on 7.01.15). Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2011. Determination of Structural Carbohydrates and Lignin in Biomass, Laboratory Analytical Procedure (LAP). Technical Report NREL/TP-510-42618, Version 0708-2011. National Renewable Energy Laboratory (NREL), Golden, CO, USA. Spurr, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26 (1), 31–43. Wang, W., Zhuang, X., Yuan, Z., Yu, Q., Qi, W., Wang, Q., Tan, X., 2012. Effect of structural changes on enzymatic hydrolysis of eucalyptus, sweet sorghum bagasse, and sugarcane bagasse after liquid hot water pretreatment. Bioresources 7 (2), 2469–2482. Xiao, L., Sun, Z., Shi, Z., Xu, F., Sun, R., 2011. Impact of hot compressed water pretreatment on the structural changes of woody biomass for bioethanol production. Bioresources 6 (2), 1576–1598. Yu, Q., Zhuang, X., Lv, S., He, M., Zhang, Y., Yuan, Z., Qi, W., Wang, Q., Wang, W., Tan, X., 2013a. Liquid hot water pretreatment of sugarcane bagasse and its comparison with chemical pretreatment methods for the sugar recovery and structural changes. Bioresour. Technol. 129, 592–598. Yu, Q., Zhuang, X., Yuan, Z., Qi, W., Wang, W., Wang, Q., Tan, X., 2013b. Pretreatment of sugarcane bagasse with liquid hot water and aqueous ammonia. Bioresour. Technol. 144, 210–215. Zhang, H., Wu, S., 2014. Impact of liquid hot water pretreatment on the structural changes of sugarcane bagasse biomass for sugar production. Appl. Mech. Mater. 472, 774–779.
