Critical Point Drying : An Effective Tool for Direct 2 Measurement of the Surface Area of Pretreated 3 Cellulosic Biomass 4

Surface area and pore size distribution of Eucalyptus samples pretreated by different 16 methods were determined by the Brunauer-Emmett-Teller (BET) technique. Three methods were 17 applied to prepare cellulosic biomass samples for BET measurements: air, freeze, and critical point 18 drying (CPD). Air and freeze drying caused severe collapse of biomass pore structures, but CPD 19 effectively preserved biomass morphology. Surface area of CPD prepared Eucalyptus samples was 20 determined to be 58–161 m2/g, whereas air and freeze dried samples were 0.5–1.3 and 1.0–2.4 m2/g, 21 respectively. Average pore diameter of CPD prepared Eucalyptus samples were 61–70Å. CPD 22 preserved Eucalyptus sample morphology by replacing water with a non-polar solvent, CO2 fluid, 23 which prevented hydrogen bond reformation in the cellulose. 24


Introduction
For efficient sugar fractionation from lignocellulosic biomass, physical contact between cellulose and cellulase enzymes is necessary.Therefore, cellulose specific surface area available for enzyme contact is one of the most important factors determining the rate and extent of enzymatic hydrolysis of biomass [1][2][3].Since the average size of cellulase enzymes is approximately 5.1 nm, internal surface of pores greater than 5.1 nm should be particularly effective for enzymatic hydrolysis [4].Various pretreatments applied for improved enzymatic digestibility also increase surface area, due not only to removal of hemicellulose and/or lignin, but also cellulose swelling.
Solute exclusion is the most widely employed method, but has several drawbacks, including relatively low accuracy and limitations on pore size ranges that can be determined.For example, given the unavailability of dextran molecule probes, only pore sizes up to 56 nm can be measured [5][6][7]15].However, non-ionic surfactant pretreatment of lignocellulosic biomass can produce pores up to 100 nm diameter [16].Inaccurate estimation can also arise from the water competition with the solute probes [1] and/or solute concentration measurement errors.Reported biomass surface area varies greatly, from 20 to over 1,500 m 2 /g [7,15].Simons' staining method has also been used to determine the feasibility of enzymatic hydrolysis of substrates [10][11][12]17], although this technique can provide only semi-quantitative information.It also has similar limitations to solute exclusion, since it also employs dye solutes for the measurement.In the other hand, NMR techniques employed for biomass surface area measurement require complicated experiment set-ups [13][14].
The Brunauer-Emmett-Teller (BET) technique employing N2 adsorption has many advantages, including high accuracy and can measure 0.4-300 nm pore sizes [18].Therefore, the BET technique is widely used to determine surface area and pore size distribution of porous materials, although it is applicable only to dried samples.Few studies have considered BET based internal surface area measurement of pretreated biomass [11,19,20].However, these indicate that BET measured surface areas differ significantly from those measured by other methods, i.e. solute exclusion, dye staining, or probes.For example, Wiman et al. compared steam pretreated spruce surface area by BET and Simmons' staining methods [11].BET measurement biomass samples were oven dried at 30°C for 24 hours to minimize structural changes.Pretreated biomass surface area was 1.3-8.2m 2 /g, far smaller than those measured by the staining method (53-64 m 2 /g).The small BET based surface area was attributed mainly to pore collapse during air drying [12,21].
To avoid pore collapse, freeze drying has been applied to cellulose and yellow poplar (Liriodendron tulipifera) wood flour pretreated by organosolv process [19,20], but the biomass surface area remains small, 5-39 and 1.8 m 2 /g for cellulose and organosolv pretreated yellow poplar, respectively.Esteghalian et al. investigated the effects of drying conditions on enzymatic hydrolysis of Douglas fir (Pseudotsuga menziesii) kraft pulp using air, oven, and freeze drying; and compared dried biomass enzymatic digestibilities.No significant differences between air and freeze dried biomass samples were evident [17].Thus, there remains no successful method that prevents pore collapse when drying cellulosic biomass.Therefore, cellulosic biomass surface area is mainly determined by in situ measuring techniques.
Critical point drying (CPD) is widely used to dry delicate samples for Scanning Electron Microscope (SEM) applications and could be a viable option for biomass sample preparation, while maintaining the original morphology.Since pore collapse is caused by removal of water, a polar solvent, from the biomass [12,21], this study attempted to prevent pore collapse by replacing water with non-polar solvents before drying.Since CPD employing a non-polar solvent (liquid CO2) is done at ~36°C, deterioration caused by high drying temperature can be minimized.There have been no previous reports on direct measurement of surface area and/or pore size distribution for CPD pretreated cellulosic biomass.
This study prepared Eucalyptus wood flour samples using three pretreatment methods to measure total surface area and pore size distribution, and drying conditions effects on surface area and pore size distribution were compared.This work will contribute to deeper understanding of the physical effects of surface area and pore size distribution on enzymatic hydrolysis rates of cellulosic biomass.Peer-reviewed version available at Polymers 2018, 10, 676; doi:10.3390/polym10060676

Pretreatments of Lignocellulosic biomass
Eucalyptus samples as lignocellulosic biomass were pretreated by dilute acid (DA), steam after NaOH impregnation, and ALEW.For DA pretreatment, 60 g (OD) of biomass sample was immersed in 160 mL of 3% (w/w) sulfuric acid and maintained at 121°C for 2 h.The slurry was allowed to stand overnight and then filtered (Whatman No. 1 glass filter) to recover insoluble solids.The recovered cellulosic biomass was washed with distilled water several times.The process of NaOH-steam pretreatment followed the procedure detailed previously [22].60 g (OD) of biomass sample was soaked in 480 mL of 3% (w/v) sodium hydroxide solution at room temperature.The slurry was allowed to stand overnight and then filtered (Whatman No. 1 glass filter) to recover insoluble solids.The recovered solids were transferred to an autoclave (working volume = 1 L) and steam pretreatment was conducted at 160°C for 12 min under 20 bar nitrogen atmosphere.For ALEW pretreatment, 60 g (OD) of biomass sample was immersed in 600 mL ALEW and maintained at 180°C for 1 h in an autoclave (working volume = 1 L) under 20 bar nitrogen atmosphere.

Drying of cellulosic biomass sample
The pretreated samples were dried by air, freeze, and critical point drying methods.For air drying (AD), approximately 5 g of each pretreated cellulosic biomass samples was dried in a vacuum oven at 50°C for 48 h.Constant weight was confirmed after drying.The freeze drying (FD) process of cellulosic biomass sample followed that detailed in the literature [19].Approximately 5 g of pretreated cellulosic biomass sample was frozen at -60°C for 48 h, and then vacuumed in a FD apparatus for 72 h.Critical point drying (CPD) was performed using a critical point dryer (13200J-AB, SPI Supplies, West Chester, PA, USA), following the procedure provided in the operation manual.Approximately 5 g of pretreated biomass samples were placed in 100 mL of 30, 50, 70, 90, 95 and 100% ethanol for 15 min, then immersed in acetone solution for a further 15 min.
After a series of solvent exchanges, acetone in the samples became replaced by liquid CO2, and the samples were then critical point dried at 36°C.

Surface area and pore size measurements of biomass sample
We used the BET method to determine surface area, average pore diameter, and total pore volume for AD, FD, and CPD biomass samples.N2 adsorption was measured using an accelerated surface area and porosity analyzer (ASAP 2420, Micromeritics Inc., Norcross, GA, USA).N2 adsorption isotherms were obtained by measuring the amount of gas adsorbed across a range of relative pressures (P/P0) at constant temperature (-196°C, liquid nitrogen phase temperature), where P and P0 are the equilibrium and saturation pressures of adsorbate gas at the temperature of adsorption, respectively.Desorption isotherms were achieved by measuring the amount of N2 gas removed as pressure decreased.Subsequently the specific surface area was calculated from the adsorption isotherms using BET theory.Total pore volume was estimated from the amount of N2 gas adsorbed at 0.98 relative pressure, under the following assumptions: pores were filled with liquid nitrogen, and adsorption average pore size was derived from 4V/A, where V is the total pore volume, and A is the surface area, corresponding to the assumed cylindrical pore model.Pore size distribution was obtained from experimental isotherms as detailed elsewhere [24].Prior to BET analysis, pre-dried samples were degassed at 90°C for 0.5 h and then again at 105°C for 4 h.

FT-IR analysis
Structural changes of raw and pretreated samples were examined with Fourier transform infrared spectroscopy (FT-IR).The samples were ground into powder and sieved through 149 μm mesh.FT-IR spectra were recorded on an FTS-175C (Bio-Rad Laboratories Inc., Hercules, CA, USA) equipped with mercury cadmium telluride detector, using KBr pellets.All spectra were collected at 4 cm -1 resolution with 32 scans in the range 4000-500 cm -1 .Surface area and pore size distribution of sample were determined by BET and the differences were compared among CPD, AD, and FD pretreatments. Figure 1 shows N2 adsorption-desorption isotherms for ALEW, DA, and NaOH-steam pretreated samples.The isotherms are typical type IV hysteresis loops (as classified by IUPAC), consistent with mesoporous materials where an adsorbate monolayer is formed on the pore surface at low pressures followed by multilayer formation.The hysteresis loop originates from capillary condensation in meso and macropores, and can have a wide variety of shapes depending on the pore geometries.Specific pore structures can often be identified from their hysteresis loop shape based on the empirical IUPAC classification.AD and FD samples also exhibited H4 type hysteresis loops, although somewhat smaller.This hysteresis type is consistent with narrow slit shaped pores and/or aggregated particles [25].All CPD samples exhibited H2 type hysteresis loops, where pore size and pore shape distribution are not welldefined, i.e., irregular.The desorption isotherm steep slope, observed for all CPD samples, typically indicates pore interconnection [26].
Figure 2 shows the corresponding biomass pore size distribution, determined by the BJH method.As reported elsewhere, AD and FD samples' large pores indicate collapse of most small pore structures during drying [26].However, CPD appears to maintain the micropore structures in the pretreated Eucalyptus samples, with average pore size approximately 6.2 nm, i.e., within the mesopore range.Table 1 shows detailed quantitative data for the ALEW pretreated Eucalyptus samples.Surface area of pretreated Eucalyptus sample varied greatly with the drying conditions.The smallest surface areas were from AD samples (0.5-1.3 m 2 /g), with FD samples having approximately twice the area (1.04-2.44 m 2 /g), although they had comparable small pore volumes (0.002-0.005 and 0.005-0.015cm 3 /g, respectively); whereas CPD sample surface area and pore volume were considerably larger (58.5-161.5 m 2 /g and 0.103-0.249cm 3 /g, respectively).
Surface area of AD Eucalyptus samples was somewhat smaller than previously reported for SO2-steam pretreated spruce (1.3-8.2 m 2 /g) [11], which is possibly due to the different feedstocks and pretreatment conditions.However, surface area of SO2-steam pretreated spruce varied greatly with pretreatment conditions tested in that study.The surface area of FD Eucalyptus samples was comparable to previously reported for yellow poplar (L.tulipifera) (1.80 m 2 /g) prepared by FD after organosolv pretreatment [20].Table 3 compares surface areas of CPD Eucalyptus sample with those previously reported.The CPD surface area for DA pretreated Eucalyptus (57.3 m 2 /g) was very close to SO2-steam pretreated spruce determined by dye staining (58.5 m 2 /g) [11].
Thus, only CPD effectively maintained the pretreated biomass morphology, although both of FD and CPD have been previously reported as effective.Both drying methods have been widely employed in SEM specimen preparation [27].The poor performance of FD for water-swollen lignocellulose reported here is probably associated with the cellulose content of feedstock, since cellulose is deformed by hornification, a consequence of irreversible changes to the cell wall structure [12].
Table 1.Mean surface areas, pore diameters and total pore volumes for ALEW pretreated Eucalyptus samples.
Although the exact hornification mechanism is unclear, one possible explanation is hydrogen bond breaking and reforming corresponding to cellulose wetting and drying, respectively.When cellulose is wet, fibers swell by hydrogen bond breakage, but shrink with reforming of hydrogen bonds upon drying.In any case, the reason for the poor FD performance remains unknown.
Possibly FD removes only free water, but not the bound water, inducing cellulosic collapse [28].
However, hydrogen reforming could be prevented if the bound water in pretreated Eucalyptus samples was replaced by a non-polar solvent prior to drying.To examine this hypothesis, we performed an FT-IR analysis to examine the hydrogen bonds in ALEW pretreated AD and CPD Eucalyptus samples, as shown in Figure 3.
The -OH stretching region of the FT-IR absorbance band, 3000-4000 cm -1 , is reported to contain information on hydrogen bonding in cellulose [29,30], and large peaks were observed between 3340-3380 cm -1 .ALEW pretreated CPD Eucalyptus sample peaks are significantly smaller than those of untreated and ALEW pretreated AD Eucalyptus.AD Eucalyptus peak height was very similar to that of untreated Eucalyptus.Thus, since peak height is proportional to hydrogen bonds in the biomass sample, CPD Eucalyptus had significantly fewer hydrogen bonds than untreated or AD Eucalyptus.Overall, hydrogen bonds in untreated Eucalyptus were broken by water during pretreatment and subsequently reconnected upon AD.However, hydrogen bond reformation upon drying was successfully prevented for CPD Eucalyptus samples, replacing bound water with a non-polar solvent, liquid CO2, prior to drying.Finally, the wide peak at 3388.9 cm -1 shifted to higher frequency after drying of ALEW pretreated Eucalyptus.This shift was more significant with AD, where hydrogen bonding became stronger than was the case for untreated sample.Biomass pore volume and surface area provide enzymes with sufficient access and adsorption to cell surfaces, thus significantly affecting enzymatic hydrolysis of cellulose.Therefore, we determined pore volumes for the different pore sizes, as shown in Table 5. ALEW pretreated Eucalyptus samples exhibited the largest pore volume accessible to enzymes, with NaOH pretreated samples exhibiting the highest pore volumes for pores smaller than 2 nm and ALEW pretreated samples having the highest pore volumes for larger pore sizes.The 25% larger surface area of ALEW pretreated samples is remarkable, even after considering the higher pore volume for larger than 2 nm pores.This larger surface area is probably due to the high lignin content, since most lignin micropores are smaller than 0.6 nm [31].

Conclusions
Critical point drying was shown to effectively prevent cellulosic pore collapse upon drying and hence enable direct determination of specific surface area and pore size distribution for pretreated Eucalyptus samples using the BET method.Comparing hydrogen bonds for the various drying methods, reformation of hydrogen bonds upon drying is mainly responsible for pore collapse.Thus, hydrogen bond reformation was successfully prevented in CPD by replacing water with liquid CO2, a non-polar solvent, before drying.
The measurement technology developed in this study will provide more detailed quantitative data on surface area and pore size distribution of water-swollen biomass.
Surface areas of CPD Eucalyptus samples were 58-161 m 2 /g, comparable to those determined by indirect measuring methods; whereas sulfuric acid pretreatment yielded considerably smaller surface area with larger average pore diameter, ALEW pretreatment produced the highest surface area, and NaOH steam pretreatment produced somewhat smaller surface area.

2. 1 .
Materials Eucalyptus (E.grandis) wood chips were supplied by Dr. Zhuang of GuangZhou Institute of Energy Conversion, Chinese Academy of Science (GIEC) in China, knife milled by Wiley mill (Mini Wiley mill, Thomas Scientific, Swedesboro, NJ, USA) and screened to a nominal size of 20-60 mesh.Alkaline electrolyzed water (ALEW) was provided by Gendocs Inc. (Daejeon, Korea) with pH = 12.2 and ORP < -795 mV.All other reagents and chemicals were analytical grade and purchased from either Sigma-Aldrich (St. Louis, MO, USA) or local suppliers in Korea.Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 25 May 2018 doi:10.20944/preprints201805.0365.v1

3. 2 .
Pretreatment conditions effects on surface area and pore size distribution Since pore volumes varied sharply with pretreatment methods (Figure4), we investigated pretreatment condition influences on pore volumes, surface area, and pore size distribution for CPD Eucalyptus samples.Alkali pretreatment yielded biomass with higher pore volume and surface area than those achieved by acid pretreatment.Swelling effects of alkali pretreatment have been reported previously.Huang et al. showed that NaOH pretreated corn cob had approximately 40% larger surface area (57.4 m 2 /g) than the corresponding sulfuric acid pretreated sample[7].In the current study, the ALEW pretreatment exhibited the strongest swelling effect on Eucalyptus samples with approximately 20% higher pore volume but similar average pore size, hence approximately 20% larger surface area than NaOH-steam pretreatment.

Figure 4 .
Figure 4. Pretreatment method effects on pore size distribution for ALEW critical point dried Eucalyptus samples.

Table 2 .
Drying method effect on BET surface area.

Table 3 .
Comparison of BET results with those determined by non-drying measurement techniques.

Table 4
[7]marizes the effects of these and the other quantified pretreatment on pore volume and surface area of Eucalyptus samples.DA pretreatment exhibits lowest pore volume and smallest surface area, approximately 45% of the values from NaOH pretreated Eucalyptus samples.The reason for the smaller surface area with DA pretreatment is unclear, although it is likely due to cellulose aggregation[7].

Table 4 .
Effects of pretreatment conditions on surface areas, average pore sizes and pore volumes of CPD Eucalyptus samples.

Table 5 .
Effects of pretreatment conditions on micropore volumes of Eucalyptus samples.