Introduction
The future depletion of fossil carbon reserves/resources and global warming has meant that biomass valorisation is required as a sustainable and green route for producing C5/C6 sugars, platform chemicals and fuels. Cellulose can be easily obtained from biomass such as grasses, wood and other agriculture residuals and can be converted to sugar monomers/oligomers via various routes such as hydrolysis (cellulolysis), pyrolysis (thermolysis) and enzymatic hydrolysis [1,2]. Among these, cellulolysis is a preferable method to obtain sugars being a mild and clean process. In cellulolysis, acid catalysts (e.g., mineral or solid acids) are commonly used to promote proton transfer to break β-1,4 glycosidic bonds [3‒7]. However, the corrosive nature of acids, as well as the associated separation issues (e.g., the liquid-phase separation of mineral acids from the soluble products and the solid-phase separation of solid catalysts from the cellulose residuals), hinder their operations at scales [8,9]. Although enzymatic hydrolysis is highly selective to glucose in reducing sugar (above 83%) [10], the long reaction time (e.g., 72 h), highly diluted system and separation issues make it impractical for biomass valorisation [2,11,12]. Microwave (MW) treatment has shown to be able to intensify various processes including hydrolysis [13‒16]. To intensify cellulolysis, especially under catalyst-free conditions, microwave irradiation was found favourable to promote the transformation of cellulose to C6 molecules with high selectivities [13].
Various theories were proposed to explain the observed phenomena in experimental studies of cellulolysis in the MW. In general, the bulk temperature phenomenon by the MW heating (regarding the heat-generating capacity of the MW system) contributed to the hydrolysis since the MW systems generally outperformed the conventional hydrothermal systems [17], especially with ionic liquids under the MW via the ionic conduction mechanism [18‒20]. Specific MW effects were also reported recently, indicating that a molecular level interaction between the MW and cellulose (via the primary alcohol groups, i.e., -CH2OH groups) is responsible for transferring the MW energy to their surrounding molecular structure to initiate the cleavage of polysaccharide chains [13]. The cellulose samples used in the hydrolysis research are largely crystalline [18,21] with amorphous cellulose found to be unstable in aqueous systems forming partially crystalline cellulose II [22]. Therefore, cellulose is very likely to be MW-transparent as the polysaccharide chains are constrained in the crystal lattice and not able to respond to the MW irradiation microscopically. The reported fast MW depolymerisation of the cellulose in the literature [13] might be accounting for the amorphous part which takes 13% of the microcrystalline cellulose (MCC). In MW chemistry, the MW-solvent interaction is responsible for MW heating (via the dielectric heating) [23], in which the high rotational velocity of the polar solvent molecules (caused by the oscillating electric field of the MW) results in molecular friction and heat generation [24,25]. Hence, under MW irradiation, the interaction between the macroscopic cellulose and the highly excited vibrating water molecules should be responsible for the effective performance observed in the cellulolysis experiments. Herein, we performed a systematic investigation of the MW-assisted catalyst-free hydrolysis of fibrous cellulose (FC) at 200°C aimed at revealing the pathway of the MW promoted cellulolysis of FC.
Materials and methods
Materials
Cellulose (fibres, medium), D-(+)-glucose (ACS Reagent), D-(–)-fructose (BioReagent), D-(+)-cellobiose (BioReagent), 1,6-anhydro-β-D-glucopyranose (levoglucosan, 99%), D-(+)-xylose (≥99%), furfural (ACS reagent, 99%) and 5-hydroxymethylfurfural (5-HMF, 99%) were all purchased from Sigma Aldrich and used as received. Sulfuric acid (95%) was obtained from Fisher Scientific. Ethanol (99.7%-100%, absolute) was purchased from VWR International. Ultrapure water (Direct-Q® Water Purification System, 18.2 MW∙cm@25°C) was used in this work.
MW-assisted and hydrothermal (HT) hydrolysis of fibrous cellulose and raw biomass
Hydrolysis of fibrous cellulose was carried out using a CEM Discover SP-D MW reactor in 35 mL Pyrex vials sealed by pressure caps (with TFM septa). The maximum pressure of the system was set as 250 PSI, and experiments were run in the Ramp-to-Temperature mode under stirring at ca. 570 r/min. A standard procedure of the cellulolysis experiment is described as: (i) FC suspended in water was prepared by stirring FC (300 mg) with the deionised water (10 mL) in the Pyrex vial (2 min); (ii) the vial containing the suspension was transferred to the MW reactor for treatment (temperature programme: room temperature to 200°C over 5 min with a maximum power input of 150 W, then at 200°C for various holding time of 2.5 min to 4 h at 50 W, the system pressure was monitored as ca. 200 PSI); (iii) the hydrolysed products was centrifuged at 4400 r/min to separate the solid residual from the liquid fraction.
The centrifuged FC were dried in the oven at 60°C for 48 h and then weighted to calculate the cellulose conversion (Xcellulose) according to Eq. (1). For the selectivity (Sn), which is the relative selectivity based on the ratio of the absolute concentration of a product (Cn) divided by the overall concentration of all identified products in the liquid phase (Ctotal) by high performance liquid chromatography (HPLC), as defined in Eq. (2).
where m0 and mt are the mass of cellulose at the initial time and sampling time, respectively. mt was corrected according to the solid mass loss due to the work-up which was measured as (7.2±2)% using blank experiments.
The obtained liquid fractions were filtered (Millex filter, 0.45 mm, sterile) prior to the HPLC analysis which was performed on a Thermo U3000 HPLC equipped with a Bio-rad Aminex HPX-87H Column, a refractive index (RI) detector (for sugar) and a UV detector (for 5-HMF and furfural, wavelength adjusted at 210 nm). Relevant HPLC conditions are: sample injection volume= 10 mL, oven temperature= 50°C, RI detector temperature= 50°C; UV detector wavelength= 210 nm; and mobile phase= 0.005 mol/L sulfuric acid aqueous solution, flow rate= 0.6 mL/min. External standard methods were used for calibrations in the HPLC analysis (Fig. S1, cf. Electronic Supplementary Material (ESM)).
For comparison, conventional HT hydrolysis of FC (300 mg in 10 mL water) was also carried out using an autoclave reactor (with a 50 mL Teflon liner) at 200°C for 1 h and 24 h (in a Memmert oven). Catalyst-free MW treatment of cellulose, xylose and cellobiose (2000 mg/L) was performed at 200°C using the same MW programme as in the cellulolysis. MW-assisted treatment of the raw biomass (i.e., maple leaves and fresh grass) were performed at 200°C without the catalyst for 1 h. Prior to the hydrolysis under the MW irradiation, maple leaves were collected and dried at 100°C and fresh grass was firstly treated in methanol to remove chlorophyll and then dried at 100°C.
Characterisation of materials
Scanning electron microscopy (SEM) images of materials were obtained by using a FEI Quanta 200 ESEM in high voltage mode of 20 kV. Platinum coating of samples was performed using an Emitech K550X sputter coater under vacuum conditions of 1 × 10-4 mbar. X-ray diffraction (XRD) and SEM characterisation of materials was performed. XRD patterns of the FC (fresh and spent ones) and microcrystalline were obtained on PANalytical X’PERT powder diffractometer using CuKa1 (l = 1.5406 Å) radiation (at 30 kV and 30 mA) with a scanning rate of 2°/min in a range of from 5° to 90°.
Results and discussion
Comparative study of catalyst-free hydrolysis of fibrous cellulose by microwave and conventional hydrothermal heating
Based on the MW input (50 W) and reaction volume (i.e., 10 mL), the MW density (rMW) of the current system is estimated as 5 kW/L. Under this specific condition, the softening temperature of FC in pure water was determined as 200°C, evidenced by the sudden conversion of FC to glucose (cf. ESM). As shown in Table S1 (cf. ESM), a significant increase of glucose yield in the liquid fraction (by ca. 2502%) was measured by raising the system temperature from 180°C to 200°C. A relatively high MW power of 150 W was used in the temperature ramp from the room temperature to 200°C (5 min). Therefore, in order to decouple the effect of 150 W power input on the cellulolysis system, an experiment with a short reaction time of 2.5 min was performed, showing no quantitative conversion of cellulose fibres (0.7%), as well as no production of C5/C6 sugars and biochemicals (Figs. 1(a) and 1(b)). Extending the MW treatment time to 1 and 4 h was beneficial to the conversion of FC into glucose, 5-HMF and furfural, as shown in Fig. 1 (relevant selectivities are 43.7%, 30.1% and 14.5%, respectively, after 4 h MW treatment). Under acid-free conditions, conventional HT treatment was found to be ineffective in the hydrolysis of FC, as observed from the comparative results of 1 h and 24 h reaction in Figs. 1(b–d) (the relevant HT conditions are as in ESM). Although a 60.7% conversion of cellulose was achieved by the HT treatment (Fig. 1(a)) at 200°C, the FC was carbonised and converted into a dark, sticky residue after 24 h (Fig. S2(a)) with no relevant products detected in the liquid fraction. These experiments demonstrated that the MW irradiation (ρMW = 5 kW/L) was needed to achieve the selective conversion of FC into C5/C6 sugars and biochemicals.
Production distribution in the liquid phase from MW-assisted catalyst-free hydrolysis of FC
Figure 2(a) shows the relationship between the conversion of cellulose and the MW irradiation time under catalyst-free conditions which is closing to linear. After 4 h MW treatment (5 kW/L), 37.2% conversion of FC was obtained. Glucose, 5-HMF and furfural were detected by HPLC (as evidenced in Figs. 2(b,c)) as the main chemicals in the liquid fractions from the MW-assisted cellulolysis, resulting in the colour change of the hydrolysis solution from light brown to dark brown by increasing MW treatment time (Fig. S2(b), cf. ESM). Figure 2(b) shows that the concentration profile of glucose and 5-HMF increased steadily as a function of the hydrolysis time. Xylose and cellobiose appeared to be intermediates in the reaction as their concentrations showed maxima over the course of MW-assisted cellulolysis. Dehydration of xylose to furfural using acid catalysts (HCl) under the MW has been reported as highly effective, resulting in the full conversion within 20 min (35 mmol/L xylose in 4 mL water, at 200°C, estimated rMW = 1.25 kW/L) [26]. In this study, MW irradiation over reaction times of 60 to 240 min, resulted in xylose conversion under catalyst-free hydrolysis of 7.28 mg (Table S2, cf. ESM), which is consistent with the furfural production over the same period during the MW treatment (Fig. 2(c)). This indicates that the MW was effective in converting xylose to furfural without acid catalysts, using relatively high MW densities.
According to previous studies, the hydrolytic depolymerisation of cellulose was enabled by the cleavage of b-1,4 glycosidic and hydrogen bonds to produce glucose and cellobiose [4,18], which are then transformed into fructose and 5-HMF. In the MW system reported, herein, xylose was detected at the beginning of the cellulolysis reaction for reaction times of<30 min with a relatively high concentration of ca. 1007.6 mg/L (at 30 min). This was much higher than the glucose production (ca. 297.6 mg/L at 30 min). Therefore, it is likely that, in the catalyst-free MW-assisted hydrolysis of FC (at 200°C), the unhindered primary alcohol groups (-CH2OH terminal groups) may react initially and result in cleavage of the C-C bonds (Fig. 3) with then cleavage of the 1,4-b-glycosidic bond depolymerising the polysaccharide chains in cellulose yielding soluble C5 (xylose) or C6 (glucose) sugars, as well as a C12 chemical of cellobiose. By extending the time of MW treatment, cleavage of the C-O-C bonds predominated, as seen by the increase in glucose yield, as shown in Fig. 2(b). Levoglucosan was formed steadily in the liquid phase as shown in Fig. 2(d) (selectivity at 4 h= 3.1%), which might be attributed to the dehydration of glucose in the polar solvent [27].
Mechanism of MW-assisted catalyst-free hydrolysis of FC
Based on the experimental findings of this work, it is proposed that the dominant pathway accounting for the depolymerisation of FC in MW-assisted cellulolysis lies in the molecular interaction between the FC and solvent molecules. In deionised water, dipolar polarisation is the main mechanism for the MW-induced heating of the cellulolysis system, in which the polar water molecules rotate with high velocities to align themselves with the alternating electric field (an MW frequency of microwaves of 2.45 GHz is optimal to enable water molecules frictional losing energy to heat up the system [25]. Under the MW conditions, rotating water molecules primarily attack the surface primary alcohol groups of the FC (by frictional forces and transferring kinetic energy to activate the C-C bonds). Upon reaching the softening temperature, the water molecules penetrate into the intersheet space of the cellulose fibres, facilitating cleavage of the C-O-C bonds and giving rise to not only xylose but also glucose and cellobiose. With the sustained MW irradiation, the intersheet bond breaking reactions dominate, whereas the hindered -CH2OH groups are likely preserved by the inter-/intra-chain hydrogen bonds as is seen in the increasing selectivity to glucose over xylose/furfural with increasing MW irradiation time.
In order to elucidate the comprehensive mechanism in the liquid phase, MW-assisted catalyst-free treatments of the primary products xylose, glucose and cellobiose (2000 mg/L) were performed under the same MW conditions at 200°C (rMW = 5 kW/L). For the reaction of glucose, MW irradiation resulted in 55.9% conversion of glucose after 0.5 h yielding mainly 5-HMF (yield= 34.5%) from dehydration of glucose, as well as small amounts of fructose (9.1%) and furfural (1.6%), as shown in Figs. 4(a) and 4(b). Hydrolysis of cellobiose under MW irradiation at 200°C formed glucose, 5-HMF, fructose, levoglucosan and furfural with yields of 51.1%, 14.1%, 6.4%, 3.3% and 0.9%, respectively (total carbon balance= 75.8%). The remaining carbon is likely to be composed organic acids and aldehydes which cannot be quantified by the HPLC method. The MW treatment of xylose revealed that the formation of furfural in the liquid phase was mainly from the dehydration of xylose. After 0.5 h MW irradiation at 200°C, xylose was quantitatively converted (conversion of xylose= 85.4%) to furfural (yield= 49.2%). Only trace amounts of xylose and furfural were detected in the solutions of glucose and cellobiose systems after MW treatment (Table S2, cf. ESM), indicating that the cleavage of -CH2OH groups from individual glucose molecules was difficult, in agreement with the literature [13].
Based on the findings above, under the MW condition (5 kW/L and 200°C), a pathway of catalyst-free hydrolysis of FC is proposed as shown in Fig. 5. Under the MW treatment above the softening temperature, the MW-induced rotation of water molecules (kinetic energy) facilitated the breaking of C-C bonds on the outer surface of cellulose, removing the -CH2OH groups exposed to the solvent and C-O-C bond breaking (inside the cellulose cavities) in the macroscopic structure of the FC, producing the first generation of cellulolysis products of xylose, glucose and cellobiose. The subsequent dehydration of these molecules in the liquid phase produced 5-HMF, furfural and levoglucosan.
A previous report performed at 220°C and a rMW of 0.8 kW/L in a monomode MW reactor proposed that -CH2OH groups function as the ‘molecular radiators’, transferring the MW energy to their nearby glucose rings to cleave the intrachain ether bonds (C-O-C) forming levoglucosan then glucose [13]. Concerning the ‘molecular radiators’ function of the -CH2OH groups in the cellulose [13], it was not conclusive based on the findings of the current work. Such an effect was claimed based on a ‘concentrated’ system using MCC in deionised water (mass ratio= 1:10). The mass ratio of FC to deionised water was also varied herein to investigate its effect on the MW-assisted hydrolysis. As shown in Fig. S3 (cf. ESM), linear correlations between the hydrolysis products and the mass ratio were all established, indicating an identical mechanism under the conditions used.
Both amorphous and crystalline phases were found in FC (Fig. 6(a), similar to that of MCC), but preferential depolymerisation of the amorphous cellulose [13] was not found at 200°C, as evidenced by the PXRD analysis of the hydrolysis residues where XRD diffractograms showed insignificant changes following MW treatment for up to 4 h (Fig. 6(b)). The pristine and hydrolysed cellulose fibres were analysed by SEM (Fig. 7), showing a reduction in fibre sizes after the MW treatment with comparable physical appearance. Interestingly, MW-hydrolysis of hydrolysed cellulose fibres under the same condition showed better efficiency than that of the pristine FC (Fig. 8) suggesting the simultaneous depolymerisation of amorphous and crystalline phases in FC under the condition used.
Finally, two types of raw biomass, namely maple leaves and fresh grass, were treated under the MW irradiation at 200°C without the catalyst for 1 h. Results presented in Fig. 9 shows that high mass loss of grass and leaves are obtained, 46.1% and 53.3% respectively, with complex product distribution in the HPLC spectra which requires further separation and identification. These results indicate that the MW-assisted catalyst-free hydrolysis is a green and efficient technology in the biomass transformation for deriving C5/C6 sugars and other biochemicals, not limited to the processed cellulose.
Conclusions
The MW-induced catalyst-free hydrolysis of FC was studied at 200°C and 5 kW/L, suggesting the specific interaction between water molecules and FC under the MW. The kinetic energy of rotating water molecules under the MW activates the C-C and intrachain ether bonds of the FC to produce C5 (xylose) and C6 (glucose) sugars, which are subsequently converted into renewable platform biochemicals, such as 5-HMF and furfural, with a ca. 96.5% total selectivity to them. The present work demonstrated an MW intensified yet clean process to convert the FC and biomass to valuable C5/C6 and according biomolecules.