Integration of Salt-Induced Phase Separation with Organosolv

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Integration of salt-induced phase separation with organosolv pretreatment for clean fractionation of lignocellulosic biomass Zhanying Zhang, William O. S. Doherty, and Ian O'Hara ACS Sustainable Chem. Eng., Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 24, 2017

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ACS Sustainable Chemistry & Engineering

Integration of salt-induced phase separation with organosolv pretreatment for clean fractionation of lignocellulosic biomass

Zhanying Zhang1,2*, William O.S. Doherty2, Ian M. O’Hara1,2 1. Syngenta Centre for Sugarcane Biofuels Development, Queensland University of Technology, Brisbane, Australia 2. Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Australia *Corresponding author. Postal address: GPO Box 2432, 2 George St, Brisbane, QLD 4001, Australia Tel: +61 7 3138 7792; Fax: +61 7 3138 4132 Email: Zhanying Zhang: [email protected]

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Abstract In this study, salt-induced phase separation was integrated with n-propanol-based pretreatment for clean fractionation of sugarcane bagasse. Firstly, biomass solid residue and pretreatment hydrolysate were separated by filtration following pretreatment. Lignins with high molecular weights (HMWs) were separated through precipitation and filtration after diluting hydrolysate with water. The filtrate was added with (NH4)2SO4 at a saturated concentration, which induced the formation of a biphasic solution: the top layer was npropanol phase containing sugar degradation products and lignins with low molecular weights (LMWs); the bottom layer was aqueous phase, rich in salt and soluble sugars. The solvent phase was directly reused for pretreatment while the aqueous phase was distilled to recycle both salt and water. Direct recycling solvent phase for pretreatment four times only led to slight decrease in glucan digestibility from 100% (fresh solvent) to 96%. Direct reuse of solvent phase also led to a significant increase in furfural concentration from 2.4 g/kg to 15.1 g/kg while recycling water and salt in aqueous phase by distillation led to a substantial increase in xylose concentration from 3.2 g/kg to 13.5 g/kg after five batches of pretreatments. In addition, the total yield of HMW lignins increased gradually from 38% to 49%. Keywords:

Organosolv

pretreatment;

Phase

separation;

lignocellulosic biomass


Fractionation;

Recycling;

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Introduction

Lignocellulosic biomass is abundant and low-cost sustainable carbon source for the production of biofuels (ethanol, n-butanol, etc.) and biochemicals (lactic acid, succinic acid, etc.) through fermentation processes

1, 2

. Pretreatment of lignocellulosic biomass is required

to improve cellulase accessibility to cellulose for enhanced fermentable sugar production 3, 4.

Organosolv-based biomass fractionation processes are one of the leading biomass pretreatment processes

3, 5

. The fractionation processes make the maximal use of each

biomass components (i.e., cellulose, hemicellulose and lignin) possible. The commonly studied organic solvents include low boiling point solvents such as ethanol (78.4 °C) and tetrahydrofuran (THF, 66.0 °C) and middle boiling point solvents such as n-butanol (117.7 °C) and methyl isobutyl ketone (MIBK, ~117-118 °C) 3, 5-15. Ethanol has been used to effectively fractionate biomass in acid-catalysed process 8, 9. In the ethanol process, celluloserich pulp is separated from pretreatment solution following pretreatment and is hydrolysed to produce glucose by cellulase enzymes. The pretreatment solution was diluted to precipitate lignins with high molecular weights (HMWs). With this process, ethanol is recovered from the diluted pretreatment solution by distillation. However, recovery and separation of soluble sugars, sugar degradation products such as furfural and soluble lignins/phenolics with low molecular weights (LMWs) are not economically feasible because of the low product concentrations. Acid-catalysed THF process is similar to the acid-catalysed ethanol pretreatment process 6, 15. THF can be distilled directly from aqueous solution, leading to the precipitation of lignin. n-Butanol and MIBK are the other two commonly used solvents for biomass fractionation 1014

. Due to the low solubility of n-butanol in water (73 g/L at 25 °C), pretreatment solution

forms biphasic solution after removal of cellulose-rich pulp by filtration 3 ACS Paragon Plus Environment

10, 11

. The organic


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solvent phase is rich in n-butanol and lignin while the aqueous phase contains soluble sugars and low concentration n-butanol. With MIBK processes, biomass is firstly cooked in an aqueous MIBK-alcohol (e.g., ethanol) solution 12-14. After cooking and solid/liquid separation, the liquid solution forms a biphasic solution with tuning the solvent ratio by adding water. The organics phase is rich in MIBK and lignin while the aqueous phase contains soluble sugars and alcohol. With these n-butanol and MIBK processes, solvent and lignin are recovered and separated by distillation of the solvent-rich phase.

Despite the progress in the fractionation of biomass using these organic solvents, recovery and separation of pretreatment by-products like soluble sugars (e.g., hemicellulose sugars), sugar degradation products (e.g., 5-hydroxymethyl furfural (HMF) and furfural) and organic acids (e.g., acetic acid) in aqueous phase are costly because of the low concentrations. Especially, the publications on biomass pulping/prereatment by aqueous n-propanol solutions are limited

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, which is due to the similar boiling points between n-propanol (97 °C) and

water (100 °C), making the separation of n-propanol from water difficult.

In this study, a salt-induced phase separation step was integrated with an alcohol solventbased pretreatment process and the whole process was developed for clean fractionation of lignocellulosic biomass. n-Propanol was selected as a model solvent for process development though this process is also adaptable to other solvents such as iso-propanol. Separation of npropanol from water was not an issue in this process because n-propanol was recovered and recycled through phase separation without distillation. More importantly, this process led to the substantial increases in the concentrations of soluble sugars in the aqueous phase as well as the accumulation of sugar degradation products and lignins with LMWs in the solvent phase.


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Methods Materials

Sugarcane bagasse was collected from Racecourse Sugar Mill (Mackay Sugar Limited) in Mackay, Australia. Sugarcane bagasse was washed with hot water at 90 °C to remove residual sugars to a negligible amount. The washed sugarcane bagasse was air-dried, and gently shaked on a sieve having an aperture size of 1.0 cm to remove pith and the residues were ground to fine particles by a cutter grinder (Retsch® SM100, Retsch GmBH, Germany). The milled bagasse was screened and particles having width range of 250 µm to 500 µm were collected and stored for pretreatment. The moisture of the sieved bagasse particles was 7.1 wt%. Bagasse particles mainly consisted of 41.6% glucan, 19.5% xylan, 3.5% arabinan, 28.3% lignin, 2.4% acetyl and 2.1% in terms of mass fraction. Ethanol, iso-propanol, n-propanol, 2butanol, n-butanol, glucose, xylose, arabinose, (NH4)2SO4 and sulphuric acid (mass fraction of 98%) were purchased from Sigma-Aldrich (US). AccelleraseTM 1000 (Batch no. 1600877126), a Danisco product (Genencor Division, Danisco Inc., US), was purchased through Enzymes Solutions Pty. Ltd (Australia). AccelleraseTM 1000 contained 30.4 mg protein/mL enzyme solution, which was measured using Bradford Protein Assay Kit purchased from Bio-Rad (US). The filter paper activity of AccelleraseTM 1000 was ~40 FPU/mL, which was measured using a method developed by the National Renewable Energy Laboratory (NREL, US)

17

. All the chemicals used in this study were of analytic grades or

above.

Solvent screening

100 g/kg to 300 g/kg alcohol solutions were prepared by mixing required amount of alcohol solvents with water. One group of the alcohol solutions contained glucose (2.5 g/kg), xylose



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(10.0 g/kg) and arabinose (2.5 g/kg). The other group of alcohol solutions contains sugar dehydration products HMF (1.0 g/kg) and furfural (10.0 g/kg). 30.0 g of each alcohol solution was transferred to a 50 mL Falcon® centrifuge tube and (NH4)2SO4 was added to the solution gradually at room temperature (24 °C) until saturated concentrations reached. Afterwards, the centrifuge tubes were centrifuged at 1000 rpm for 5 min in a Beckman centrifuge (GS-6R, Beckman, US). Except for ethanol solution containing 100 g/kg ethanol, all the other solutions formed biphasic solutions. The bottom phase (aqueous phase) was removed by a syringe and the weights of both aqueous phase and organic solvent phase were recorded. The concentrations of alcohols in bottom phase, and sugars in both organic solvent and aqueous phases were determined by a high performance liquid chromatography (HPLC) system as described in section of “HPLC analysis”. The concentrations of alcohols in solvent phase could not be determined by the HPLC system because of the high alcohol concentrations affecting the accuracy of the test. However, they were calculated based on the mass of alcohol solvent phase, the mass of total alcohol and the mass of alcohol in aqueous phase. The phase separation experiment was conducted in duplicate and the results were the means of the duplicate data.

Biomass fractionation

Sugarcane bagasse was fractionated in a process shown in Figure 1. For pretreatment, 5.0 g of sugarcane bagasse was transferred to a 100 mL pressure resistant glass flask (Ace Glass Inc., US) containing 50.0 g aqueous n-propanol solution with 588 g/kg n-propanol, 400 g/kg water and 12 g/kg H2SO4. The flask was sealed with a screw lid and immersed in an oil bath preheated to 140 °C. The reaction was conducted at 140 °C for 90 min under magnetic stirring at 250 rpm. A total of five flasks were used in the first batch pretreatment to collect enough materials for later use. Biomass fractionation by acidified aqueous ethanol (588 g/kg


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ethanol, 400 g/kg water and 12 g/kg H2SO4) was included as control. The pretreatment conditions of biomass fractionation were the same as those with n-propanol. After pretreatment, the flasks were cooled in iced water and all the reaction solutions from the same batch pretreatment (five flasks) were mixed and filtered by a Whatman 541 filter. The solid residue was washed with 500 mL water (5 × 100 mL) and was filtered. The wash solution and the original pretreatment solution were mixed to precipitate lignin followed by filtration to separate the diluted pretreatment solution and lignin. Lignin was collected and washed with 200 mL (2 × 100 mL) water. The diluted pretreatment solution was saturated with adding 400 g (NH4)2SO4 to promote the formation of a biphasic solution. Undissolved (NH4)2SO4 was filtered and collected. After phase separation, the weights of solvent phase and aqueous phase were recorded. 1 mL sample from solvent phase and 1.0 mL sample from aqueous phase were withdrawn for HPLC analysis to determine the concentrations of npropanol, sugars and sugar degradation products. The solvent phase was equally divided to five portions and transferred to flasks for next batch pretreatment. Water was distilled from aqueous phase at 80 °C under vacuum pressure and collected. Fresh water was added to offset the water distilled from aqueous phase to make the final mass of 500 g, which was used for washing solid residues generated in the next batch pretreatment. Stillage ((NH4)2SO4, sugars and ~25% water) and the previous undissolved (NH4)2SO4 was reused to induce biphasic solution in processing next batch pretreatment solution.

The second batch pretreatment was also conducted in five flasks with the same reaction conditions (bagasse loading, preteratment temperature and time) as those for the first bathc pretreatment. Prior to the second batch pretreatment, recovered n-propanol was replenished with a small amount of n-propanol depending on the n-propanol concentration in solvent phase determined by HPLC to make the final concentration of n-propanol in the solvent solution same as that (588 g/kg) in the first batch pretreatment. 12 g/kg H2SO4 (final acid 7 ACS Paragon Plus Environment


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concentration) was added to the solvent solution. A small amount of fresh water was also added to maintain the solvent composition ratio to be the same as that for the first batch pretreatment. Following the second batch pretreatment, the pretreatment mixture was processed in the same manner as that for the first batch pretreatment. After lignin separation, (NH4)2SO4 collected from the first batch pretreatment plus 50 g of fresh (NH4)2SO4 was used to saturate dilute pretreatment solution from the second batch pretreatment. The pretreatments were repeated four times and the procedure for processing pretreatment solution following each pretreatment was similar to that for processing the second batch pretreatment solution. Another 50 g of fresh (NH4)2SO4 was added in processing the pretreatment solution from the third batch pretreatment. In the fourth and fifth batch pretreatments, no more fresh (NH4)2SO4 was used since the amount of (NH4)2SO4 recovered previously was sufficient to saturate the pretreatment solution and to promote biphasic solution.

A portion of biomass pulp collected after each batch pretreatment and all the lignins recovered were vacuum-dried at 45 °C 48 h for biomass compositional analysis by a method developed by the National Renewable Energy Laboratory, US

18

. The remaining biomass

pulp was stored at 4 °C for enzymatic hydrolysis. The pretreatment was conducted in duplicate and the results were the means of the duplicate data.

Enzymatic hydrolysis of bagasse pulp

Enzymatic hydrolysis was carried out in a 20 mL glass vial containing 5 g solution. The cellulose loading was 2 wt% and the reaction solution also contained 0.05 M citrate buffer to maintain pH 4.8 and 0.02 wt% sodium azide to prevent the growth of microorganisms. The dosage of Accellerase for enzymatic hydrolysis was ~20 FPU/g cellulose (0.5 mL Accellerase solution/g cellulose). The reaction was carried out at 50 °C for 72 h in a rotary incubator (Ratek OM 11 Orbital Mixer, Australia) with shaking speed of 150 rpm. The sampling times 8 ACS Paragon Plus Environment

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were 0, 6, 24 and 72 h and sample volume was 0.2 mL each time. The samples were centrifuged at 9,000 g for 5 min. 0.1 mL supernatant was diluted 10 times by de-ionized water. The diluted sample was filtered through 0.22 µm disk filter prior to sugar analysis by HPLC. All the enzymatic hydrolysis experiments were conducted in duplicate.

HPLC analysis A HPLC system with a Bio-Rad Aminex HPX-87H column and Waters refractive index detector and UV detector (280 nm) was used to quantify solvent and sugar degradation products such as 5-hydroxymethylfurfural (HMF) and furfural in samples. The mobile phase was 5 mM H2SO4 at a flow rate of 0.6 mL/min. The column temperature was 65 °C. A Phenominex RPM monosaccharide column was used to determine the sugars during enzymatic hydrolysis. The column temperature was 85 °C and the mobile phase was water at a flow rate of 0.5 mL/min.

Results and discussion

Comparison of sugar distribution with different biphasic solutions

Five aqueous alcohol solutions were tested for their capacity to form biphasic solutions in the presence of saturated (NH4)2SO4. All the alcohol solutions formed biphasic solutions with alcohol concentrations of 100 – 300 g/kg except the one with 100 g/kg ethanol. Table 1 shows solvent distribution at an initial alcohol concentration of 200 g/kg containing sugars. All the alcohol solvents were enriched in the top layers (organic solvent phase) but to different extents. Distribution coefficient was used to indicate the solubility difference of a compound in solvent phase and aqueous phase, which was the ratio of concentrations of the compound in solvent phase and aqueous phase at equilibrium. In addition, mass distribution of each compound in solvent phase and aqueous phase was calculated considering the 9 ACS Paragon Plus Environment


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concentration of the compound in each phase and the amount of the corresponding phase. Solvent distribution coefficient of n-butanol was the highest, followed by 2-butanol, npropanol and iso-propanol, respectively while it was the lowest for ethanol. The trend of phase separation abilities of ethanol, n-propanol and iso-propanol was in line with the previous study 19. As a result, n-butanol concentration and recovery in the solvent phase was the highest, followed by n-propanol, iso-propanol and ethanol. 2-butanol and n-propanol concentrations in the solvent phase were 901.3 g/kg with a recovery of 94.6% and 837.4 g/kg with a recovery of 92.4%, respectively.

Table 2 shows sugars distribution in solvent phase and aqueous phase. Distribution coefficients of sugars were much lower than those of alcohols because of the much lower solubilities of sugars in alcohol solvent phase than those in aqueous phase. The majority of each sugar was distributed to aqueous phase. Recoveries of sugars in aqueous phase with ethanol solvent were the lowest but still close to 90% while with n-butanol they were the highest, close to 100%. The recoveries of sugars in aqueous phase with n-propanol and 2butanol solvents were greater than 98%. It was also observed that recovery of glucose in aqueous phase with each alcohol was highest, followed by arabinose and xylose at the given initial concentrations. Alcohol-based aqueous biphasic systems have been used to extract bioproducts such as proteins 20, nucleic acids 21 and 2,3-butanodiol 22. However, the information on distribution of sugars especially C5 sugars in alcohol-based biphasic systems is limited. The present results clearly show that salt-induced alcohol-based biphasic systems can be used to extract sugars (to aqueous phase).

Aqueous alcohol solutions containing sugar degradation products such as HMF and furfural were also induced to biphasic solutions. The alcohol solvent distribution in the biphasic solutions were similar to those containing sugars (data not shown). In contrast to sugar


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distribution, the majorities of HMF and furfural were distributed to alcohol solvent phases after in the biphasic solutions (Table 3). In general, distribution coefficient of furfural was higher than that of HMF in each biphasic solution at the given initial concentrations. The distribution coefficients of HMF and furfural in ethanol biphasic solution were much lower than those in other alcohol biphasic solutions.

Although pretreatments by biphasic organic solvent systems have been reported previously, the detailed information on how the soluble sugars and sugar degradation products distributed in organic solvent phase and aqueous phase is very limited. This study shows that the distributions of these compounds vary depending on the solvents. The results also show it is possible to recover water miscible alcohols, soluble sugars and sugar degradation products through the formation of biphasic solutions induced by a salt. Compared to the ethanol biphasic system, iso-propanol, n-propanol and 2-butanol systems (1) have high concentrations and recoveries of solvents in solvent phase, (2) have recoveries of high sugar degradation products in alcohol solvent phase and (3) have high sugar recoveries in aqueous phase. Compared to n-butanol (solubility of ~73 g/kg water at 25 °C) and 2-butanol (solubility of ~184 g/kg water at 25 °C), iso-propanol and n-propanol are miscible with water. If a 60% alcohol solution is used for pretreatment, HMW lignins can be precipitated out with diluting iso-propanol or n-propanol pretreatment solution with any ratio of water prior to adding salt. However, when n-butanol and 2-bunaol are used, large amounts of water are required to prevent self-formation of biphasic solutions to separate lignins prior to the formation of biphasic solutions by adding salts. Iso-propanol and n-propanol were only occasionally used for biomass pulping 16, 23, possibly due to the high cost of solvent. In addition, the boiling point of n-propanol (97 °C) is too close to that of water, which makes its recovery more difficult by distillation. With the salt-



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induced biphasic solutions, iso-propanol and n-propanol can be recovered efficiently from their aqueous solutions. In this study, further investigations have been focused on n-propanol as it is expected that iso-propanol will have similar properties to n-propanol in formation of biphasic solutions and biomass fractionation.

The effects of (NH4)2SO4 concentrations on the distribution of n-propanol and sugars were also examined with 200 g/kg n-propanol solution (Tables S1 and S2). At a salt concentration of 50 g/kg, biphasic solutions did not form. At salt concentrations of 100 g/kg and above, biphasic solution formed and recoveries of solvent (in solvent phase) and sugars (in aqueous phase) increased with increasing salt concentrations. Saturated salt concentration resulted in the highest recoveries of solvent (in solvent phase) and sugars (in aqueous phase).

Fractionation of sugarcane bagasse

Fractionation process description

Figure 1 shows the process for fractionation of sugarcane bagasse using aqueous n-propanol. In this process, pulp was separated following pretreatment and was converted to glucose by enzymatic hydrolysis. Lowering solution pH and dilution are well known methods for lignin precipitation following soda pulping and alcohol pulping of lignocellulosic biomass, respectively

24, 25

. In this study, the pretreatment solution was diluted approximately three

times and the pH was not adjusted since the pH of the dilute solution was already low (~2.5) (Figure S1). After dilution, the n-propanol concentration was about 200 g/kg solution.

After removal of HMW lignins by dilution and filtration, (NH4)2SO4 was added to promote the formation of a biphasic solution (Figure S1). N-propanol was enriched in the top phase and was directly reused in the next batch pretreatment. The aqueous phase was almost colourless, indicating a good extraction of lignins with low molecular weight (soluble 12 ACS Paragon Plus Environment

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lignin/phenolics) into the solvent phase. Instead of recovering n-propanol by distillation following each pretreatment like other organosolv processes

9, 26

, water and salt were

separated by distillation, recovered and reused in the present process, which led to a gradual increase in concentrations of soluble sugars.

After a number of recycles, the concentrated sugar solutions with salt can be separated through ion chromatography technology, e.g., simultaneous moving bed chromatography (SMBC), which will be similar to the process for separation of sugars and ion liquid using SMBC 27. Alternatively, salt-rich xylose solution may be used for the production of furfural in a biphasic system

28

.

Fractional distillation of solvent can be used to recover the

accumulated LMW lignins, acetic acid and furfural, respectively after a number of solvent recycles.

Characterisation of bagasse pulp

Table 4 shows bagasse pulp yield, composition and glucan digestibility with five batches of pretreatments. Bagasse pretreated by aqueous ethanol solution was also included as a control. Slightly higher pulp yield, xylan and lignin contents but lower glucan content were found with bagasse sample pretreataed by acidified aqueous ethanol compared to those with bagasse pretreated by acidified aqueous n-propanol (first batch), indicating that acidified aqueous npropanol was slightly more effective in removal of xylan and lignin. As a result, glucan recovery in bagasse pretreated with aqueous n-propanol was lower than that in bagasse pretreated with ethanol. From 1st batch to 5th batch, lignin content and recovery in pretreated bagasse increased gradually. The colour of pretreated bagasse also darkened with increasing pretreatment batch numbers. The increased lignin content is likely caused by lignin condensation due to the content of soluble lignins in recycled n-propanol solution, lignin



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precipitation due to water wash of pulp, and/or reduced pretreatment efficiency with the use of recycled lignin-containing n-propanol solutions.

Figure 2 shows the glucan digestibility at 6, 24 and 72 h. At 6 h, glucan digestibility decreased with increasing pretreatment batch number. This is possibly due to the increased lignin content, which negatively affects cellulose accessibility by formation of lignin barrier and/or non-productive adsorption of cellulase by lignin

29-31

. Nevertheless, after 24 h

hydrolysis, the gaps in glucan digestibility between the 1st batch pulp and subsequent batch pulps were narrowed. At 24 h glucan in 1st batch pretreated biomass was completely converted to glucose. Further increase in enzymatic hydrolysis time to 72 h only slightly increased glucan digestibilities of subsequent batches of pulps. After 72 h hydrolysis, glucan in bagasse samples from 1st and 2nd batch pretreatments were completely hydrolysed to glucose while glucan digestibility decreased slightly to 96% with increasing pretreatment batch number to five. Glucan digestibility of bagasse pretreated with n-propanol (1st batch) was slightly higher than that of bagasse pretreated with ethanol, indicating that pretreatment by acidified aqueous n-propanol was slightly more efficient in improving glucan digestibility than that by aqueous ethanol. Table 4 shows that glucose yields were the highest with bagasse pretreated in the 1st batch and 2nd batch but decreased slightly in the subsequent pretreatments due to the decrease in glucan digestibility. In general, the glucose yields of the five pretreatments with n-propanol and recycled n-propanol were lower than that of the ethanol pretreatment due to the low glucan recoveries. This indicates that the pretreatment conditions with n-propanol were a little bit severe, leading to the hydrolysis of higher portions of glucan to pretreatment solutions than those with ethanol. Nevertheless, the glucose yield did not decrease


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significantly and was greater than 83% after five batches of pretreatments. Optimisation of pretreatment conditions will be able to improve the glucose yield in future studies.

Lignin yield

Figure 3 shows distribution and yield of lignin after each batch pretreatment concentration (Figure 3a) and estimated LMW lignin concentration in solvent phase (Figure 3b). It is worth noting the total lignin distribution was calculated based on the amount of accumulative lignins at the end of each batch. For example, the total lignin in the biomass for the first batch was 28.3 g with the use of 100 g bagasse while it is 56.6 g (28.3 × 2) with the use of the same amount of bagasse in the 2nd batch pretreatment. The recovered HMW lignin yield after first batch pretreatment was 38% and increased to 49% after five batches of pretreatments. Although increasing dilution times with water could lead to the precipitation of more lignin 32

, more salt will be required to induce the formation of a biphasic solution and more energy

will be consumed to recover water from aqueous phase. Therefore, in this study three times of dilution were used in this study to precipitate reasonable amount of lignin. In contrast, the yield of soluble lignins/phenolics with LMWs in solvent phase decreased from 50% to 35% (Figure 3a) but the concentration increased from 19 g/kg to 60 g/kg (Figure 3b) after five batches of pretreatments. The increase in recovered HMW lignin yield is likely attributed to the increase in soluble lignin concentration in solvent phase, which limits the dissolution of more lignin (the increase in soluble lignin concentration was 16 g/kg solvent from 1st batch to 2nd batch and was 7 g/kg solvent from 4th batch to 5th batch). Lignin in solid residue increased slightly from 12% to 16% after five batches of pretreatmens (Figure 3a and Table 4), which explained the slight drop of glucan digestibility followed repeated pretreatment (Figure 2 and Table 4).



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Removal of HMW lignin from solvent solution reduces the viscosity of the solvent thus increases the mixing, mass and heat transfer during pretreatment, leading to improved pretreatment effectiveness 33. Although lignin concentration has increased from 19 g/kg to 60 g/kg after five batches of pretreatments, the results show that pretreatment effectiveness in terms of glucan digestibility only decreased slightly (Table 4). It is expected that the solvent can be recycled for more batches without significantly decreasing glucan digestibility. After a number of pretreatment batches, high concentration of LMW lignins can be recovered by fractional distillation.

Solvent concentration and distribution

Table 5 shows concentration and mass distribution of n-propanol in the biphasic solution after each batch pretreatment. The initial n-propanol concentration in the dilute solution before phase separation was about 196 g/kg, close to that previously used in the synthetic alcohol solutions (200 g/kg). However, compared to the synthetic solution, the concentration of n-propanol in the solvent phase of the biphasic solution following pretreatment was 170 g/kg to 200 g/kg lower (632 – 672 g/kg vs 842 g/kg). The solvent distribution coefficient of the biphasic solution formed from the pretreatment solution was generally lower than that of synthetic solution. These results indicate the presence of other impurities such as soluble lignin and phenolics may have affected the solvent distribution. Other components which may also have affected solvent distribution were possibly propyl glycosides. Alkyl glycosides are the major products when alcohols and sugars are incubated in the presence of acid catalysts at elevated temperatures

34, 35

. Nevertheless, the concentration of n-propanol in the

solvent phase was in a range of 632 g/kg to 672 g/kg, higher than the initial solvent concentration (588 g/kg) used for pretreatment. Therefore, the solvent phase can be reused directly for next batch pretreatment with slight dilution. After five batches of pretreatments,


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n-propanol concentration was 632 g/kg, still higher than that used for the 1st pretreatment. In addition, the glucan digestibility after five batches of pretreatments was still high (96%), indicating more recycles can be conducted before recovery of n-propanol from solvent phase by distillation.

The aqueous phase contained only 9 g/kg n-propanol. Since the aqueous phase is distilled to separate water (and the residual n-propanol) and salt for recycle, the loss of n-propanol will be negligible. Table 5 shows that after five pretreatments the majority of n-propanol was still distributed to the solvent phase while only a small portion of n-propanol was distributed to aqueous phase.

Distribution of soluble sugar and sugar degradation products

Table 6 shows the soluble sugar and sugar degradation product distribution in biphasic solution after each pretreatment. Arabinose was not detected in the solvent phase due to its low concentration. Compared to the synthetic solution, sugar distribution coefficients with pretreatment solutions were higher indicating the solvent phase has stronger affinity to sugars. This is likely due to the formation of propyl glycosides, which mainly existed in solvent phase and led to an increased sugar distribution coefficient. Nevertheless, the total amounts of glucose and xylose present in aqueous phase with aqueous n-propanol solution after pretreatment were still very high (> 95%). Mass factions of HMF and furfural in alcohol solvent phases were also high, 89% and 94% respectively, after five batches of pretreatments.

Figure 4 shows accumulations of sugars in aqueous phase and sugar degradation products (HMF and furfural) in solvent phase. Soluble glucose concentration increased from 0.7 g/kg to 2.7 g/kg, xylose from 3.2 g/kg to 13.5 g/kg and arabinose from 0.4 g/kg to 1.6 g/kg with increasing pretreatment batches from one to five due to the recycle use of salt stillage



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(containing sugars). Higher sugar concentrations are expected with increasing numbers of recycles. Sugars and salts can be separated and recovered by SMBC after a number of recycles. Alternatively, these xylose- and salt-rich solutions can be used to produce value added chemicals such as furfural in salt-induced biphasic system 28. HMF concentration in solvent phase increased from 0.15 g/kg to 0.30 g/kg whereas furfural concentration increased from 2.4 g/kg to 15.1 g/kg with increasing pretreatment batches from one to five. The significant increase in furfural is attributed to the presence of xylose and significant amounts of n-propyl xylosides in solvent phase, which were dehydrated to furfural during the recycle of solvent and repeated pretreatment. The high concentration of furfural in solvent phase could reduce the recovery cost. The HMF and furfural concentrations in aqueous phase after five batches of pretreatments were only 0.01 g/kg and 0.2 g/kg, respectively (data not shown). Although acetic acid is another major by-product from hydrolysis of acetyl groups in lignocellulosic biomass

36

, it can not be precisely determined by HPLC used in this study.

This is possibly due to the formation of n-propyl acetate by esterification of acetic acid with n-propanol under acid conditions 37. It is expected that n-propyl acetate is mainly distributed to n-propanol solvent phase due to its low solubility in water and accumulate with recycling the solvent. Therefore, n-propyl acetate can also be recovered after a number of repeated pretreatments.

Conclusions A clean biomass fractionation process consisting of biomass pretreatment using aqueous npropanol solution and salt-induced phase separation was developed and demonstrated. Unlike the other organosolv fractionation processes, this clean fractionation process did not recover the organic solvent by distillation; instead, the solvent was recovered and reused directly after each pretreatment following phase separation. In contrast, in the present process, water and 18 ACS Paragon Plus Environment

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salt used to induce the formation of the biphasic solution are recovered and recycled by distillation following each pretreatment. As a result, this novel clean fractionation process leads to the accumulation of sugars especially hemicellulose sugars in aqueous phase as well as the accumulation of LMW lignins and sugar degradation products in solvent phase. After a number of repeated pretreatments, these components can be recovered at higher concentrations, which improves the process economics for recovery of by-products.

Supporting Information. Supporting information is available. Tables 1S and 2S provide supporting data on solvent and sugar distribution at different salt concentrations; Figure 1S illustrates how the pretreatment solution was processed.

Acknowledgements

The authors gratefully acknowledge the financial support by Syngenta Biotechnology Inc. (US) and Sugar Research Australia.

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Legends of Tables and Figures Tables Table 1 Alcohol distribution in biphasic solutions Table 2 Sugars distribution in biphasic solutions Table 3 HMF and furfural distribution in biphasic solutions Table 4 Bagasse pulp yield, composition, glucan digestibility and glucose yield Table 5 Concentration, distribution and yield of n-propanol in biphasic solutions after each batch pretreatment Table 6 Soluble sugars distribution in biphasic solutions after each batch pretreatment1 Figures Figure 1 Sugarcane bagasse fractionation process using aqueous n-propanol solution Figure 2 Glucan digestibility at 6 h, 24 h and 72 h. Pretreatment was conducted at 140 °C for 90 min using pretreatment solutions containing 58.8% alcohol and 1.2% H2SO4. Figure 3 Lignin distribution after each batch pretreatment (a) and concentration of LMW in solvent phase after phase separation (b). LMW lignin concentration = (total amount of lignin in untreated bagasse (g) – the amount of lignin in solid residue after pretreatment (g) – the amount of HMW lignin recovered from precipitation (g))/the amount of solvent phase (kg) Figure 4 Accumulation of sugars in aqueous phase (a) and sugar degradation products in solvent phase (b)


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Table 1

Ethanol

6.3±0.3

Alcohol concentration, g/kg Solvent Aqueous phase3 phase4 470.6±14.3 74.6±2.2

Iso-propanol

38.4±1.7

772.4±13.2 20.1±0.4

86.6±0.6

13.4±0.6

n-Propanol

76.6±3.8

842.4±10.8 11.0±0.4

92.4±0.3

7.6±0.3

2-Butanol

114.1±5.6

901.3±15.3

7.9±0.3

94.6±0.2

5.4±0.2

n-Butanol

475.3±20.1 950.6±14.0

2.0±0.1

98.7±0.0

1.3±0.0

Solvent

1

D

2

Alcohol mass distribution (%)5 Solvent Aqueous phase phase 51.8±1.7 48.2±1.7

1. Initial solvent concentration before phase separation was 20 wt%. 2. D, distribution coefficient: component concentration (g/kg) in solvent phase/component concentration (g/kg) in aqueous phase. 3. Calculated values: (total amount of a component (g) – the component amount in aqueous phase (g, component concentration in aqueous phase (g/kg) × the amount of aqueous phase (kg))/total amount of solvent phase (kg). 4. Results determined by HPLC analysis. 5. Alcohol mass distribution: total amount of alcohol in each phase (g)/total amount of alcohol in both phases (g) × 100%.



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Table 2

Solvent

Glucose D1

Ethanol

0.43±0.02

Glucose mass distribution (%)2 Solvent Aqueous phase phase 7.9±0.5 92.1±0.5

0.61±0.02

Xylose mass distribution (%)2 Solvent Aqueous phase phase 10.8±0.5 89.2±0.5

Iso-propanol

0.04±0.00

0.9±0.0

n-Propanol

0.02±0.00

2-Butanol n-Butanol

0.59±0.03

Arabinose mass distribution (%)2 Solvent Aqueous phase phase 10.6±0.6 89.4±0.6

99.1±0.0

0.11±0.00

2.4±0.1

97.6±0.1

0.08±0.00

1.8±0.0

98.2±0.0

0.4±0.0

99.6±0.0

0.05±0.00

1.1±0.0

98.9±0.0

0.04±0.00

0.9±0.0

99.1±0.0

0.01±0.00

0.2±0.0

99.8±0.0

0.03±0.00

0.4±0.0

99.6±0.0

0.02±0.00

0.3±0.0

99.7±0.0

0.01±0.00

0.1±0.0

99.9±0.0

0.02±0.00

0.3±0.0

99.7±0.0

0.01±0.00

0.2±0.0

99.8±0.0

Xylose D1

Arabinose D1

1.

D, distribution coefficient: component concentration (g/kg) in solvent phase/component concentration (g/kg) in aqueous phase.

2.

Sugar mass distribution: the amount of a sugar in each phase (g, sugar concentration in each phase (g) × the amount of each phase (kg))/total amount of the sugar in both phases × 100%.

26

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Table 3

Ethanol

HMF mass distribution (%)2 HMF D1 Solvent Aqueous phase phase 14.7±0.6 76.2±1.1 23.8±1.1

21.6±0.7

Furfural mass distribution (%)2 Solvent Aqueous phase phase 82.4±0.9 17.6±0.9

Iso-propanol

44.2±2.1 89.1±0.4 10.9±0.4

79.5±3.6

93.6±0.3

6.4±0.3

n-Propanol

45.4±1.3 88.9±0.4 11.1±0.4 101.5±3.8 94.7±0.2

5.3±0.2

2-Butanol

38.5±1.6 86.6±0.5 13.4±0.5

88.4±3.2

93.7±0.2

6.3±0.2

n-Butanol

35.7±1.3 85.7±0.4 14.3±0.4

95.5±3.1

94.1±0.0

5.9±0.0

Solvent

Furfural D1

1.


2.

Mass distribution: the amount of a component in each phase (g, component concentration in each phase (g) × the amount of each phase (kg))/total amount of the component in both phases × 100%.



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Table 4 Content (%)2

Recovery (%)3

Batch no.

Pulp yield (%)1

72 h glucan digestibility (%)4

Glucose yield (%)5

Glucan

Xylan

Lignin

1st

40.9±0.6

87.1±1.1

1.8±0.1

8.3±0.3

85.6±2.3 3.7±0.2 12.0±0.5

100.6±0.1

86.1±0.5

2nd

41.5±1.0

86.8±2.3

2.0±0.0

8.7±0.3

86.3±1.9 4.2±0.2 12.7±0.8

100.3±1.0

86.6±1.4

3rd

41.0±1.0

87.6±1.8

1.6±0.1

9.6±0.2

85.9±1.0 3.5±0.1 13.9±0.4

97.3±2.1

83.6±1.6

4th

41.6±2.1

86.4±0.7

1.7±0.0

10.2±0.5

85.9±0.9 3.5±0.0 15.0±0.8

96.4±0.4

82.9±0.5

5th

42.1±0.8

86.1±1.3

2.0±0.2

10.8±0.6

86.7±2.1 4.3±0.1 16.1±0.5

96.3±2.0

83.5±1.9

Ethanol (1st) Untreated bagasse

44.0±0.5

85.3±2.1

4.0±0.3

9.2±0.3

89.8±0.3 9.0±0.4 14.3±0.6

98.4±2.4

88.4±1.5

100

41.6±0.9

19.5±0.6

28.3±1.1

6.5±0.2

6.5±0.2

Glucan

100

Xylan

100

Lignin

100

1.

Pulp yield (%) = the amount of solid residue after pretreatment (g)/ the amount of bagasse used for pretreatment (g) × 100%.

2.

Component content was calculated based on the dry mass.

3.

Component recovery (%) = component content in solid residue after pretreatment (%) × pulp yield (%)/component content in biomass prior to pretreatment (%).

4.

Glucan digestibility (%) = total amount of glucose after enzymatic hydrolysis (g)/(total amount of glucan in solid residue after pretreatment (glucan content (%) × the amount of solid residue (g)) × 1.11) ×100%.

5.

Glucose yield (%) = glucan recovery (%) × glucan digestibility (%)/100.

28


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Table 5

71.9±3.5

Solvent phase2 654.7±13.0

Aqueous phase3 9.1±0.4

Alcohol mass distribution (%)4 Solvent Aqueous phase phase 93.8±0.2 6.2±0.2

2nd

73.9±4.0

671.6±15.8

9.1±0.6

92.6±0.3

7.4±0.3

3rd

77.6±2.6

664.3±10.2

8.6±0.2

92.8±0.3

7.2±0.3

4th

73.8±3.6

646.1±14.4

8.8±0.6

92.9±0.2

7.1±0.2

5th

67.2±2.9

631.9±15.7

9.4±0.7

92.4±0.3

7.6±0.3

Alcohol concentration, g/kg

Batch no.

D1

1st

1. D, distribution coefficient: component concentration (g/kg) in solvent phase/component concentration (g/kg) in aqueous phase. 2. Calculated values: (total amount of n-propanol (g) – n-propanol amount in aqueous phase (g, n-propanol concentration in aqueous phase (g/kg) × the amount of aqueous phase (kg))/the amount of solvent phase (kg). 3. Results determined by HPLC analysis. 4. Alcohol mass distribution: total amount of alcohol in each phase (g)/total amount of alcohol in both phases (g) × 100%.



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Table 6

0.25±0.01

Glucose mass distribution (%) Solvent Aqueous phase phase 5.0±0.2 95.0±0.2

Xylose mass HMF mass distribution (%) distribution (%) 2 HMF D Solvent Aqueous Solvent Aqueous phase phase phase phase 0.22±0.01 4.4±0.2 95.6±0.2 25.9±1.1 84.4±0.6 15.6±0.6

89.9±1.6

Furfural mass distribution (%) Solvent Aqueous phase phase 94.9±0.3 5.1±0.3

2nd

0.24±0.01

3.9±0.1

96.1±0.1

0.18±0.01 3.0±0.1 97.0±0.1 38.6±1.6 86.7±0.4

13.3±0.4

103.2±3.9

94.6±0.3

5.4±0.3

3rd

0.20±0.01

3.2±0.1

96.8±0.1

0.16±0.00 2.7±0.1 97.3±0.1 42.3±1.2 87.5±0.5

12.5±0.5

100.9±3.2 94.3±0..3

5.7±0.3

4th

0.17±0.01

2.9±0.0

97.1±0.0

0.16±0.01 2.7±0.1 97.3±0.1 43.9±1.4 88.6±0.4

11.4±0.4

95.5±3.0

94.4±0.1

5.6±0.1

5th

0.16±0.00

2.7±0.1

97.3±0.1

0.14±0.01 2.5±0.1 97.5±0.1 43.7±1.7 88.8±0.3

11.2±0.3

84.6±2.8

93.9±0.2

6.1±0.2

Batch no.

Glucose D2

1st

Xylose D2

Furfural D2

1.

Arabinose was not detected in solvent phase.

2.


3.

Component mass distribution: the amount of a component in each phase (g, component concentration in each phase (g) × the amount of each phase (kg))/total amount of the component in both phases × 100%.

30


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Sugarcane bagasse

Pulp

Pulp Wash

Enzymatic hydrolysis

Glucose Fermentation

Biofuels

Water Pretreatment

Salt and sugars

Liquid

Liquid Dilution

Phase separation

Aqueous phase

M recycles Distillation

Concentrated sugars and salt

Acid 1.

HMW lignins

N recycles Distillation

Solvent phase

Figure 1

31


2. 3.

HMF and acetic acid (bp: 114-120 °C) Furfural (bp 162 °C) LMW Lignins (distillation residue)


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Figure 2


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a

b

Figure 3



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a

b

Figure 4


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Table of Contents

Integration of salt-induced phase separation with organosolv pretreatment for clean fractionation of lignocellulosic biomass

Zhanying Zhang1,2*, William O.S. Doherty2, Ian M. O’Hara1,2 *Corresponding author. Email: Zhanying Zhang: [email protected]

A clean organosolv fractionation process integrating salt-induced phase separation was developed for producing sugars and chemicals from lignocellulosic biomass.

Solid for glucose

Organosolv pretreatment

Liquid

Water

HMW lignins

Salt & sugars

Solvent phase (increased HMF, furfural, LMW lignins)

Aqueous phase (increased sugars) Distillation


Integration of Salt-Induced Phase Separation with Organosolv

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