Ionic liquid pretreatment of lignocellulosic biomass with ionic liquid

water mixtures

Agnieszka Brandt,a,b Michael J. Ray,b,c Trang Q. To,a David J. Leak,b,c Richard J. Murphyb,c

and Tom Weltona*

Abstract

Ground lignocellulosic biomass (Miscanthus giganteus, Pine (Pinus sylvestris) or Willow

(Salix viminalis)) was pretreated with ionic liquid-water mixtures of 1-butyl-3-

methylimidazolium methyl sulfate and 1-butyl-3-methylimidazolium hydrogen sulfate. A solid

fraction enriched in cellulose was recovered, which was subjected to enzymatic hydrolysis.

Up to 90% of the glucose and 25% of the hemicellulose contained in the original biomass

were released by the combined ionic liquid pretreatment and the enzymatic hydrolysis. After

the pretreatment, the ionic liquid liquor contained the majority of the lignin and the

hemicellulose. The lignin portion was partially precipitated from the liquor upon dilution with

water. The amount of hemicellulose monomers and their conversion into furfurals was also

examined. The performance of ionic liquid water mixtures containing 1,3-dialkylimidazolium

ionic liquids with acetate, methanesulfonate, trifluoromethanesulfonate and chloride anions

was investigated. The applicability of the ionic liquid 1-butylimidazolium hydrogensulfate for

lignocellulose pretreatment was also examined. It was found that ionic liquid liquors

containing methyl sulfate, hydrogen sulfate and methanesulfonate anions were most

effective in terms of lignin/cellulose fractionation and enhancement of cellulose digestibility.

Introduction

The rising demand for liquid transportation fuels is placing increasing demands on finite oil

reserves, raising prices and encouraging the search for oil in more remote locations, often in

fragile ecosystems. In addition, the planet’s climate is affected by carbon dioxide emitted

from the use of fossilised carbon as an energy source. The production of many chemicals

and materials is also reliant on fossil fuel resources.

Lignocellulose, essentially the cell wall material of woody plants, is a porous micro-structured

composite mainly consisting of cellulose, hemicellulose and lignin. It has been projected that

lignocellulosic biomass has the potential to be a large-scale, low-cost and sustainable

feedstock for renewable fuels and chemicals.1 Compared to starch or vegetable oil

a Department of Chemistry, Imperial College London, London SW7 2AZ, UKb The Porter Alliance, Imperial College London, London SW7 2AZ, UKc Division of Biology, Imperial College London, London SW7 2AZ, UK

1

substrates that are currently used as biofuel and biomaterial feedstocks, significantly higher

biomass yields per unit area of land are expected, while requiring less energy and material

input for its production.2 Various plant species have been suggested as being suitable

dedicated biofuel crops, with properties such as rapid growth, low fertiliser input, and short

harvest cycles. Currently favoured crops include grasses (miscanthus, switchgrass),

hardwoods (willow, poplar, eucalyptus) and softwoods (pine, fir, spruce). Careful

implementation of lignocellulose based technology as a substitute for fossil resources could

help reduce man-made carbon dioxide emissions in a sustainable way.3

Proposed routes for transforming lignocellulosic biomass into useful products are the

microbial fermentation of the glucose and other carbohydrates contained in the biomass and

the thermo-chemical conversion of the lignocellulose via pyrolysis or gasification. For the

fermentation route, deconstruction of the lignocellulosic matrix is necessary before the

carbohydrates are released. A typical deconstruction sequence producing fermentable

carbohydrates is: size reduction to chips, a pretreatment that solubilises the hemicellulose

and alters/removes lignin,4 followed by detoxification and neutralisation. The pretreated

biomass is subsequently processed using hydrolytic enzymes (saccharification) to produce

sugar monomers.

The pretreatment step is responsible for a significant portion of the energy consumption and

cost of the biofuel production process and improvements are required.5 A large number of

pretreatment options are defined in the literature, such as dilute acid, concentrated acid,

ammonia fibre expansion (AFEX), lime and organolv pretreatment. Different plant groups

exhibit distinct tissue structures and varying cell wall composition, which leads to a variable

resistance to deconstruction.

This paper explores the potential of certain ionic liquids as pretreatment solvents, in

particular their mixtures with water. Ionic liquids are a diverse group of salts that are liquid at

ambient temperatures or melt at slightly elevated temperatures. In the last two decades,

ionic liquids containing organic cations with quaternised ammonium, phosphonium and

sulfonium cores have enjoyed increasing popularity in many fields of research.6

Many ionic liquids have negligible vapour pressures under process-relevant conditions and

the nature and combination of cation and anion can be tuned to suit a particular application.

Cellulose and lignocellulose processing are only two out of many recently explored

applications for these alternative solvents.7 Ionic liquids are polar solvents with varying

degrees of hydrogen-bonding ability.8 It has been found that the ionic liquid needs to contain

anions with high hydrogen-bond basicity such as chloride, phosphates, phosphonates and

carboxylates in order to be able solubilise cellulose.9 The hydrogen-bond acidity also plays a

role. If a hydrogen-bond acidic functionality is incorporated into the ionic liquid structure, it

will compete for the hydrogen-bond basic site on the anion and reduce cellulose

2

solubilisation.10, 11 Water also decreases the solubility of cellulose,12 probably for a similar

reason. The empirical Kamlet-Taft solvent descriptors can be used to predict cellulose

solubility.13 Cellulose can be reconstituted by adding a protic antisolvent, such as water or

alcohols, and spun into fibres or films. A variety of homogenous derivatisations of cellulose

dissolved in ionic liquids can be accomplished.14

Ionic liquids were initially used in cellulose processing15 before their application was

extended to lignocellulose processing. The solubility of lignocellulose in ionic liquids has

been reported in various hydrogen-bond basic ionic liquids, suggesting that ionic liquids

which are cellulose solvents are also suitable for lignocellulose processing.16, 17 Reduced

crystallinity of the cellulose contained in lignocellulose was observed upon precipitation with

an antisolvent.18 A correlation between the hydrogen-bond basicity of the anion and the ionic

liquid’s ability to swell and partially dissolve wood chips has been observed.19 The solubility

of lignin in ionic liquids also seems to depend on the anion.17, 20 It has been shown that Kraft

pulp lignin has a very high solubility in the ionic liquids 1,3-dimethylimidazolium methyl

sulfate, [C1C1im][MeSO4], and 1-butyl-3-methylimidazolium methyl sulfate, [C4C1im]

[MeSO4].20

Enhanced glucose release from ionic liquid pretreated wood has also been observed, mainly

with dialkylimidazolium ionic liquids containing acetate, chloride and dimethyl phosphate

anions.21-23 However, the sugar release by hydrolytic enzymes was often less than 80-90%

(which is expected for an effective pretreatment operation). Recently, the impact of ionic

liquid pretreatment on biomass composition has received attention. It was noted that lignin

and hemicellulose are partially removed during pretreatment with 1-ethyl-3-

methylimidazolium acetate, [C2C1im][MeCO2].17, 24-26 A correlation between lignin removal and

cellulose digestibility was suggested.17, 21

Various publications concluded that application of methyl sulfate containing ionic liquids in

lignocellulose pretreatment did not enhance cellulose digestibility,17, 21, 24, 27 despite their

ability to dissolve large amounts of lignin.

Water reduces not only cellulose solubility in ionic liquids,12 but also the effectiveness of ionic

liquid pretreatment with [C2C1im][MeCO2].21, 28 Biomass contains significant quantities of

water, 2-300% relative to the oven-dried weight. In addition, ionic liquids are hygroscopic

and will absorb significant quantities of moisture when exposed to air.29 The drying of ionic

liquids requires heat and vacuum, particularly when the ionic liquids are strongly hydrogen

bond basic, like [C2C1im][MeCO2]. Therefore, an ionic liquid pretreatment that tolerates

moisture would be beneficial for the over-all energy and cost balance of a lignocellulose

processing system using ionic liquids.

An advantage of ionic liquid pretreatment could be the recovery of a separate lignin fraction

which could be converted to aromatic, value-added chemicals. Lignin recovery from ionic

3

liquids has been achieved after treatment of sugar cane bagasse with 1-butyl-3-

methylimidazolium alkylbenzenesulfonate, [C2C1im][ABS], an ionic liquid mixture containing

aromatic sulfonate anions, mainly xylenesulfonate.30 Lignin recovery has also been observed

after pretreatment with [C2C1im][MeCO2], when the regeneration solvent was a mixture of

water and acetone.20, 26

This study investigates the influence of water on the effectiveness of ionic liquid

pretreatment. We have devised a notation to indicate the amount of the ionic liquid contained

in the pretreatment solvent/liquor. This involves a subscript being added to the usual ionic

liquid notation indicating the ionic liquid content in volume percent (vol%), with the remainder

being water. An example is [C4C1im][MeSO4]80%, which is a mixture of 80 vol% [C4C1im]

[MeSO4] and 20 vol% water. Conversions of vol% into weight percent (wt%) and mole

percent (mol%) were calculated and are listed in Table 1. When allowing [C4C1im][MeSO4] to

equilibrate with the moisture in the laboratory air a water content of 70,400 ppm or 7.0 wt%

was measured (last entry of Table 1). Although the moisture content of air is variable, the

measurement demonstrates the highly hygroscopic nature of this ionic liquid.

Table 1: Ionic liquid concentration in aqueous pretreatment liquors.Mixture Volume percent

(vol%)Weight percent

(wt%)Molar percent

(mol%)[C4C1im][MeSO4]98% 98 98 81[C4C1im][HSO4]95% 95 96 64[C4C1im][MeSO4]90%

[C4C1im][HSO4]90%90 92

4446

[C4C1im][MeSO4]80%

[C4C1im][HSO4]80%

[C4C1im][MeSO3]80%*[C2C1im][MeCO2]80%

[C4C1im]Cl80%*[C4C1im][OTf]80%

80

838382828184

262726323024

[C4C1im][MeSO4]60%

[C4C1im][HSO4]60%60 65 12

[C4C1im][MeSO4]40%

[C4C1im][HSO4]40%40 45 6

[C4C1im][MeSO4]20%

[C4C1im][HSO4]20%20 23 2

[C4C1im][MeSO4]wet n.a. 93 49*These ionic liquids are solid at room temperature. Therefore vol% and wt% were calculated using the density at 80°C.

The aim of this work is to investigate the effect of the composition of the ionic liquid liquor on

the pretreatment. Solid recovery, pulp composition, its enzymatic digestibility, the

precipitation of a lignin-containing fraction and the production of furfurals in the liquor were

investigated. The application of an ionic liquid with a monoalkylated imidazolium cation was

also examined. Pretreatment of different feedstocks was carried out to assess their

recalcitrance towards pretreatment with ionic liquid water mixtures.

4

Results and Discussion

Tissue softening of Miscanthus chips

In preliminary experiments, we observed substantial disintegration of Miscanthus cross

sections immersed in the ionic liquid 1-butyl-3-methylimidazolium methyl sulfate,[C4C1im]

[MeSO4], when heated above 80°C. This encouraged us to investigate the application of this

ionic liquid for biomass pretreatment. The use of [C4C1im][MeSO4], dried to a water content

below 0.3 wt%, resulted in formation of a degraded biomass-ionic liquid composite that was

not enzymatically digestible. In contrast, using a mixture of 80 vol% ionic liquid and 20 vol%

water yielded a solid fraction that was separable from the (intensely coloured) ionic liquid

fraction and highly digestible. It was concluded that a certain amount of water was necessary

for successful pretreatment with [C4C1im][MeSO4]. In the “dry” sample, 0.3 wt% water was

contained in the ionic liquid as residual moisture and 0.7 wt% was introduced with the air-

dried biomass containing 8 wt% moisture, supplying 1.1 wt% or 15 mol% water in total. This

was apparently not sufficient to obtain an enzymatically digestible pulp.

Influence of the water content on the saccharification yield after ionic liquid

pretreatment with [C4C1im][MeSO4]

A range of ionic liquid water mixtures were used for pretreatment of Miscanthus to explore

the effect of the water content in more detail. The effect of water on the enzymatic release of

glucose and hemicellulose is shown in Figure 1. The yields are calculated based on the

glucose and hemicellulose content found in the untreated Miscanthus feedstock (on an

oven-dry basis), which were 43.6 wt% and 24.3 wt%, respectively. In preliminary

experiments, it was shown that the only detectable hemicellulose sugar released during

saccharification was xylose.

5

0102030405060708090

100

Glucose [MeSO4]-

Xylose [MeSO4]-

Xylose [HSO4]-

Glucose [HSO4]-

Ionic liquid content (vol%)

Sacc

harifi

catio

n yi

eld,

% o

f max

. pos

sible

Figure 1: Sugar yields obtained from Miscanthus pulp after pretreatment with [C4C1im][MeSO4] or [C4C1im][HSO4] water mixtures at 120°C. The [C4C1im][MeSO4] pretreatment was carried out for 22 h, while [C4C1im][HSO4] pretreatment lasted 13 h, and the saccharification 96 h.

The best saccharification yields were obtained after pretreatment with mixtures containing

60-90 vol% ionic liquid. Pretreatment with [C4C1im][MeSO4]90%, resulted in the release of 92%

of the glucose originally contained in the biomass. Pretreatment with [C4C1im][MeSO4]80% and

[C4C1im][MeSO4]60%,resulted in the release of 89% and 87% based on the original glucan

content. Glucose yields decreased when the ionic liquid content was higher or lower. The

hemicellulose yield was significantly lower than the glucose yield, regardless of the mixture

composition; 24% of the hemicellulose sugars (based on the initial hemicellulose content)

were released after [C4C1im][MeSO4]60% pretreatment. Similar yields were obtained with

mixtures containing 40-90 vol% [C4C1im][MeSO4].

Water sensitivity of [C4C1im][MeSO4]

When attempting to recycle [C4C1im][MeSO4], we found that the ionic liquid anion was

partially hydrolysed. After recording a mass spectrum of the recovered ionic liquid, a high

abundance of a negatively charged species at m/z=97 was detected, which was ascribed to

the hydrogen sulfate, [HSO4]-, anion. This led to the conclusion that the ester bonds in

methyl sulfate anions are hydrolytically unstable under the conditions of the pretreatment

and mixtures of the ester and the hydrolysed form are produced.

The extent of anion hydrolysis depended on the water content of the liquor (Figure 2). The

more water was present in the mixture, the greater the anion hydrolysis, with exception of

mixtures where the water content was higher than 90 mol%. These results suggest that

6

without extreme precautions to protect [MeSO4]- containing ionic liquids, [HSO4]- will be

present and other studies using these ionic liquids should be interpreted in this light.20

20 30 40 50 60 70 80 90 1000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Water content (mol%)

[MeS

O4]

/[C4

C1im

]

Figure 2: Ratio of [MeSO4]- anions to ionic liquid cations in the recycled ionic liquid after pretreatment of Miscanthus (detected by 1H-NMR), the remaining anions being [HSO4] -.

Influence of the water content on the enzymatic saccharification of [C4C1im][HSO4]

treated miscanthus

With the knowledge that the binary 1-butyl-3-methylimidazolium methyl sulfate water

mixtures turned into quaternary mixtures of two ionic liquids plus two molecular solvents

(water and methanol) we set out to identify the active component(s). Miscanthus was

pretreated with aqueous mixtures of [C4C1im][HSO4], which allowed us to exclude methyl

sulfate and methanol. The saccharification yields obtained from the pulps pretreated with

various [C4C1im][HSO4] water mixtures are shown in Figure 1. The glucose yields were

almost identical to the glucose yields obtained with the quaternary mixtures. The pattern of

hemicellulose release was also similar, however, after [C4C1im][HSO4]40%-80% pretreatment,

less hemicellulose was recovered than after treatment with the equivalent methyl sulfate

containing mixtures.

A glucose recovery of 90% after ionic liquid pretreatment is a substantial improvement

compared with the saccharification yields reported after pretreatment with other ionic liquids.

It has been reported that 74% glucose was enzymatically released from ground maple wood

after [C4C1im][MeCO2] treatment at 90°C for 24 h.21 70% glucose was released from maple

wood after [C2C1im][MeCO2] treatment at 90°C for 24 h. Li et al. reported only 15% glucose

release from ground Eucalyptus, pretreated with 1-allyl-3-methylimidazolum chloride,

[C=C2C1im]Cl, at 120°C for 5 h,22 while 55% of the glucose was released after 1-ethyl-3-

methylimidazolium diethyl phosphate, [C2C1im][Et2PO4], pretreatment of ground wheat straw

at 130°C for 30 min.23 It should be noted that saccharification yields obtained from ball-milled

7

lignocellulose samples were not considered for this listing because fine milling can have a

considerable effect on cellulose digestibility.22 The use of ground material reduces the

economic viability,31 so using fine powders obtained by ball-milling is of very little relevance

for an industrial process. Studies using 3,5-dinitrosalicylic acid (DNS) for the determination

of glucose yield were also not considered. The test is not specific for glucose and therefore

glucose yields from lignocellulose are often overestimated.

Effect of pretreatment time on the enzymatic saccharification

Next, we were interested in the optimisation of the pretreatment time. Figure 3 shows the

saccharification yields for both [C4C1im][MeSO4]80% and [C4C1im][HSO4]80% pretreatment after

various lengths of time. It can be seen that the enhancement of the cellulose and

hemicellulose digestibility mainly occurred within the first 4 h. This was also the period of

where the mass loss increased significantly (data shown in ESI). The pretreatment was

practically complete after 8 h, achieving around 80% glucose and 30% hemicellulose

release. When prolonging the pretreatment, the glucose yield slightly increased to above

85%, but the hemicellulose yield decreased to just over 20%. This experiment shows that

the presence or absence of methyl sulfate in the pretreatment mixture does not significantly

influence the speed of the pretreatment. It is anticipated that the pretreatment time can be

shortened by the application of higher temperatures, but it must be balanced with the ionic

liquid stability and potential side reactions.30

0 5 10 15 20 25 300

10

20

30

40

50

60

70

80

90

100

Xylose [HSO4]-

Xylose [MeSO4]-

Glucose [HSO4]-

Glucose [MeSO4]-

Time (h)

Sacc

harifi

catio

n yi

eld,

% o

f max

. pos

sible

Figure 3: Glucose and hemicellulose yields after enzymatic hydrolysis of Miscanthus pretreated with [C4C1im][MeSO4]80% and [C4C1im][HSO4]80% at 120°C.

8

The effect of [C4C1im][MeSO4]80% and [C4C1im][HSO4]80% pretreatment on biomass

composition

The composition of untreated Miscanthus and pretreated pulp is shown in Table 2 and

Figure 4. The untreated biomass contained 43.6% glucose, 24.3% hemicellulose and 26.5%

lignin. After pretreatment with [C4C1im][MeSO4]80% for 2 h, the main effect was a reduction of

the lignin content. Treatment with [C4C1im][HSO4]80% for 2 h resulted in the removal of lignin

and hemicellulose. After an extended pretreatment for 22 h, most of the lignin and

hemicellulose was solubilised and the glucan content increased from 44% in the untreated

biomass to 85% in the pretreated biomass. 91% of the original glucan was still present in the

pulp. The biomass recovery after 22 h was less than 46%, showing that more than half of the

wood had been solubilised in the ionic liquid. Tan et al. reported a mass recovery between

46% and 55% after pretreatment with [C2C1im][ABS] at 170-190°C, indicating that this ionic

liquid mixture might be capable of similar biomass fractionation.30 The simultaneous removal

of lignin and hemicellulose has also been reported for [C2C1im][MeCO2],24, 25 albeit less

complete than seen in this study with [C4C1im][HSO4]80%.

Table 2: Composition of untreated Miscanthus and Miscanthus pretreated with [C4C1im][MeSO4] and [C4C1im][HSO4].

Glu Xyl Ara Man Gal Lignin Ash Extractives

Mass loss

untreated 43.6 18.3 3.4 1.1 2.4 26.5 1.3 4.7 0[MeSO4] 2 h 45.4 18.3 2.1 1.3 2.2 19.3 1.1 - 10[HSO4] 2 h 44.5 8.6 0 0 3.4 14.9 0.6 - 28[HSO4] 22 h 39.5 3.3 0 0 1.1 1.9 0.6 - 56

0102030405060708090

100

HSO4 22h HSO4 2h MeSO4 2h untreated

Mass lossExtractsAshLigninArabinanGalactanMannanXylanGlucan

wt%

Figure 4: Composition of Miscanthus before and after pretreatment with [C4C1im][HSO4]80% and [C4C1im][MeSO4]80% at 120°C for 2 h or 22 h.

Production of solubilised sugars and furfurals

As seen above, the hemicellulose was removed from the biomass during treatment with

[C4C1im][HSO4] and [C4C1im][MeSO4] water mixtures. It is likely that under the conditions of

the pretreatment, (partial) hydrolysis of solubilised hemicellulose occurred. Therefore the

9

concentration of monomeric carbohydrates in the pretreatment liquor was investigated.

Figure 5 shows the relative amount of hemicellulose sugars and glucose in [C4C1im]

[HSO4]80% and [C4C1im][MeSO4]80% liquors at different time points.

The amount of hemicellulose monomers in the liquor increased within the first 4 h. The

increase was more pronounced in the [C4C1im][HSO4]80% liquor. The maximum amount of

hemicellulose monomers was detected around 4-8 h. This coincided with a major increase of

cellulose digestibility after 4-8 h of treatment (Figure 3). Subsequently, the hemicellulose

concentration in the pretreatment liquor decreased, suggesting that conversion of

carbohydrate monomers into furfurals was occurring.

0 5 10 15 20 25 300

2

4

6

8

10

12

14

16

Glucose [MeSO4]-

Hemicellulose [HSO4]-

Glucose [HSO4]-

Hemicellulose [MeSO4]-

Time (h)

% o

f max

imum

pos

sible

Figure 5: Amount of glucose and hemicellulose monomers found in [C4C1im][HSO4]80% and [C4C1im][MeSO4]80% liquors during pretreatment at 120°C.

Furfural was detected in the ionic liquid liquors and quantified for selected mixtures (Figure

6).

The glucose content was significantly lower than the hemicellulose sugar content and hardly

changed over time. The smaller amount of solubilised glucose is ascribed to the slow

hydrolysis of cellulose under the conditions of the pretreatment and the decomposition of

glucose to HMF. The small amount of HMF might be due to its decomposition to other

degradation products in the presence of water.

10

0

5

10

15

20

25

30

35

[HSO4]-, 22h [HSO4]-, 2h [MeSO4]-, 2h

Glucose HemicelluloseFurfuralHMF

% o

f max

imum

pos

sible

Figure 6: Solubilised carbohydrates (monomers only) and the fraction converted to furfural after pretreatment with [C4C1im][HSO4]80% and [C4C1im][MeSO4]80% liquors.

Lignin recovery

We attempted to recover lignin from the liquor (Figure 7), as this has been successfully

demonstrated for other ionic liquids.26, 30 It was found that diluting the ionic liquid liquor with

water precipitated a fine powder. The powder was characterised by IR spectroscopy and

comparison with a spectrum of a reference lignin (alkaline lignin) showed that the precipitate

is likely to be mostly lignin (see ESI). When methanol was used for washing the pulp, instead

of water, the majority of the precipitate remained in solution and a 15-20% improvement of

precipitate recovery was observed. Therefore washing the pulp with methanol was preferred.

The final protocol consisted of washing the pulp with methanol, drying the combined ionic

liquid fractions by evaporating the methanol, and precipitating the lignin by diluting the dried

ionic liquid liquor with water. The precipitate was washed with copious amounts of water and

dried before the yield was determined. The data (Figure 7) show that the yield of precipitate

was up to 50% of the Klason lignin content of the untreated biomass. More precipitate was

obtained when the ionic liquid content in the pretreatment liquor was high.

11

0%10%

20%30%

40%50%

60%70%

80%90%

100%0

10

20

30

40

50

60

Ionic liquid content

Lign

in y

eld

(%)

Figure 7: Yield of precipitate (relative to Klason-lignin content of the untreated biomass) after pretreatment of Miscanthus with [C4C1im][HSO4] water mixtures at 120°C for 13 h.

We also examined the time dependency of the precipitate yield and observed that the yield

of precipitate plateaued within 8 h (Figure 8). The yield was slightly higher from [C4C1im]

[HSO4]80% compared to [C4C1im][MeSO4]80%..

0 5 10 15 20 25 3005

101520253035404550

[C4C1im][HSO4]80%

[C4C1im][MeSO4]80%

Time (h)

Yiel

d, %

of m

axim

um p

ossib

le

Figure 8: Time course of lignin recovery after pretreatment of Miscanthus with [C4C1im][MeSO4]80% and [C4C1im][HSO4]80% at 120°C. The lignin was isolated from the liquor by precipitation with water.

The effect of the ionic liquid cation

The use of ionic liquids with mono-alkylated imidazolium cations (1-alkylimidazolium, [CnHim]+) is advantageous from an industrial point of view, as the ionic liquids are easier to

synthesise and thus cheaper to produce.7 Therefore an exemplary pretreatment of

Miscanthus with 1-butylimidazolium hydrogen sulfate, [C4Him][HSO4], was carried out. The

sugar yields after treatment with [C4Him][HSO4]80% and a subsequent enzymatic

saccharification are shown in Figure 9. After 4 h pretreatment, 69% of the original glucose

12

and 10% of the original hemicellulose were enzymatically released. The yield was somewhat

improved by prolonging the treatment to 20 h, when 75% of the glucose was recovered.

However, the xylose yield was reduced to only 3%. Pretreatment with [C4Him][HSO4]95%

resulted in significantly reduced glucose yields (44%).

80%, 4 h 80%, 20 h 95%, 20 h Water0

10

20

30

40

50

60

70

80

GlucoseXylose

Sacc

harifi

catio

n yi

eld,

% o

f max

. pos

sible

Figure 9: Enzymatic saccharification yields obtained from Miscanthus after pretreatment with [C4Him][HSO4]95% and [C4Him][HSO4]80%. Saccharification was carried out for 96 h.

The results of the compositional analysis and the mass loss of [C4Him][HSO4] treated

Miscanthus are presented in Table 3 and Figure 10. 80-93% of the lignin and more than 95%

of the hemicellulose wereremoved. The thorough removal of hemicellulose is reflected by

the low xylose yields obtained during saccharification. Treatment with [C4Him][HSO4]95% not

only resulted in the solubilisation of lignin and hemicellulose, but also in a substantial

removal of the cellulose fraction (51% of the glucan), explaining the reduced glucose yield

shown in Figure 9. The results indicate that pretreatment with [C4Him][HSO4] was harsher

than with [C4C1im][HSO4] under comparable conditions, potentially due to the increased

acidity of the [C4Him][HSO4] compared to [C4C1im][HSO4].

Table 3: Composition of Miscanthus pretreated with [C4Him][HSO4]80% and [C4Him][HSO4]95% at 120°C. Values are given in %; Glu=glucan, Xyl=xylan, Man=mannan, Gal=galactan, Ara=arabinan.IL content, treatment time Glu Xyl Man Gal Ara Lignin Ash Mass

loss80%, 4 h 40.9 2.9 0 0.7 0.2 5.0 0.8 49.580%, 20 h 37.7 1.0 0 1.0 0 5.4 0.6 54.299%, 20 h 22.4 0.6 0 0.6 0 1.9 0.4 74.2

13

80%, 4 h 80%, 20 h 95%, 20 h0

10

20

30

40

50

60

70

80

90

100

Mass lossExtractsAshLigninArabinanGalactanMannanXylanGlucan

%

Figure 10: Composition of Miscanthus after pretreatment with [C4Him][HSO4] water mixtures at 120°C.

It was also possible to obtain a precipitate upon dilution of the ionic liquid liquor. For the

[C4Him][HSO4]80% liquor, the yield was nearly 100% of the lignin content. For the 95% liquor,

the amount of precipitate was almost double the amount of the lignin content. We explain the

unusually high precipitate yield with the formation of pseudo-lignin. The formation of water-

insoluble carbohydrate degradation products has been observed during biomass

pretreatment under severe acidic conditions and found to obscure the Klason lignin yield.

Therefore it has been termed pseudo-lignin.32, 33 The formation of such degradation products

is undesirable and optimisation of the pretreatment conditions is required to minimise this

80%, 4 h 80%, 20 h 95%, 20 h0

20406080

100120140160180200

Lignin removed (%)

Recovered precipitate (% of lignin)

% o

f max

imum

pos

sible

Figure 11: Lignin removal and precipitate yield after pretreatment of Miscanthus with [C4Him][HSO4] water mixtures at 120°C.

The effect of the ionic liquid anion on the composition of ionic liquid treated Miscanthus

The effect of [C4C1im][HSO4]80% was compared with the effect 20/80 vol% water

dialkylimidazolium ionic liquid mixtures. The anions that we examined were

trifluoromethanesulfonate, [OTf]-, methanesulfonate, [MeSO3]-, chloride, Cl-, and acetate,

14

[MeCO2]-. It should be noted that the acetate containing ionic liquid, [C2C1im][MeCO2], was of

commercial quality.

Table 4: Composition of pretreated Miscanthus after treatment with 80/20% ionic liquid water mixtures at 120°C for 22 h. Values are given in %; Glu=glucan, Xyl=xylan, Man=mannan, Gal=galactan, Ara=arabinan.Ionic liquid anion Glu Xyl Man Gal Ara Lignin Ash Mass

loss[MeCO2]- 41.9 7.9 0 4.0 3.4 11.6 0.5 30.6Cl- 44.5 17.8 0 2.3 2.7 22.5 0.7 9.5[MeSO3]- 37.1 4.3 0 2.3 0 8.5 1.0 46.8[HSO4]- 39.5 3.3 0 1.1 0 1.9 0.6 53.6[OTf]- 43.6 13.7 0 5.1 4 24.3 1.0 8.3

Mass loss

0.00

102030405060708090

100

[MeCO2] Cl [MeSO3] [HSO4] [OTf] untreated

Mass lossExtractivesAshLigninArabinoseGalactoseMannoseXyloseGlucose

%

Figure 12: Effect of the ionic liquid anion on the mass loss and the composition of the recovered pulp after pretreatment of Miscanthus with 80% ionic liquid water mixtures at 120°C for 22 h. The data are ordered (left to right) according to the hydrogen-bond basicity of the ionic liquid, which is, in case of 1,3-dialkylimidazolium ionic liquids, a property of the anion.

Figure 12 and Table 4 show that the nature of the anion has a profound effect on mass loss

and pulp composition. [C4C1im][HSO4]80% removed lignin and hemicellulose most thoroughly,

followed by [C4C1im][MeSO3]80% and then by [C2C1im][MeCO2]80%. Hardly any change of the

composition was observed when the biomass was treated with [C4C1im]Cl80% and [C4C1im]

[OTf]80%, despite the fact that high solubility of Kraft lignin has been reported for both ionic

liquids(in anhydrous form).17, 20 The contradiction could be resolved if lignin solubilisation and

lignin extraction (which usually involves chemical modifications) were regarded as different

properties.

The effect of the anion on the saccharification yield

Enzymatic saccharification of Miscanthus treated with the ionic liquid liquors was also carried

out (Figure 13). In general, the enzymatic glucose release appeared to reflect the extent of

compositional change/mass loss achieved during ionic liquid pretreatment. The highest

glucose yield was observed after [C4C1im][MeSO3]80% and [C4C1im][HSO4]80% pretreatment.

The hemicellulose yield behaved slightly differently. The xylose yield was the highest after

15

pretreatment with [C2C1im][MeCO2]80%. The yield was significantly lower after [C4C1im]

[MeSO3]80% and [C4C1im][HSO4]80% pretreatment. Comparatively high hemicellulose yields

after [C4C1im][MeCO2] treatment can also be found in the literature.21 The increased

hemicellulose recovery after [C2C1im][MeCO2]80% treatment could be due to a buffering effect

exerted by the basic acetate anion. Its ability to combine with protons to form acetic acid

may limit the acid-catalysed hydrolysis of hemicellulose polymers. Inhibition of the hydrolysis

of cellobiose by [C4C1im][MeCO2] has been observed in mixtures of the ionic liquid, water

and catalytic amounts of strong acid.34 Binder et al., have also observed inhibition of

cellulose depolymerisation in [C4C1im][MeCO2], despite addition of catalytic amounts of

HCl.35 The methanesulfonate anion appears to have a less protective effect and acid-

catalysts which are released from the biomass (acetic acid and hydroxycinnamic acids) can

aid xylan hydrolysis. Hydrogensulfate increases the amount of available protons, which

could explain the particularly low xylan content in the pulp. The glucose and xylose yields

obtained after treatment with [C4C1im]Cl80% and [C4C1im][OTf]80% were low, despite their

ability to dissolve cellulose and lignin preparations (in case of triflate only lignin solubility).

0

10

20

30

40

50

60

70

80

90

100

[MeCO2] Cl [HSO4][MeSO3] [OTf] nottreated

Glucose

Hemicellulose sugars

Sacc

harifi

catio

n yi

eld,

% o

f max

. pos

sible

Figure 13: Impact of the ionic liquid anion on glucose and hemicellulose yields after enzymatic saccharification of Miscanthus pulp pretreated with 80/20 vol% ionic liquid water mixtures at 120°C for 22 h.

The effect of the anion on delignification and precipitate recovery

The yield of precipitate seems to be related to the ability of the liquor to extract lignin (Figure

14). The best delignification and the highest precipitate yield was obtained with [C4C1im]

[HSO4]80%, followed by [C4C1im][MeSO3]80% and then[C2C1im][MeCO2]80%. This supports the

notion that the precipitate comprises lignin, although it was shown in Figure 11 that pseudo-

lignin also precipitate upon dilution of the ionic liquid liquor.

16

0102030405060708090

100

[MeCO2] Cl [HSO4] [MeSO3] [OTf]

Delignification

Lignin recovery% o

f max

. pos

sible

Figure 14: Effect of the anion on the lignin removal and precipitate yield after pretreatment of Miscanthus with 80/20 vol% ionic liquid water mixtures. The higher yield from [HSO4]- containing liquors (compared to Figure 7 and Figure 8) is ascribed to the larger quantity of ionic liquid and biomass used in this experiment. Values are relative to the lignin content of the untreated biomass.

The effect of the anion on the formation of soluble degradation products

The quantities of carbohydrate monomers and dehydration products solubilised in the

pretreatment liquors are shown in Figure 15. The [C4C1im][HSO4]80% and [C4C1im][MeSO3]80%

liquors contained approximately 45% of the total hemicellulose as either sugar monomers or

furfural. In [C4C1im][HSO4]80%, the majority of the largest fraction was furfural. Conversion of

pentoses into furfurals was also observed in [C4C1im][MeSO3]80%, but to a lesser extent. This

is ascribed to the non-acidic nature of this ionic liquid. Only small quantities of monomers

were detected in the acetate containing liquor, which is probably due to fact that the

solubilised carbohydrates are mostly in oligomeric form. No furfural was formed in [C2C1im]

[MeCO2]80% in our experiment. It is likely that the acidity of the liquor is responsible for the

varying concentrations of sugar monomers and furfural found in the liquor. Like the

hydrolysis of glycosidic bonds, the rate of furfural formation depends on the acid

concentration.36 Since the acidity/basicity of 1,3-dialkylimidazolium ionic liquids is determined

by the anion, its nature should have a profound impact on the fate of the solubilised

hemicellulose. The amount of solubilised glucose and HMF were small in all cases. This is

ascribed to the enhanced stability of the cellulose fraction towards hydrolysis under

pretreatment conditions and the propensity of HMF to react to formic and levulinic acid in the

presence of water.

17

0

5

10

15

20

25

30

35

40

45

[OTf] [HSO4] [MeSO3] Cl [MeCO2]

GlucoseHemicelluloseFurfuralHMF

% o

f max

. pos

sible

Figure 15: Sugar monomers and furfurals solubilised in liquors containing 80 vol% 1,3-dialkylimidazolium ionic liquid with various anions after treatment of Miscanthus at 120°C for 22 h.

The effect of the biomass type: pretreatment of willow and pine

Pretreatment with [C4C1im][HSO4]80% was also performed on ground willow (a hardwood

species) and pine (a softwood species). For comparison, willow and pine were also

pretreated with [C2C1im][MeCO2]80%. The effect of the pretreatment on the biomass

composition is shown in Table 5 and Figure 16.

Table 5: Composition of untreated willow and pine and the pulps after treatment with [C4C1im][HSO4]80% and [C4C1im][MeCO2]80%.

Glu Xyl Man Gal Ara Lignin Ash Extractives

Mass loss

Willow 46.7 16.8 3.6 1.9 2.5 24.1 0.7 3.7 0Willow, [MeCO2] 36.3 6.4 2.9 2.7 1.9 19.9 0.7 - 29Willow, [HSO4] 39.1 3.4 0 0.8 0.9 3.6 0.5 - 52Pine 45.8 2.5 12.0 2.6 3.4 25.5 1.3 4.3 0Pine, [MeCO2] 40.4 2.5 16.1 3.4 2.7 21.1 0.6 - 13Pine, [HSO4] 37.9 3.2 4.6 0 0 8.8 0.2 - 45

18

0102030405060708090

100

Willowuntreated [HSO4][MeCO2]

Pineuntreated [HSO4][MeCO2]

Mass lossExtractsAshLigninArabinanGalactanMannanXylanGlucan

%

Figure 16: Composition of willow (3 bar graphs on the left) and pine (on the right) before and after pretreatment with [C4C1im][HSO4]80% and [C4C1im][MeCO2]80% for 22 h at 120°C.

For both substrates, lignin and hemicellulose removal were more extensive after [C4C1im]

[HSO4]80% pretreatment than after treatment with [C2C1im][MeCO2]80%. The degree of

cellulose enrichment after [C4C1im][HSO4]80% pretreatment of willow was almost as good as

the enrichment observed for Miscanthus pulp. A precipitate could be recovered from all

samples. Significantly higher yields were obtained from the [C4C1im][HSO4]80% liquors (see

ESI). The glucose yields obtained via enzymatic saccharification are shown in Figure 17.

More than 80% of the original glucose was released from [C4C1im][HSO4]80% pretreated

willow pulp, approaching the saccharification yields obtained from Miscanthus pretreated

with this liquor. However, enzymatic saccharification of pine pulp only released up to 30% of

the glucose; the type of ionic liquid playing a minor role. The generally higher yields obtained

after [C4C1im][HSO4]80% pretreatment could be due to the improved lignin and hemicellulose

removal by the hydrogen sulfate containing liquor, as observed for Miscanthus.

19

0 10 20 30 40 50 60 70 80 90 1000

102030405060708090

100

Miscanthus, [HSO⊝]

Willow, [HSOÁ]

Willow, [MeCO⊇]

Pine, [HSO⊝]

Pine, [MeCO⊇]

Saccharifcation time (h)

Gluc

ose

yiel

d, %

of m

ax. p

ossib

le

Figure 17: Enzymatic saccharification of lignocellulosic feedstocks after pretreatment with [C4C1im][HSO4]80% or [C2C1im][MeCO2]80% for 22 h at 120°C.

Ionic liquid solvent properties and biomass digestibility

We measured the Kamlet Taft polarity of [C4C1im][HSO4] and [C4C1im][MeSO3] (Table 6), as

it has not been reported in the literature. Three parameters are used to determine the

strength of solvent solute interactions. The parameter describes the hydrogen-bond acidity

of the solvent, the hydrogen-bond basicity and * the polarisability. Our measurements

showed that the parameter of [C4C1im][HSO4] is the same as the value for [C4C1im]

[MeSO4]. The hydrogen-bond acidity is very different, in fact, the value cannot be

determined for [C4C1im][HSO4], because it protonates one of the dye probes.

We would like to point out that the high glucose yields were achieved without complete

solubilisation of the biomass. This is due to the relatively low values of [C4C1im][MeSO4],

[C4C1im][HSO4] and [C4C1im][MeSO3], which do not enable cellulose solubilisation. The

parameters are lower than the values of [C4C1im][MeCO2] (=1.20), 1-butyl-3-

methylimidazolium dimethyl phosphate, [C4C1im][Me2PO4], (=1.12) and [C4C1im]Cl

(=0.83).19 Although [C2C1im][MeCO2] can dissolve cellulose when it is anhydrous, the

presence of 20 vol% water prevents cellulose solubility.

Table 6: Kamlet-Taft parameters of selected ionic liquids used in this work. *

[C4C1im][MeSO3] 0.44 0.77 1.02

[C4C1im][MeSO4]19 0.55 0.67 1.05

[C4C1im][HSO4] - 0.67 1.09

We also attempted to correlate the glucose yields with the ionic liquids’ hydrogen-bond

basicity. While it is clear that the nature of the anion affects the saccharification yield, it could

not be correlated with the ionic liquid’s value.

20

Conclusions

It has been demonstrated for the first time that the ionic liquids [C4C1im][HSO4], [C4C1im]

[MeSO3] and the ionic liquid mixture [C4C1im][MeSO4]/[HSO4] can be used to pretreat

lignocellulosic biomass. These ionic liquids functioned effectively in the presence of

significant quantities of water, eliminating the need for anhydrous conditions during

pretreatment. Commercial [C2C1im][MeO2] was also effective in the presence of 20 vol%

water, but the saccharification yield was lower. Lignin and hemicellulose were solubilised

during pretreatment, leaving behind a solid residue that was highly enriched in cellulose. The

enzymatic saccharification of Miscanthus pulp pretreated at 120°C with liquors containing 80

vol% ionic liquid resulted in glucose yields of ca. 90%. The hemicellulose was partially

recovered with the solid and readily hydrolysable during enzymatic saccharification.

However, a significant portion of the hemicellulose remained in the pretreatment liquor as

sugar monomers and was partially converted dehydration products. The amount of furfurals

generated during ionic liquid pretreatment arises from the acidity of the ionic liquid liquors. In

the presence of 20 vol% water, treatment with [C4C1im]Cl and [C4C1im][OTf] had little effect

on the biomass, showing that the anion of 1,3-dialkylimidazolium ionic liquids plays an

important role in determining the effectiveness of ionic liquid pretreatment and the tolerance

towards water. We could not find a correlation between the pretreatment effectiveness and

the anion basicity, as previously found for cellulose solubility or wood chips swelling. While

the enzymatic sugar release from the grass and hardwood pulps was very good, yields from

softwood pulp were only moderate. Upon dilution with water a precipitate was recovered that

is likely to contain lignin as well as pseudo-lignin. This study also suggests that mono-

alkylated imidazolium ionic liquids, such as [C4Him][HSO4], are in principle promising,

industrially relevant alternatives to dialkylimidazolium ionic liquids. The next challenge is to

optimise these processes.

Acknowledgements

We gratefully acknowledge the help of Julian Gianuzzi with pretreatment and

saccharification experiments and funding of the studentship for Agnieszka Brandt by the

Porter Institute.

Experimental

Materials

The lignocellulosic feedstocks used in this study were pine sapwood (Pinus sylvestris,

variety SCOES) from East Sussex, de-barked mixed willow (Salix viminalis, variety TORA)

stems and Miscanthus giganteus whole stems. The biomass was air-dried, ground and

sieved (0.18-0.85 mm, -20+80 of US mesh scale) before use. The moisture content of

21

untreated lignocellulose was 8.0% (Miscanthus), 8.9% (Pine) and 7.6% (Willow) based on

oven-dry weight. The biomass was stored in plastic bags at room temperature.

1-butyl-3-methylimidazolium methyl sulfate (Basionic AC01) was purchased from Sigma-

Aldrich. 1-ethyl-3-methylimidazolium acetate (Basionic BC01) was a gift from BASF AG,

Ludwigshafen. 1-butyl-3-methylimidazolium chloride and 1-butyl-3-methylimidazolium

trifluoromethanesulfonate were synthesised as described previously.19 The syntheses of 1-

butyl-3-methylimidazolium hydrogen sulfate, 1-butylimidazolium hydrogen sulfate and 1-

butyl-3-methylimidazolium methanesulfonate are described in the ESI. The ionic liquids were

dried to a water content <0.3 wt%, with exception of [HC4im][HSO4] which had a water

content of 1 wt%.

Measurement of Kamlet-Taft parameters

Kamlet-Taft parameter were measured as reported previously.19

Recycling of [C4C1im][MeSO4]

The ionic liquid liquor obtained after lignin precipitation was dried under vacuum at 40°C. A

sample of the dried ionic liquid was submitted to mass spectrometry. Part of the recovered

ionic liquid was dissolved in DMSO-d6 and a 1H NMR spectrum recorded. The peaks of

the methyl group at 3.40 ppm and of the C-2 ring hydrogen were used to determine the

anion to cation ratio. The pretreatment was carried out in capped vessels, so it is

reasonable to assume that the water content did not change substantially during the

pretreatment. The water introduced by the ionic liquid and the air-dried biomass was taken

into account, but not water consumed in hydrolytic reactions.

Lignocellulose pretreatment and isolation of pulp

0.500 g biomass (on oven-dry weight basis) was placed in wide-mouthed Pyrex culture

tubes with screw cap and Teflon-lining. 5 ml dried ionic liquid or the equivalent volume of

ionic liquid plus water were added. Mixing effects were neglected. The samples were

incubated without stirring in an oven at 120°C. After the pretreatment was finished, the

samples were cooled to room temperature and mixed with 10 ml methanol. After 2 h, the

suspension was filtered through hardened cellulose filter papers (Whatman 541 or

equivalent). The supernatant was set aside for precipitation and analysis of the monomer

and furfural content. The pulp was washed with methanol from a wash bottle, placed in a vial

and incubated with 10 ml fresh methanol overnight. The suspension was filtered again,

rinsed with methanol and air-dried on the filter paper overnight. The air-dried weight was

recorded and the samples transferred into re-sealable air-tight sample bags. In order to

obtain enough material for compositional analysis the pretreatment experiments were scaled

up 2-3x.

22

Lignin recovery

The supernatant obtained after pretreatment was dried under mild vacuum at 40°C to

remove the organic wash solvent. 10 ml water was added per 5 ml of original liquor. The

suspension was centrifuged and the precipitate washed 3x with 10 ml distilled water, air-

dried for several days and dried under vacuum at room temperature. The precipitate yield

was calculated based on the Klason lignin content of untreated biomass using Equation 1.

Part of the precipitate may be pseudo-lignin.

Lignin yield (% )=m precipitate

mKlasonlignin∙100% Eq. 1

The precipitate was characterised by IR spectroscopy using a Spectrum 100 IR machine

(Perkin-Elmer) equipped with a universal ATR sampling accessory with diamond crystal.

Determination of moisture content

100-200 mg air-dried biomass was wrapped in aluminium foil of known weight and heated at

105°C overnight. The samples were transferred into a desiccator and the weight determined

after 5 min. The moisture content was calculated according to Eq. 2 and used to determine

the oven-dried weight of untreated and pretreated biomass. The moisture content of the air-

dried biomass was in the range 5-12%.

moisture (%)=mair dried−moven dried

movendried∙100% Eq. 2

Enzymatic saccharification

Enzymatic saccharification was performed according to LAP “Enzymatic saccharification of

lignocellulosic biomass” (NREL/TP-510-42629). 150 mg untreated or pretreated air-dried

lignocellulosic biomass was used per saccharification experiment. If a pretreatment condition

was run in duplicate or triplicate, one saccharification experiment was performed per sample.

If the pretreatment condition was not replicated, the saccharification was performed in

duplicate. The enzyme preparations used for the saccharification were Celluclast (cellulase

mix from T. reseei) and Novozyme 188 -glucosidase which also contain hemicellulolytic

activity and can therefore hydrolyse xylan (both from Sigma). Glucose and hemicellulose

yields were calculated based on the glucose and hemicellulose content of the untreated

biomass, respectively.

Compositional analysis

The compositional analysis (lignin, carbohydrates, ash) was performed according to

laboratory analytical procedure (LAP) “Determination of structural carbohydrates and lignin

23

in biomass” (NREL/TP-510-42618). 300 mg of oven-dried sample were used per

experiment. Extractives from untreated pine and willow flour were removed by a one-step

automated solvent extraction with 95% ethanol using the ASE 300 accelerated solvent

extractor (Dionex) according to the LAP “Determination of extractives” (NREL/TP-510-

42619). Extractives from untreated Miscanthus were removed by a two-step solvent

extraction using deionised water and subsequently 95% ethanol according to the same LAP.

HPLC analysis of glucose and hemicellulose sugars was performed on an Agilent 1200

system equipped with an Aminex HPX-87P column, a de-ashing column and a Carbo-P

guard column (all Biorad). The mobile phase was de-ionised water, the column temperature

80°C and the flow rate 0.6 ml/min. The content of carbohydrates, Klason lignin, ash and

extractives (where applicable) was expressed as a fraction of the sum (normalised to 100%).

Quantification of solubilised sugars and furfurals

200 μl pretreatment liquor was mixed with 600 μl deionised water in 1.5 ml plastic cup,

vortexed and centrifuged with a table-top centrifuge (Biofuge 13, Heraeus) at maximum

speed for 10 min. The supernatant was transferred into a clean cup and centrifuged for 10

min. The supernatant was transferred into HPLC sample vials and analysed on a Jasco

HPLC system equipped with an Aminex HPX-87H column (Biorad) using a 10 mM sulfuric

acid mobile phase. The column oven temperature was 55°C, the flow rate 0.6 ml/min and

the acquisition time 55 min. Standard concentrations of 2-furaldehyde (furfural) and 5-

(hydroxymethyl)-2-furaldehyde (HMF) standards were prepared in deionised water to

concentration of 0.01, 0.02, 0.1, 0.2 and 0.4 mg/ml. The factor fHPLC(S) was obtained from the

respective calibration curve. The relative yield of solubilised sugar monomers and furfurals,

wt%(S), was calculated using Eq. 3. The molecular mass transformation factor FT was 1.37

for furfural, 1.28 for HMF and 0.91 for glucose and 0.88 for hemicellulose sugars. The mass

fraction factor FC was 0.243 for hemicellulose sugars and furfural and 0.436 for glucose and

HMF.

wt% (S )=AHPLC ∙ FD ∙V PLFHPLC (S)∙mbiomass

∙FC ∙ FT Eq. 3

AHPLC: area of HPLC peak, FHPLC(S): HPLC calibration factor for substance S, FD: dilution

factor, VPL: Volume of pretreatment liquor in ml, mbiomass: biomass (oven-dried weight) in mg,

FC: fraction of glucan or hemicellulose sugars in untreated biomass as determined by

compositional analysis (in %), FT: Transformation factor accounting for molecular mass

differences between starting material and product

24

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