Quantitative glucose release from softwood after pretreatment with low-cost ionic liquids

Article abstract of DOI:10.1039/c8gc02155d

Softwood is an abundantly available feedstock for the bio-based industry. However, achieving cost-effective sugar release is particularly challenging, owing to its guaiacyl-only lignin. Here, we report the highly effective pretreatment of the softwood pine (Pinus sylvestris) using ionoSolv pretreatment, a novel ionic liquid-based lignocellulose fractionation technology. Three protic, low-cost ionic liquids, 1-butylimidazolium hydrogen sulfate, triethylammonium hydrogen sulfate and N,N-dimethylbutylammonium hydrogen sulfate, were used to fractionate the biomass into a carbohydrate-rich pulp and a lignin. The carbohydrate-rich pulp was hydrolysed into fermentable sugars by enzymatic saccharification. Under the most successful pretreatment conditions, quantitative glucose release from the pulp was achieved, which equates to a projected glucose release of 464 mg per gram of pine wood entering the process. We further intensified the process by increasing the solid to solvent ratio up to 1:2 g g^-1 while maintaining saccharification yields of 75% of the theoretical maximum. We also demonstrate for the first time that N,N-dimethylbutylammonium hydrogen sulfate, [DMBA][HSO₄], is an excellent low-cost pretreatment solvent, surpassing the pretreatment effectiveness of its symmetrically substituted analogue triethylammonium hydrogen sulfate. This shows that ionoSolv pretreatment with protic hydrogen sulfate ionic liquids is a truly feedstock-independent pretreatment option, further increasing the commercial potential of this pretreatment technology.

Full text of DOI:10.1039/c8gc02155d

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Quantitative Glucose Release from Softwood after
Pretreatment with Low-cost Ionic Liquids
Cite this: DOI: 10.1039/x0xx00000x
Florence J. V. Gschwend, Clementine L. Chambon, Marius Biedka, Agnieszka
Brandt-Talbot, Paul S. Fennell and Jason P. Hallett*
Received 00th January 2012,
Accepted 00th January 2012
Softwood is an abundantly available feedstock for the bio-based industry, however, achieving
cost-effective sugar release is particularly challenging owing to its guaiacyl-only lignin. Here,
we report the highly effective pretreatment of the softwood pine (Pinus sylvestris) using
ionoSolv pretreatment, a novel ionic liquid-based lignocellulose fractionation technology.
Three protic, low-cost ionic liquids, 1-butylimidazolium hydrogen sulfate, triethylammonium
hydrogen sulfate and N,N-dimethylbutylammonium hydrogen sulfate, were used to fractionate
the biomass into a carbohydrate-rich pulp and a lignin. The carbohydrate-rich pulp was
hydrolysed into fermentable sugars by enzymatic saccharification. Under the most successful
pretreatment conditions, quantitative glucose release from the pulp was achieved, which
equates to a projected glucose release of 464 mg per gram of pine wood entering the process.
We further intensified the process by increasing the solid to solvent ratio up to 1:2 g/g while
maintaining saccharification yields of 75% of the theoretical maximum. We also demonstrate
DOI: 10.1039/x0xx00000x
www.rsc.org/
4
for the first time that N,N-dimethylbutylammonium hydrogen sulfate, [DMBA][HSO ] is an
excellent low-cost pretreatment solvent, surpassing the pretreatment effectiveness of its
symmetrically substituted analogue triethylammonium hydrogen sulfate. This shows that
ionoSolv pretreatment with protic hydrogen sulfate ionic liquids is a truly feedstock-
independent pretreatment option, further increasing the commercial potential of this
pretreatment technology.
competitiveness of bio-derived products compared to cheap,
highly optimised petroleum-derived fuels and chemicals.2,4 To
Introduction
Bioenergy and biorefining research has attracted widespread minimize unsustainable land use changes associated with
attention in recent years as many countries seek to minimise feedstock production for biorefining, emphasis has shifted
their reliance on oil imports, reduce their carbon emissions and towards utilisation of lignocellulosic biomass, which refers to
boost their agricultural and forestry sectors. Petroleum is the woody and thus inedible parts of plants. Fuels and
currently relied upon for the production of the majority of the chemicals produced from lignocellulose are referred to as
1
world’s liquid fuels, plastics, solvents and chemicals. A shift second-generation; promising lignocellulosic crops are
towards the use of biomass instead of petroleum will require dedicated energy crops (i.e. Miscanthus and short-rotation
significant investment in the development of novel process coppice willows), agricultural residues (corn stover, sugarcane
technologies.2
bagasse and wheat straw) or forestry residues, the latter often
3
Considering the importance of liquid fuels worldwide within being composed of softwoods such as pine and fir. Although
the transportation sector, there are increasing opportunities for lignocellulose is an abundant and relatively inexpensive source
biofuels to be incorporated into the energy mix. Biofuels can of sugars, its greater structural complexity means that the
contribute towards energy security, if produced sustainably, sugars are much harder to isolate than sucrose or starch. In
however, a number of factors currently limit expansion. These order to release sugars, a pretreatment step is required to
include concerns surrounding competition for land with food increase accessibility of cellulose prior to hydrolysis of the
production, or with endangered habitats with high carbon- sugars contained within, a process that adds substantial costs.5
storage capacity.3 A major barrier is also the economic Negative effects on downstream processes by side-products
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8
generated during pretreatment pose further challenges. These experiments. Softwood is produced sustainably for timber and
issues currently present a technical and economic obstacle to paper production and widely available as a forestry residue, in
establishing cellulosic sugar based biorefineries.
the form of sawdust at timber and pulp mills, or as construction
Ionic liquid pretreatment is a comparatively new biomass waste wood.25 According to NNFCC Bioeconomy Consultants,
deconstruction approach. Two strategies during the ionic liquid around 1.3 million tonnes of softwood forest waste arise in the
pretreatment of lignocellulosic biomass are being followed: UK annually, which can be removed from forests sustainably at
decrystallization of cellulose6,7 and extraction of lignin (i.e. a cost of between £18-50/dry tonne vs. around 140 thousand
ionoSolv pretreatment).8,9 The Dissolution pretreatment option tonnes of Miscanthus produced in the UK (including 16%
relies on ionic liquids with strongly hydrogen-bond basic moisture) at a cost of £45-70/tonne.26 An advantage of
anions, such as 1-ethyl-3-methylimidazolium acetate softwood is the high content of hexoses (Figure 1), due to the
[
EMim][OAc], which dissolve cellulose in the absence of glucomannan dominated hemicellulose.27 Hexose sugars are
water.10 Regenerated cellulose and hemicellulose exhibit a readily fermented by conventional yeasts,28 while many
better and faster digestibility (ca. 50 times higher enzymatic fermentative organisms are not adapted to the conversion of
hydrolysis rate) due to lower crystallinity11,12 and enlarged pentose sugars (xylose and arabinose), which make up the
surface area, but these ionic liquids suffer from low thermal majority of hemicellulose sugars in grasses and hardwoods.27
stability (limiting their recyclability),13 requirement for dry Softwoods are known to be more recalcitrant than grasses and
conditions, and high solvent cost.14 The ionoSolv pretreatment hardwoods due to a higher lignin content (up to 30%)27 and due
extracts the majority of lignin from biomass such as to the lignin consisting predominantly of guaiacyl (G) units
Miscanthus8,15,16 sugarcane bagasse17 and willow18 in the (95%).29 Guaiacyl units result in a higher content of biphenyls
presence of 10-40 wt% water. Although the cellulose is still and diphenyl ethers in the lignin,8 and more easily form
crystalline, saccharification is enhanced and accelerated up to condensed structures during processing, therefore presenting a
4
0 times compared to untreated biomass due to increased greater challenge to delignify, creating a barrier to quantitative
surface area and the removal of lignin. The ionoSolv sugar release and requiring high (Costly) enzyme loadings for
pretreatment is therefore in its function similar to Organosolv saccharification.30 Due to this, low-cost aqueous pretreatment
pretreatment.8,19,20 The extracted lignin can be recovered via such as steam explosion31 and dilute acid32 are largely
addition of water as an anti-solvent, while the cellulose can be ineffective in pretreatment of this recalcitrant feedstock. Better
hydrolysed to glucose via enzymatic saccharification and used results can be obtained with Organosolv33 and alkaline
in subsequent fermentations or chemical transformations. peroxide34 pretreatments, however high capital and chemical
During ionoSolv pretreatment, β-O-4’ linkages in the lignin are recovery cost limit their applicability.
broken, leading to fragmentation;21 however, lignin A limited number of studies have applied ionic liquid in
condensation reactions may also occur. It has been discovered softwood pretreatment. A study using dissolution pretreatment
that protic ILs, such as 1-butylimidazolium hydrogen sulfate with the ionic liquid 1-butyl-3-methylimidazolium chloride
[
HBim][HSO
4
]
and triethylammonium hydrogen sulfate [BMim]Cl has shown that a highly digestible pulp can be
[
TEA][HSO
4
]16,22 demonstrate similar efficacy as their obtained from pine wood chips with a glucose yield of around
dialkylimidazolium counterparts,23 and the alkylammonium IL 78%.35 Cox et al. used the protic ionic liquid
[TEA][HSO4] can be recycled multiple times with a slight 1-methylimidazolium chloride [HMim]Cl, obtaining up to 50%
increase in saccharification yield observed upon recycling.16 glucose release from pine chips.36 Both studies, however,
This is significant, as protic ionic liquids can be prepared via employed low, economically unattractive biomass to solvent
one-step acid-base reactions using readily available amines and ratios of 1:20 and ca. 3:100 g/g, respectively.
mineral acids. A recent study has suggested prices of $1.24 kg-1
for triethylammonium hydrogen sulfate and between $2.96-
5.88 kg-1 for 1-methylimidazolium hydrogen sulfate, if
1.3
0.8
$
synthesised on a bulk scale.24 This compares well to pricing for
4
3.4
13.3
common organic solvents such as acetone or ethyl acetate,
27.6
80%
4.5
-1
which cost between $1.30-$1.40 kg . Recently, we showed that
the pretreatment of Miscanthus with [TEA][HSO ] can be
4
4
.5 4.6
carried out at 180°C for 15 min without any yield penalty,
leading to commercially relevant process intensification.22 We
0
%
20%
40%
60%
100%
4
have further demonstrated that [TEA][HSO ] can be re-used at
least 4 times for pretreatment of Miscanthus while maintaining
Glucan
ASL
Mannan
AIL
Xylan
Ash
Arabinan
Extractives
high saccharification yield and no ionic liquid degradation was
detected through proton NMR.16
Figure 1 Composition of pine wood used in this study. ASL: acid-soluble lignin,
AIL, acid insoluble lignin.
Thus far, Miscanthus16,22 sugarcane bagasse17 and willow18
have been the main feedstocks investigated for deconstruction
and fractionation with ionoSolv ionic liquids, while softwoods
have received little attention apart from preliminary
This study investigates the application of protic, hydrogen
sulfate based ionic liquids for the fractionation of pine, a
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commonly grown softwood with wide geographical
abundance.37
We optimise pretreatment conditions
temperature and time) according to a bench-scale protocol
1
20°C
(
100
developed in our laboratory,15 applying the protic ionic liquid
N-butylimidazolium hydrogen sulfate. Enzymatic glucose
release was used as the key indicator. We also analysed the
composition of the cellulose-rich pulp and chemical
characteristics of the isolated lignin. We then applied two low-
cost alkylammonium hydrogen sulfate ionic liquids at the
optimised conditions. For the first time, an asymmetric
trialkylammonium hydrogen sulfate ionic liquid was used for
ionoSolv fractionation, and its effectiveness was compared with
the alkylimidazolium and symmetric tirethylammonium
analogues. The ions making up the three ILs used in this study
are displayed in Figure 2.
8
0
60
4
0
20
0
0
2
4
6
8
Hours of Pretreatment
1
50°C
1
00
8
6
0
0
N
NH
40
HSO4
N
NH
2
0
H
Figure 2 Structures of the ionic liquid ions used in this study: the hydrogen
0
+
+
sulfate anion, and the 1-butylimidazolium, [HBim] , triethylammonium, [TEA] ,
0
2
4
6
8
+
and N,N-dimethylbutylammonium, [DMBA] , cations.
Hours of Pretreatment
1
70°C
1
00
Results and discussion
8
0
EFFECT OF TEMPERATURE AND TIME ON PRETREATMENT
WITH [HBIM][HSO
4
]
60
40
Fractionation effectiveness and enzymatic saccharification yield
A set of experiments was carried out using the protic ionic
2
0
liquid 1-butylimidazolium hydrogen sulfate [HBim][HSO
three different oven temperatures (120, 150 and 170°C) with a
:10 g/g biomass to solvent ratio. As we discussed in our study
on process intensification of Miscanthus fractionation using
triethylammonium hydrogen sulfate [TEA][HSO
],22 the
4
] at
0
0
1
2
3
4
1
Hours of Pretreatment
4
Glucan recovery
Delignification
designated reaction temperature inside the pretreatment vessel
is only reached after around 30 min. This should be borne in
mind when interpreting results for pretreatment times of 30 min
or less.
Lignin precipitate
Saccharification yield
Hemicellulose removal
Figure 3 shows how a number of indicators for pretreatment
and fractionation effectiveness perform over time. The glucan
recovery, delignification and (non-glucan) hemicellulose
removal were derived by combining compositional analysis
Figure 3 Key indicators of fractionation effectiveness for pine wood
pretreated at 120°C, 150°C and 170°C with [HC im][HSO ] containing 20
wt% water. The biomass to solvent ratio was 1:10 g/g
4
4
data and pulp yields; the precipitated lignin yield was since we observed high glucose release when performing
determined gravimetrically. The enzymatic glucose release additional experiments with wet pulp, which are detailed
(
saccharification yield) was determined for air-dried pine pulps. towards the end of the manuscript. After the initial decrease,
The saccharification yield is a key performance indicator, as saccharification yields increased beyond that released from
glucose is a key substrate for fermentations or chemical untreated biomass for all investigated temperatures. For 120°C,
transformations producing bio-based chemicals and fuels.
the saccharification yield did not reach a peak within the
For the saccharification yield, we observed an initial decrease timeframe studied, while for experiments at 150 and 170C,
compared to untreated pine at all three temperatures. We there was a peak saccharification yield at 60 and 30 min,
attribute this to hornification (irreversible pore collapse) respectively. The highest observed glucose releases were 44, 62
occurring during the air-drying of the pulp after washing,38 and 71% for 120, 150 and 170°C, respectively, showing that a
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higher pretreatment temperature is essential for effective industrial-scale protocol (we anticipate a washing step with
enzymatic cellulose hydrolysis. The peak saccharification recycled ionic liquid instead) and less redeposition is therefore
yields occurred at similar pretreatment conditions identified for expected under scaled-up conditions. An extensive study of
Miscanthus,22 sugarcane bagasse17 and willow18 pretreatment ionoSolv softwood pretreatment up to the 200 L scale has been
with [TEA][HSO
4
].
performed and will be reported in the near future.
Peak delignification was 79% at 150°C and 81% at 170°C, and We further note that no minimum for the lignin content was
it appeared to occur slightly later than peak saccharification. found within the studied timeframe at 120°C for pine, while a
We also observed that saccharification yield and lignin removal minimum can be detected for other feedstock types. For pine,
correlated strongly, as can be seen from Figure 4. A delignification and saccharification yield was limited to 66%
relationship
saccharification yield was reported previously for saccharification yield for Miscanthus pretreated with
Miscanthus.16,22
[TEA][HSO ], showing that softwood requires higher
between
delignification
and
enzymatic and 44% after 8 h compared to a 88% delignification and 77%
4
temperatures in order to achieve good pretreatment results.16
The reason for this may be that lignin extraction is facilitated
above the glass transition temperature of lignin (thought to be
in the range 130-150°C).41 We hypothesise that fast extraction
from the cellulose pulp is particularly beneficial for softwood
delignification, as guaiacyl-rich lignin is prone to forming
stable crosslinks in acidic systems via condensation reactions.42
Glucan recoveries were fairly constant under mild conditions
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
(i.e. at 120°C), while hemicellulose was removed over time.
For 120°C, we observed that hemicellulose removal from pine
was faster than hemicellulose removal from Miscanthus using
0
20
40
60
80
100
[
TEA][HSO
4
],16 with 64% of hemicelluloses removed in the
Delignification (%)
first hour of pretreatment compared to 44% in the case of
Miscanthus. High temperatures accelerated hemicellulose
removal. Near-quantitative hemicellulose removal was
Figure 4 Correlation of enzymatic glucose release and delignification of pine after
pretreatment with [HC im][HSO ] with a water content of 20 wt% and a pine
4
4
wood to solvent ratio of 1:10 g/g at different temperatures and times. achieved after 4 h and 45 min at 150 and 170°C, respectively,
Percentage of the theoretical maximum according to compositional analysis is
shown.
which is similar to what was observed for Miscanthus
pretreated with [TEA][HSO
4
].22 The recovery and valorisation
of the dissolved hemicellulose portion from ionoSolv solutions
remains a challenge. One suggested option is to produce
furfural.16
At 150 and 170°C, the amount of lignin associated with the
pulp increased after reaching a minimum (2 h at 150°C and
3
0/45 min at 170°C). This has been observed in the past for
For the temperatures above 120°C, we observed that
pretreatment effectiveness declined past the optimum point,
which we attribute to reduced glucan recovery and deposition
of condensed lignin and pseudo-lignin on to the surface of the
cellulose fibrils. This was confirmed by compositional analysis
of the pulps (numerical values can be found in Table S1 in the
ESI). In an extreme case, the glucan recovery dropped to zero
after 4 h of pretreatment at 170°C. We note that glucan
appeared to be less stable in this study compared to Miscanthus
pretreatment of Miscanthus with [TEA][HSO ] at temperatures
4
of 150-180°C.22 The increase in the amount of lignin present in
the pulp at the later stages of pretreatment has been attributed to
both condensation reactions within lignin fragments and the
formation of pseudo-lignin from hemicellulose which
contribute to the formation of high molecular weight substances
which can precipitate onto the pulp surface.39 While the
compositional analysis technique fails to distinguish between
actual lignin and pseudo-lignin, formation of the latter is
strongly suspected under conditions where the lignin precipitate
exceeds the measured delignification (e.g. after more than 1 h at
pretreatment in [TEA][HSO
nearly 100% at 150°C and 4 h,22 compared to 70% glucan
recovery after 4 h of pretreatment of pine with [HBim][HSO ].
4
], where glucan recovery was
4
1
70°C). Evidence for condensation reactions was found when
It is currently unclear if the differences in cellulose and
hemicellulose stability are due to the nature of the feedstock or
the ionic liquid. An initial decrease in glucan recovery in the
pulp has been attributed to removal of glucose containing
hemicelluloses, such as galactoglucomannans and xyloglucans,
rather than degradation of cellulose.43 Galactoglucomannans
constitute up to 15% of softwood.44
analysing the precipitated lignin, which is discussed below.
The re-precipitation of condensed lignin and also that of
pseudo-lignin on to the cellulose surface are thought to have a
negative effect on saccharification yields and kinetics by
making the cellulose substrate less accessible.40 It should
further be noted that the work-up procedure used here involves
diluting the IL with ethanol after pretreatment to facilitate its
separation from the pulp. Visual observations suggested that
ethanol-induced lignin precipitation was responsible for some
of the lignin reprecipitation at prolonged pretreatment times.
We note that dilution with ethanol is unlikely to be part of an
Lignin characterisation
As was shown in the previous section, there is a clear link
between residual lignin content in the pulp, and saccharification
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yields (Figure 4). In order to improve the economics of
cellulosic biorefineries, not only the cellulose/sugar yield needs
to be optimised, but also the characteristics of the lignin. We
therefore analysed the isolated lignins for their subunit
composition and linkages and the molecular weight to gain
insight into the reactions taking place. Pine lignin (and more
generally softwood lignin) consists predominantly of guaiacyl
(G) units (95%) and around 45-50 β-O-4’ linkages per 100
aromatic rings.29 The second most abundant linkage in
softwood lignin is the phenylcoumaran (β-5’) linkage with
around 10-13 linkages per 100 aromatic units. In smaller
numbers, resinol (β- β’) and diarylpropane (β-1’) linkages are
also present.45,46 Figure 5 shows the most important linkages
and subunits in pine lignin.
Figure 6 shows the aromatic and the oxygenated regions of a
representative HSQC NMR spectrum for one of the isolated
pine lignins. Peaks for the three unsubstituted positions of the
guaiacyl ring as well as certain linkages were assigned. We
note that there are a number of peaks in the aromatic region of
the spectrum which are currently unassigned, as they do not
match any of the commonly found structures in ball-milled
lignin. They probably correspond to new structures formed
during the ionic liquid extraction. We also observed a peak at
4
.2/67 ppm, which we assign to the plasticiser bis(2-ethylhexyl)
phthalate (DEHP). It has been observed in a previous study16
and is presumed to have been leached from the seals of the
pressure tubes used for heating the biomass with the ionic
liquid and subsequently precipitated with the lignin fraction
during antisolvent addition.
Figure 6 HSQC NMR spectra of pine lignin isolated after extraction with
[HC
water content of 20 wt%. The side chain region (top) and aromatic region
bottom) are shown. PL is plasticizer. Two signals for residual butylimidazolium
4 4
im][HSO ] at 170°C for 30 min with a biomass to solvent ratio 1:10 g/g and a
(
are marked in red. Grey peaks could not be assigned.
RO

O
The pulse sequence used in these experiments only gives semi-
quantitative information, since equilibration of the 13C
excitation is incomplete within the programmed relaxation time

5

'
HO




HO

'
O
O
O
O



'

'
'
(in order to achieve manageable acquisition times), and carbon
O
nuclei will relax at different rates in different chemical
environments. However, the observed peak volume is linear
with respect to the concentration of the analyte, so time course
trends can be established with a suitable internal standard. It
A
B
C

-aryl ether (-O-4')
resinol (-')
phenylcoumaran (-5')
2
has been suggested in previous literature that the G signal can
be used as such an internal standard in the case of softwoods, as
it is not thought to participate in condensation reactions or
overlap with signals of degradation products.45 It should
therefore be representative of the total number of aromatic units
OH
OH
1
'

6

2

OH
5
O
in the lignin. However, the G
undergo chemical modification, leading to a G2,cond peak, hence
the sum of G and G2,cond is used here as a reference integral.
2 6 5
signal shifts if G or G sites
O
2
D
G
With these assumptions, we compared the signal intensities for
the four ether linkages shown in Figure 5 and the different
positions of the guaiacyl unit found in the precipitated lignin, in
order to discuss characteristics of the isolated lignins, and lignin
reactivity more generally.
diarylpropane (-1')
guaiacyl
Figure 5 The most prominent ether bonds and lignin subunit in pine lignin.
Figure 7 shows the relative HSQC volume integrals for pine
lignin isolated after pretreatment for 1 h at 120, 150 and 170°C.
It can be seen that the majority of β-O-4’ ether linkages (45-
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5
0%), the most abundant lignin linkages in pine wood,45 were
broken after pretreatment for 1 h. The data suggest that the
ether bonds contained in the less abundant β-β’ and β-5’
linkages break at a slower rate, which has been observed
previously.22 Similarly, the aromatic ring signals provided
evidence for substitution reactions occurring on the aromatic
rings (lignin condensation). The degree of condensation was
higher at high pretreatment temperatures, as evidenced by the
80
6
4
0
0
20
0
lower G
for the 150°C lignin, and even more so for the 170°C lignin.
We note that there was an unexpected increase in the G signal
6 2
and G signal intensities and the higher G2.cond signal
5
intensity. We have preliminary evidence that this is due to
carbohydrate-derived degradation products which are generated
under acidic conditions and precipitate with the lignin. We will
provide more data on this topic in a future publication.
4
h 120°C
1 h 150°C
0.5 h 170°C
Figure 8 Abundance ether linkages and unsubstituted aromatic ring sites in lignin
precipitates obtained after pretreatment of pine wood with [HBim][HSO ] with
0 wt% water. The lignin and saccharification yield are shown for reference. The
4
2
lignins selected here were similar in composition, while indicators of
pretreatment effectiveness were markedly different.
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
EFFECT
OF
BIOMASS LOADING
ON
PRETREATMENT
EFFECTIVENESS
Lignocellulose pretreatment is a capital-intensive process, with
the pretreatment reactor(s) expected to represent ca. 20% of the
capital expenditure (CAPEX) in a typical cellulosic ethanol
plant.47,48 Since the pretreatment reactor size and also solvent
cost will scale approximately inversely with solids loading
β-β' β-O-4' β-1'
β-5'
G6
G5
G2
cond
G2
(
more precisely with total volume), a higher solids loading will
intensify the process and decrease CAPEX by approximately
0% when doubled, according to standard power laws
exponent 0.7) for economies of scale.22 In order to improve the
1
h 120°C
1 h 150°C
1 h 170°C
4
(
Figure 7 Abundance of ether linkages and C-H
isolated pine lignin, obtained after pretreatment with [HBim][HSO
n
sites on the guaiacyl ring of
4
] with 20 wt%
economic viability of ionoSolv pretreatment of pine wood, high
solids loadings are therefore important.49
water for 1 h.
We thus assessed the pretreatment efficacy for pine at loadings
ranging from 5 to 50 wt%, corresponding to biomass to solvent
ratios from 1:20 to 1:2 g/g. These experiments were carried out
using the optimal conditions for glucose release from the pulp
We also compared the characteristics of the lignin isolated at
the optimum pretreatment conditions. Figure 8 shows the
relative size of the ether linkage and guaiacyl ring signals for
lignins isolated after 0.5 h at 170°C, 1 h at 150°C and 4 h at
identified for [HBim][HSO
shows the pulp, lignin and saccharification yields
saccharification conducted after air-drying of the pulps) as a
4
] (170°C for 30 min). Figure 9
1
20°C, as well as the corresponding lignin precipitate and
saccharification yield of the pulp (as a percentage of the
maximum possible). The data show that these lignins had a
similar degrees of condensation and quantities of unhydrolysed
ether linkages, while lignin and saccharification yields were
substantially different. We have previously shown that lignin
isolated from Miscanthus at optimum conditions using
(
function of the biomass loading. Pulp yields increased from
around 41% to 56% when increasing the loading from 5 to 50
wt%. Lignin yields remained stable at around 55% for the 5-
25% solid loading range, while for the 35 and 50 wt% loadings,
] are different in chemical characteristics.22 Here,
a drop in the lignin yield to around 30% was observed. The
saccharification yield was highest at the lowest loading, giving
[TEA][HSO
4
we observe that lignin extraction and yield can be increased
with temperature without altering the degree of condensation
and the degree of hydrolysis in the precipitated lignin. This
could be due to the different compositions of grass and
softwood lignins.
7
5
8% of the theoretical yield, decreasing to below 40% for the
0 wt% pine wood loading.
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ARTICLE
lower M
w
at higher loadings. This is consistent with the
apparent trend towards more ether cleavage with increasing
biomass loading. Especially the decrease in the PDI is thought
to stem from an upper cut-off for M during ethanol dilution at
w
high loadings, resulting in a narrower range of molecular
weights in the isolated lignin.
These findings suggest that delignification and the structure of
the extracted lignin are not altered in a significant way at higher
loadings, but lignin yield and molecular weight of the isolated
lignin is. The lignin and saccharification yield, and the pulp
recovery, may be improved, and higher loading allowed with
an adapted washing protocol that avoids re-precipitation and/or
removes re-precipitated lignin from the pulp surface.
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
Biomass Loading (wt%)
Pulp recovered
Lignin precipitate
Saccharification yield
9
0
Figure 9 Effect of biomass loading on pulp yield, lignin yield and glucose release
from the pulp via enzymatic saccharification after pretreatment of pine with
80
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
[
4
HBim][HSO ] (20 wt% water) for 30 min at 170°C.
The increase in pulp yield and the decrease in lignin yields with
higher loading could be due to limitations of particle wetting in
the ionic liquid solution. It is also possible that the lignin was
extracted and precipitated during the pulp washing with
ethanol, discussed in more detail below.
We also investigated how lignin properties are affected by
increasing the biomass loading. Figure 10 shows the HSQC
NMR signal intensities, as well as molar mass of the lignins as
a function of biomass loading. The HSQC NMR analysis
showed only small changes in the chemical functionality of the
recovered lignins with increasing biomass loading. It appears
that the ether linkage content in the isolated lignin decreased
slightly with increased loadings, and the degree of condensation
and pseudo-lignin content also rose, which could be due to
increased concentration of dissolved lignin fragments and
hemicellulose in the ionic liquid solution. The observed
differences are small, however, and they may not be significant.
Overall, the compositions of the lignins isolated at various
loadings were broadly similar.
β-β' β-O-4' β-1'
10%
β-5'
15%
G2
cond
G2
G5
50%
G6
5
%
25%
35%
1
0000
6
4
2
0
8
6
000
000
4000
2
000
0
0
20
40
60
Biomass Loading (%)
n w
The GPC data demonstrate a decrease in M and M with
higher loading, although the values found for the 15 and 25
wt% loading experiments were inconsistent with the trend.
Furthermore, the PDI of the isolated lignin was lower at higher
loadings. As mentioned earlier, the observed differences may
Mn
Mw
PDI
Figure 10 (Top) Abundance of chemical functionalities in isolated pine lignin as a
function of biomass loading according to HSQC NMR spectroscopy. Signal
intensities are relative to 100
2
G +G2,cond. (Bottom) Molar masses and
be due to the pulp isolation method (employing ethanol polydispersity of recovered lignins as a function of biomass loading. Pine was
4
pretreated with [HBim][HSO ] (20 wt% water) for 30 min at 170°C.
washing), resulting in a bias in the fraction recovered rather
than the lignin structure being affected by the higher loading
directly. This is supported by the fact that sulfate based ionic Effect of pulp drying and hornification on saccharification yields
liquids have been shown to dissolve up to 70 wt% lignin50 and
it is therefore unlikely that we were witnessing saturation even
be subjected to enzymatic hydrolysis directly after washing,
at the highest biomass loadings. However, ethanol has a much
lower lignin solubility,51 and so its addition is likely to
laboratory procedure in order to facilitate sample handling).
precipitate larger fragments out of solution during the washing
step. Since the solubility of polymers of equivalent chemical
hornification of pretreated lignocellulose which negatively
functionality is governed by molecular size, we expect that the
In an industrial process, the cellulose-rich pulp is expected to
rather than undergoing an air-drying step (which is part of our
However, we note that there is evidence that air-drying leads to
affects enzymatic saccharification yields,38 hence we
cut-off for larger fragments in ethanol would be at slightly
investigated whether omitting the air-drying would improve
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Page 8 of 13
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glucose release from pine pulps. For this, pulp samples were outcome than a more expensive alkylimdiazolium hydrogen
submerged in water directly after Soxhlet extraction with sulfate. Lignin recovery correlated well with saccharification
ethanol. Moisture contents of wet pulps obtained after solvent yields. The highest lignin recovery was observed for
exchange were around 90 wt%. Wet saccharification was [DMBA][HSO
carried out for pulps obtained at 10 or 50 wt% loading (1:10 saccharification yield (30 and 36%, respectively) were obtained
and 1:2 g/g biomass to solvent ratio), using the optimised when using [TEA][HSO ]. This was less than half of the yields
pretreatment conditions of 30 min at 170°C for [HBim][HSO ]. achieved by the best-performing [DMBA][HSO ].
The results in Table 1 show that the sugar release from wet [TEA][HSO ] has previously been shown to effectively
4
](72%), while the lowest lignin recovery and
4
4
4
4
pulps was substantially higher. At 10% loading, we observed delignify Miscanthus,16 sugarcane bagasse17 and willow18
quantitative glucose release from the wet pulp, while at 50% biomass, resulting in saccharification yields of up to 80% from
loading, 75% of the glucose was released, representing an air-dried pulps,14,16 although slightly higher yields at optimised
increase of 57 and 120% vs. the dried pulps, respectively. This conditions have been typically observed for imidazolium based
represents a significant improvement. Even with the lower yield hydrogen sulfate ILs (up to 90%).23 It is unclear at present why
at 50% biomass loading, we estimate the reduction in reactor [TEA][HSO
size would reduce CAPEX per tonne of glucose obtained by [HBim][HSO
4
]
was so drastically outperformed by
] and [DMBA][HSO ] in this case. We note that
4
4
5
7%.
the order of fractionation performance mirrors how fast the
three ionic liquids hydrolyse β-O-4 linkages, as measured by
Table 1 Saccharification yield obtained from air-dried and wet pine pulps. Pine was
pretreated at 170°C for 30 min with a solid to solvent ratio of 1:10 and 1:2 g/g in
Gregorio et al.53 We also note that [DMBA][HSO
] and
4
TEA][HSO ] have the same molar mass and it is therefore
4
[
[
4
HBim][HSO ].
unlikely that the difference in performance is linked to an effect
of molar ratio of ionic liquid to water or biomass. Work on
correlating the effect of ionic liquid cation substitution pattern
with pretreatment efficacy is currently underway and will be
presented in due course.
Saccharification yield
Loading
0%
0%
with air drying
without air drying
102.2±1.9%
1
5
64.8±3.5%
34.4±3.2%
74.9±2.9%
SELECTION OF IONIC LIQUID
The imidazolium-based ionic liquid [HBim][HSO ] can be
4
produced at relatively low-cost compared to many imidazolium
ILs, due to being a protic IL that can be synthesised from
inexpensive sulfuric acid and the imidazole precursor in one
step. However, alkylammmonium hydrogen sulfates would be
more cost-effective, as the 1-butylimidazole precursor is still
fairly expensive. Hence we tested two ILs made from low-cost
amines, triethylammonium hydrogen sulfate, [TEA][HSO
N,N-dimethylbutylammonium hydrogen sulfate,
DMBA][HSO ], under the optimised pretreatment conditions
of 30 min at 170°C.
TEA][HSO
4
] and
[
4
Figure 11 Saccharification yield (from air-dried pulps) and lignin and pulp
[
4
]
has been successfully applied for the recovery after pretreatment at 170°C for 30 min with three protic hydrogen
fractionation of Miscanthus,16,22 sugarcane bagasse,17 willow18
sulfate ILs, each containing 20 wt% water. Pine was pretreated at a solid to
solvent ratio of 1:10 g/g.
and switchgrass,14 while the asymmetric [DMBA][HSO
never been used as a fractionation solvent. We were particularly
interested in applying [DMBA][HSO ] due to it being a liquid
4
] has
4
HSQC NMR spectroscopy of the lignins isolated from different
at room temperature, even in the absence of water, and hence it ionic liquids showed a decrease in the quantity of ether bonds
having a low viscosity, which facilitates handling in the lab and in the lignin in the order [TEA]>[HBim]>[DMBA], with
at scale.52 It has also been shown to be very effective in
hydrolysing β-O-4 linkages in a lignin model compound.53 We
volume integral sizes for the β-O-4’ bond of 13.6 vs 10.4 and
6.9%, respectively (Figure 12), which again is in line with the
have previously demonstrated that the optimum pretreatment
conditions for 1-butylimidazolium and triethylammonium
hydrogen sulfate solutions are the same, if the acid base ratio is
the same.16,23
The saccharification yields and lignin and pulp recoveries
obtained for 10 % solid loadings are shown in Figure 11. We
obtained the highest saccharification yields for [DMBA][HSO
4
]
(74%), showing that a lower-cost trialkylammonium hydrogen
sulfate IL can achieve similar or an even better pretreatment
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ARTICLE
decreasing rate constant observed for the three ILs. Table 2 Molar mass and polydispersity of lignins recovered after pretreatment of pine
wood at 170°C for 30 min with three low-cost hydrogen sulfate ILs. A solid to solvent
ratio of 1:10 g/g was used.
IL
M
n
M
w
PDI
3.7
3.9
5.1
[TEA][HSO
[HBim][HSO
[DMBA][HSO
4
]
922
967
775
3452
3810
3949
4
]
4
]
w n
M : Average molar masss, M : number average molar mass, PDI:
Polydispersity index
Experimental
Materials
Starting materials for ionic liquid synthesis were purchased
from Sigma Aldrich and used as received, unless stated
otherwise. H NMR spectra were recorded on a Bruker 400
The degree of condensation in the lignin, evidenced by a more
intense G2,cond signal, increased in the same order. The lignin
isolated using [DMBA][HSO ] can therefore be regarded as the
4
most modified lignin. Together with the high lignin precipitate
yield obtained with this IL, these data suggest that lignin
extraction was faster, probably due to a combination of
improved mass transfer due to the low viscosity of
1
MHz spectrometer. Chemical shifts (δ) are reported in ppm, the
1
13
DMSO signal at 2.500 ( H dimension) and 39.520 ( C
dimension). Electrospray mass spectrometry experiments were
carried out by Dr Lisa Haigh (Imperial College London,
Chemistry Department) on a Micromass Premier spectrometer.
The Karl-Fisher titrator used in this work was a V20 volumetric
Titrator (Mettler-Toledo) and the analytical balance a Sartorius
CPA 1003 S balance (±0.001 g).
[
DMBA][HSO
the acidic ionoSolv solution) and the higher reactivity of lignin
in [DMBA][HSO ]. A similar trend was observed for the
4
] (giving rise to longer lignin residence times in
4
average molar masses for the isolated lignins, as determined by
gel permeation chromatography (Table 2). Pretreatment with
Synthesis of 1-hydrogen-3-butylimidazolium hydrogen sulfate
[
DMBA][HSO
than applying the other two ILs. As a result, a high PDI of 5.1
was found for this IL vs. PDIs of 3.7 and 3.9 for [TEA][HSO
and [HBim][HSO ], respectively. We note that the lignin
obtained under optimised conditions with [DMBA][HSO ] has
a notably higher PDI than those observed with the other two
ILs, or for Miscanthus pretreated with [TEA][HSO ] at
optimised conditions,22 however, willow pretreatment with
TEA][HSO ] has resulted in similarly high PDIs of 4.6 up to
4 n w
] yielded a lower M and a higher M lignin
[
HBim][HSO
M H SO (322 mL, 1.61 mol) was cooled with an ice bath.
Under stirring, 200 g of freshly distilled N-butylimidazole
1.61 mol) was added dropwise. The water was removed under
4
].
5
2
4
4
]
4
(
4
reduced pressure and the product dried in vacuum at 50°C
overnight. The ionic liquid was recovered as a clear, pinkish,
4
1
viscous liquid. H NMR: δH (400 MHz, DMSO-d
6
)/ppm: 9.14
(
s, 1H, N-CH-N), 7.79 (s, 1H, N-CH), 7.68 (s, 1H, N-CH), 4.26
(br, N-H, HSO ), 1.77 (m, 2H,
-), 4.19 (t, J = 7.2 Hz, 2H, N-CH
N-CH -CH ), 1.23 (m, 2H, N-CH -CH -CH ), 0.88 (t, J = 7.4
Hz, 3H, N-CH -CH -CH -CH
). 13C NMR: δC (101 MHz,
DMSO-d )/ppm: 135.72 (N-CH-N), 122.46 (N-CH), 120.45 (N-
CH), 48.68 (N-CH
CH ), 13.75 (N-CH
[
4
over 6 for optimised sugar release.18
4
2
2
2
2
2
2
2
2
2
3
6
2
), 31.90 (N-CH
-CH -CH -CH
3
2
-CH
).
2
), 19.29 (N-CH
2
-CH
2
-
2
2
2
2
+
+
-
MS (Magnet FB ) m/z: 125 ([HBim] , 100%), (Magnet FB )
m/z: 79 ([HSO
4
]-, 100%).
Synthesis of triethylammonium hydrogen sulfate [TEA][HSO4].
Triethylamine (75.9 g, 750 mmol) was cooled with an ice bath
in a round-bottom flask. 150 mL 5 M H SO (750 mmol) was
2 4
added dropwise while stirring. The water was removed using a
rotary evaporator and the product dried using the Schlenk line
at 40°C overnight. The ionic liquid was recovered as a white,
Figure 12 Abundance of ether linkages and C-H motives on the guaiacyl ring in hygroscopic solid.
pine lignin recovered after pretreatment at 170°C for 30 min with three protic
hydrogen sulfate ILs containing 20 wt% water, according to HSQC NMR
spectroscopy. Pine wood was pretreated at a solid to solvent ratio of 1:10 g/g.
1
-,
4
H NMR: δH (400 MHz, DMSO-d
6
)/ppm: 3.39 (s (br), HSO
N-H ), 3.10 (q, J = 7.3 Hz, 6H, N-CH ), 1.20 (t, J=7.3 Hz, 9H,
N-CH -CH
). 13C NMR: δC (101 MHz, DMSO-d
N-CH ), 9.15 (N-CH -CH ).
+
2
2
3
6
)/ppm: 46.21
(
2
2
3
+
+
-
MS (Magnet FB ) m/z: 102 ([TEA] , 100%), (Magnet FB )
m/z: 79 ([HSO
4
]-, 100%).
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Synthesis of N,N-dimethylbutylammonium hydrogen sulfate
Saccharification assays were carried out according to a protocol
published by the NREL55 in triplicates with blanks (also
triplicates). All reagents were purchased from Sigma Aldrich.
The detailed procedure can be found in the ESI.
Hydrolysis was run for 7 days and Novozymes experimental
enzyme mixture NS-22201 was used as the enzymes. Analysis
was performed on a Shimadzu HPLC system with RI detector
and an Aminex HPX-87P column (BioRad, 300 x 7.8 mm) with
purified water as mobile phase (0.6 mL/min).
[
DMBA][HSO
N,N-dimethylbutylamine (75.9 g, 750 mmol) was cooled with
an ice bath in a round-bottom flask. 150 mL of 5 M H SO
750 mmol) was added dropwise while stirring. The water was
4
].
2
4
(
removed using the rotary evaporator and the product dried
using the Schlenk line at 70°C overnight. The ionic liquid was
recovered as a clear, viscous liquid.
1H NMR: δH (400 MHz, DMSO-d
6
)/ppm: 9.24 (s, 1H, N-H),
), 2.76 (d, J = 4.3 Hz, 6H,
), 1.64 – 1.51 (m, 2H, N-CH -CH ), 1.29 (h, J = 7.4
Hz, 2H, N-CH -CH -CH ), 0.89 (t, J = 7.4 Hz, 3H, N-CH
CH -CH -CH )/ppm:
). 13C NMR δC (101 MHz, DMSO-d
6.62 (N-CH
CH -CH -CH
3
.02 (dt, J = 12.9, 5.0 Hz, 2H, N-CH
2
Lignin characterisation
N-(CH
)
3 2
2
2
2
2
2
2
-
HSQC NMR spectroscopy
2
2
3
6
Circa 20 mg of isolated lignin was dissolved in 0.25 mL of
DMSO-d and the solution transferred to a Shigemi tube.
6
HSQC NMR spectra were recorded on a Bruker 600 MHz
spectrometer (pulse sequence hsqcetgpsi2, spectral width of 10
5
2
), 42.48 (N-CH
), 13.71 (N-CH
3
), 25.82 (N-CH
2
-CH
).
2
), 19.40 (N-
2
2
2
2
-CH -CH -CH
2
2
+
3
+
-
MS (Magnet FB ) m/z: 102 ([DMBA] , 100%), (Magnet FB )
m/z: 79 ([HSO
4
]-, 100%).
1
13
ppm in F2 ( H) with 2048 data points and 160 ppm in F1 ( C)
with 256 data points, 16 scans and 1 s interscan delay). Spectra
were analysed using MestReNova (Version 8.0.0, Mestrelab
Research 2012). All spectra were referenced to the DMSO peak
Feedstock
Pinus sylvestris was obtained from Metla (Finnish Forest
Research Institute). It was air-dried, ground and sieved (180-
1
13
at 2.500 ppm ( H) and 39.520 ppm ( C). Integrals were
obtained for spectra of the same series of experiments
simultaneously to ensure that the same areas were integrated.
All relevant spectra were copied into one file and selected them
while integrating the relevant area in one spectrum. Integration
areas were selected visually according to peak assignments
8
50 µm, 20 + 80 US mesh scale) prior to use and stored in
plastic bags at room temperature in the dark.
Fractionation of Biomass
Pretreatments, determinations of oven dried weight and ionic
liquid water content measurements were conducted according
found in literature.21 For ether linkages, the C-H
integrated. Integral sizes are reported with respect to 100
+G2,cond) signals. All spectra can be found in the ESI.
α
-signals were
1
5
to the standard operating procedures from our laboratory , in
triplicate. A biomass to solvent ratio of 1:20 to 1:2 g/g was used
(G
2
(ODW basis). Hydrogen sulfate ionic liquids with 20wt% water
were used as the solvent, using between 2 and 10 g of ionic Gel permeation chromatography
liquid-water mixture and 1 g of biomass, except for the 1:20 g/g
GPC measurements were performed using an Agilent 1260
Infinity instrument equipped with a Viscotek column set
AGuard, A6000M and A3000M). The Agilent 1260 Infinity
RID detector was used for detection. GPC grade DMSO
containing LiBr (1 g/L) was used as eluent at a flow rate of 0.4
mL/min at 60°C. Samples were prepared by dissolving 20 mg
lignin in 1 ml eluent and filtering through a 0.2 µm syringe
filter. Ten pullulan standards (Agilent calibration kit, 180 < Mp
experiment, where only 0.5 g of biomass and 10 g of IL-water
mixture was used. A detailed description of the procedure can
be found in the ESI.
(
For experiments without air-drying of the pulp, the procedure
was followed unchanged until completion of the Soxhlet
extraction step. After the extraction, the pulp containing
thimbles were transferred from the Soxhlet adapter into 50 mL
centrifuge tubes and DI water was added immediately. This was
incubated for at least an hour. The thimble was removed from
the plastic tube and the pulp transferred back to the centrifuge
tube using a spatula. The suspensions were centrifuged for 30
<
780,000) were used to calibrate the instrument.
Conclusions
min at 2000G and the supernatant decanted. The wet pulps We have demonstrated the successful application of easily-
were weighed and their moisture content determined synthesised protic ionic liquids for the pretreatment of the
immediately. Thereafter, the pulps were stored in the fridge at recalcitrant softwood, pine. A highly digestible cellulose pulp is
4
°C.
obtained, giving rise to quantitative glucose yields after only
0 min of pretreatment at 170°C. Quantitative glucose release
was achieved at a solid to liquid ratio of 1:10 g/g using 1-
3
Compositional Analysis
Compositional analysis was carried out according to a butylimidazolium hydrogen sulfate, when the pulp drying step
published procedure by the National Renewable Energy was omitted. High saccharification yields were correlated with
Laboratory (NREL).54 The detailed procedure can be found in
extensive lignin removal from the pine wood.
the ESI.
Carbohydrate free lignins were isolated through antisolvent
(
water) addition and were found to be depolymerised and
Saccharification Assay.
partially condensed.
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ARTICLE
Furthermore, we show that high solids loading of up to 1:2 g/g
achieve good pine fractionation, especially if the cellulose-rich
pulp is not air-dried. It may be possible to devise an adapted 11
Stavila, J. Pelton, H. Blanch, B. a. Simmons and A. George, Green
Chem., 2014, 16, 1236–1247.
S. H. Lee, T. V Doherty, R. J. Linhardt and J. S. Dordick,
Biotechnol. Bioeng., 2009, 102, 1368–76.
washing protocol that reduces re-precipitation and/or removes
1
1
1
2
3
4
X.-F. Tian, Z. Fang, D. Jiang and X.-Y. Sun, Biotechnol. Biofuels,
re-precipitated lignin from the pulp surface, further improving
the performance of ionoSolv pretreatment of softwood at high
solids loadings.
2
011, 4, 1–14.
M. T. Clough, K. Geyer, P. a Hunt, J. Mertes and T. Welton, Phys.
Chem. Chem. Phys., 2013, 15, 20480–95.
4
The ultra low-cost ionic liquid [DMBA][HSO ] proved to be a
A. George, A. Brandt, K. Tran, S. M. S. N. S. Zahari, D. Klein-
Marcuschamer, N. Sun, N. Sathitsuksanoh, J. Shi, V. Stavila, R.
Parthasarathi, S. Singh, B. M. Holmes, T. Welton, B. A. Simmons
and J. P. Hallett, Green Chem., 2015, 17, 1728–1734.
more effective pretreatment solvent than [HBim][HSO
the conditions studied.
4
] under
The potential to use low-cost ionic liquids as effective
pretreatment media for softwoods is very encouraging, as
ionoSolv pretreatment has the potential not only to be
economically viable but also to be feedstock independent,
which is a unique combination.
1
1
5
6
F. J. V. Gschwend, A. Brandt, C. L. Chambon, W.-C. Tu, L.
Weigand and J. P. Hallett, J. Vis. Exp., 2016, e54246.
A. Brandt-Talbot, F. J. V. Gschwend, P. S. Fennell, T. M.
Lammens, B. Tan, J. Weale and J. P. Hallett, Green Chem., 2017,
1
9, 3078–3102.
Acknowledgements.
17
C. L. Chambon, T. Y. Mkhize, P. Reddy, A. Brandt-Talbot, N.
Deenadayalu, P. S. Fennell and J. P. Hallett, Biotechnol. Biofuels,
The authors wish to acknowledge financial support from the
Grantham Institute for Climate Change and the Environment
and Climate-KIC for a studentship for FJVG, and the
Engineering and Physical Sciences Research Council (EPSRC;
EP/K014676/1 and EP/K038648/1) for funding for ABT.
CLC is grateful for funding in the form of an Imperial College
President’s Scholarship (EPSRC).
2
018, 11, 247.
1
1
2
2
2
2
2
8
9
0
1
2
3
4
L. Weigand, S. Mostame, A. Brandt-Talbot, T. Welton and J. P.
Hallett, Faraday Discuss., 2017, 202, 331–349.
A. Brandt, J. Gräsvik, J. P. Hallett and T. Welton, Green Chem.,
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