One-pot integrated biofuel production using low-cost biocompatible protic ionic liquids

Article abstract of DOI:10.1039/c7gc01179b

The transformation of biomass into liquid fuels is of great importance. Previous work has demonstrated the capability of specific ionic liquids (ILs), such as 1-ethyl-3-methylimidazolium acetate ([C₂C₁Im][OAc]) and cholinium lysinate ([Ch][Lys]), to be effective biomass pretreatment solvents. Using these ILs for an integrated biomass-to-biofuel configuration is still challenging due to a significant water-wash related to the high toxicity of [C₂C₁Im][OAc] and pH adjustment prior to saccharification for the highly basic [Ch][Lys]. In this work, we demonstrate, for the first time, that a one-pot integrated biofuel production is enabled by a low cost (～$1 per kg) and biocompatible protic IL (PIL), ethanolamine acetate, without pH adjustments, water-wash and solid-liquid separations. After pretreatment, the whole slurry is directly used for simultaneous saccharification and fermentation (SSF) with commercial enzyme cocktails and wild type yeast strains, generating 70% of the theoretical ethanol yield (based on switchgrass). The structure-performance relationships of PILs in terms of lignin removal, net basicity, and pH value are systematically studied. A technoeconomic analysis (TEA) revealed that an integrated biorefinery concept based on this PIL process could potentially reduce the minimum ethanol selling price by more than 40% compared to scenarios that require pH adjustment prior to SSF. Improvement of the economic performance will be made by reducing the dilution and enzyme loading during SSF as identified by TEA. This study demonstrates the impact of a biocompatible IL in terms of process optimization and conversion efficiency, and opens up avenues for realizing an IL based efficiently integrated biomass conversion technology.

Full text of DOI:10.1039/c7gc01179b

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DOI: 10.1039/C7GC01179B
One-pot integrated biofuel production using low-cost
biocompatible protic ionic liquids
Jian Sun1,2, N.V.S.N. Murthy Konda1,3#, Ramakrishnan Parthasarathi1,2#, Tanmoy Dutta1,2,
Marat Valiev4, Feng Xu1,2, Blake A. Simmons1,3, and Seema Singh1,2*
1 Deconstruction Division, Joint BioEnergy Institute, Emeryville, CA, USA.
2 Biomass Science and Conversion Technology Department, Sandia National Laboratories,
Livermore, CA, USA.
3 Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory,
Berkeley, CA, USA.
4 Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory,
Richland, WA, USA.
*Corresponding author: E-mail: seesing@sandia.gov
# Equal contribution as the co-second author.
Abstract: Transformation of biomass into liquid fuels is of great importance. Previous
work has demonstrated the ability of specific ionic liquids (ILs), such as 1-ethyl-3-
methylimidazolium acetate ([C2C1Im][OAc]), and cholinium lysinate ([Ch][Lys]), to be
effective biomass pretreatment solvents. Using these ILs for an integrated biomass-to-
biofuel configuration is still challenging due to significant water-wash related to high
toxicity of [C2C1Im][OAc] and pH adjustment prior to saccharification for highly basic
[Ch][Lys]. In this work, we demonstrate, for the first time, that a one-pot integrated
biofuel production enabled by a low cost (~$1/kg) and biocompatible protic IL (PIL),
ethanolamine acetate, without pH adjustments, water-wash and solid-liquid separations.
After pretreatment, the whole slurry is directly used for simultaneous saccharification and
fermentation (SSF) with commercial enzyme cocktails and wild type yeast strains,
generating 70% of theoretical ethanol yield (based on switchgrass). The structure-
performance relationships of PILs in terms of lignin removal, net basicity, and pH value
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are systematically studied. Techno-economic analysis (TEA) revealed that an integrated
biorefinery concept based on this PIL process could potentially reduce minimum ethanol
selling price by more than 40% compared to scenarios that require pH adjustment prior to
SSF. Improvement of the economic performance will be made by reducing dilution and
enzyme loading during SSF as identified by TEA. This study demonstrates the impact of
a biocompatible IL in terms of process optimization and conversion efficiency, and opens
up avenues for realizing an IL based efficiently integrated biomass conversion
technology.
1. Introduction
Lignocellulosic biomass has long been recognized as a potential significant source of
mixed sugars and aromatics for the production of biofuels, renewable chemicals, and
other biomaterials.1, 2 The recalcitrance of lignocellulose is the major hurdle in terms of
conversion into fermentable sugars using thermochemical or enzymatic approaches.3
Among the various pretreatment technologies developed to overcome biomass
recalcitrance, ionic liquid (IL)-based technology has been shown to have excellent
performance due to unique solvation properties and superior performance of certain ILs
when compared to other classes of solvents.4 The IL pretreatment process can be
feedstock agonistic,5, 6 efficient with high biomass loadings,5 efficiently scaled-up,7 and
operated in a continuous mode.8 The main challenges of the IL pretreatment process in
terms of commercial viability are cost-competitiveness and reduction in process
complexity. An IL-based integrated process that combines biomass pretreatment,
enzymatic hydrolysis and microbial fermentation has been recently demonstrated to
address some aspects of these challenges.9, 10
However, there remain several challenges related to IL toxicity, pH compatibility and
IL cost, that need to be addressed prior to the realization of and affordable and scalable
IL-based biomass conversion technology. The toxicity of most of the ILs that are
2

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effective biomass solvents is detrimental to enzymes and microorganisms used
downstream. There have been several efforts on the development of engineered IL-
tolerant enzymes and microbes, to address this challenge.11, 12 However, removal of ILs
from the pretreated slurry is still commonly practiced to reduce toxicity, but the excessive
use of water and corresponding water treatment pose an economic challenge for the
scale-up of water-wash based IL pretreatment technology.13 Another challenge is pH
compatibility between the three unit operations (biomass pretreatment, saccharification
and fermentation), especially in the case of ILs (e.g. cholinium lysinate, 1-ethyl-3-
methylimidazolium lysinate and cholinium glycine) that generate high pH values (>10) to
benefit pretreatment.10, 14, 15 In this context, pH adjustment using a mineral acid before
saccharification and fermentation is generally required, which significantly impacts IL
recovery and reuse. The third challenge is the concern about the cost of pretreatement
process, because ILs remain to date the most expensive research-grade solvents under
investigation for the dissolution of biomass.16 To address this challenge, one proven way
is through the use of protic ILs (PILs).4 This class of ILs that are synthesized through
simple neutralization of organic amine with mineral acid, have been explored for various
applications.17-19 As one example, the bulk production of triethylammonium hydrogen
sulfate could be as low as $1.24 kg−1,20 and its performance on lignocellulosic biomass
pretreatment was tested in water washing process.21 However, given all the advantages of
new ILs, process consolidation for cellulosic biofuel production enabled by developing
low toxic, pH compatible and low-cost IL is still highly desirable.
In this work, for the first time, we demonstrated the potential of low-cost PILs in
addressing the above challenges for cellulosic ethanol production via consolidated
bioprocessing. These ILs contain ions derived from simple amino bases (e.g.,
ethanolamine, diethanolamine, triethanolamine, and cholinium hydroxide) and acids (e.g.,
acetic acid, sulfuric acid, phosphoric acid, hydrochloric acid and formic acid). Enabled by
high lignin removal capacity, low toxicity and pH compatibility of the optimized PIL, the
pretreated slurry is directly converted into ethanol (70% of theoretical yield) via
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simultaneous saccharification and fermentation (SSF) using commercial enzyme and wild
type microbes. This integrated approach represents a promising route that includes
several salient features: 1) employs inexpensive, biocompatible and easily-synthesized
PILs; 2) allows high biomass loading pretreatement; 3) is water-wash free; and 4) does
not require pH adjustment or solid/liquid separations.
2. Results and Discussion
2.1 Identifying and screening of ILs
The continued search for an IL with the desirable physical, chemical and biological
properties for biomass pretreatment is difficult without the insight of fundamental
understanding and predictive development. It has been indicated that solubility of major
biomass components (e.g. cellulose and/or lignin), is critical for effective biomass
pretreatment.6 The inherent intricacy of these biomass components associated to the
many different types of possible interactions allows very selective dissolution in wide
range of different ILs. It is reported that solvent parameters such as basicity (β) correlates
well with an IL’s ability to dissolve lignocellulose,22, 23 and that net basicity correlates
with an IL’s ability to dissolve cellulose.23, 24
In PILs, the proton transfer from the acid to the base leads to the presence of proton-
donor and proton-acceptor sites, which can be used to establish hydrogen-bond
networks.25 The cations of the PILs studied are presented in Fig. 2A, including simple
and inexpensive primary, secondary or tertiary alkylamines such as ethanolamine (EOA),
diethanolamine (DEOA), triethanolamine (TEOA), and triethylamine (TEA), while the
anions were derived from standard organic and mineral acids such as acetic acid, lactic
acid, sulfuric acid, formic acid and hydrochloric acid. The synthesized PILs are named as
ethanolamine acetate ([EOA][OAc]), ethanolamine lactate ([EOA][La]), ethanolamine
formate ([EOA]F), ethanolamine bisulfate ([EOA][HSO4]), ethanolamine chloride
([EOA]Cl, diethanolamine acetate ([DEOA][OAc]), diethanolamine formate ([DEOA]F),
triethanolamine acetate ([TEOA][OAc]), triethanolamine formate ([TEOA]F), and
triethylamine bisulfate ([TEA][HSO4]). Cholinium acetate ([Ch][OAc]) and 1-ethyl-3-
4

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methylimidazolium acetate ([C2C1Im][OAc]) were also investigated for comparing the
impact of different combinations of anions and cations.
We calculated the molecular acidity, basicity, and net basicity values (Fig. 2B) as well
as optimized geometries (Fig. S1, ESI) for these ILs using a set of density functional
theory (DFT, see descriptors in ESI) based global descriptors such as chemical potential
(µ) and chemical hardness (η) from the standard working equations. Fig. 2B shows the
predicted solvent parameters of ILs investigated in this work. Comparison of the
experimental screening results with the calculated solvent parameters of these ILs
indicates that effective pretreatment requires an IL with high net basicity. Notably,
ethanolamine acetate ([EOA][OAc]) has the highest net basicity value (1.7) as compared
with the other ILs selected for screening including [C2C1Im][OAc] (0.69), [Ch][OAc]
(0.29), [EOA]Cl (0.52), [EOA][HSO4] (0.46) and [TEA][HSO4]) (0.51). Predictably,
more acidic anion counterpart lowers the net basicity values of PILs and increasing the
number of ethyl groups in cations gradually decreases the ILs net basicity ([EOA][OAC]
vs. [DEOA][OAC] vs. [TEOA][OAC] is 1.7 vs. 1.64 vs. 0.91); ([EOA]F vs. [DEOA]F
vs. [TEOA]F is 1.63 vs. 1.57 vs. 0.86). Also it can be seen that net basicity values of
[EOA][OAc] (1.7), [DEOA][OAc] (1.64) and [EOA]F (1.63) are higher than that of
+
+
[C2C1Im][OAc] (0.69). In comparison with the –N(CH3)3 group in [Ch][OAc], -NH3
group in [EOA] framework tends to have stronger hydrogen bonding interactions with
lignocellulosic components, which enhances biomass solvation.26,27
The corresponding PILs were synthesized through a stoichiometric neutralization
reaction of certain acids and bases (Fig. 3A), and verified by NMR analysis (1H NMR
13
and C NMR spectrum are provided in Figs. S2-S11, ESI). Next, one-pot pretreatment
and saccharification efficiency of these PILs were evaluated using switchgrass (SG) as
the model biomass and the results were summarized in Fig. 3B. Among all of the PILs
synthesized, [EOA][OAc] displayed the best performance, and the sugar yields (glucose,
57.9%; xylose 18.7%) were comparable or even better than [C2C1Im][OAc] (glucose,
54.4%; xylose 10.4%). The acetate or formate anion with a variety of cations
demonstrated the strong dependence of the performance on the nature of cation.
Compared to [EOA] cation, the cations such as [DEOA] and [TEOA], led to lower
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glucose yields as 32, 20 and 5.6%, respectively. The lower sugar yields generated in the
cases of [DEOA] and [TEOA] based PILs are possibly due to the lower net basicity of
these PILs compared to those in the case of [EOA] based ILs (Fig. 2B). Also as
previously discussed,25, 28 primary amine cation appears to have a stronger preference for
the formation of AnB aggregates (A=acid, B=base, n>1) than secondary and tertiary
amine cations, with more free amine present at the 1:1 composition which can contribute
to biomass pretreatment efficiency (Table S1, ESI). The anion of PILs also exhibited
obvious effect on the sugar yield, with acetate as the top performer. The PILs derived
from strong acids such as sulfuric acid, hydrochloric acid, and formic acid, result in low
sugar yields. The result is consistent with the report that acetate based ILs have good
biomass pretreatment efficiencies.29 Based on the enzymatic saccharification results,
[EOA][OAc] was selected for further systematic investigation for the development of an
integrated one-pot process.
2.2 Structure-performance relationship of IL in one-pot process
To further understand the performance of the ILs studied in a one-pot process,
especially in the case of [EOA][OAc], the composition of SG before and after IL
pretreatment in terms of cellulose crystallinity and lignin removal as well as the pH of
ILs were studied. The degree of cellulose crystallinity and lignin removal have been
considered as important factors in determining the hydrolysis rates of relatively refined
cellulosic substrates.30 First, the proportions of crystalline/amorphous cellulose and the
disordered components (i.e. amorphous cellulose, hemicelluloses and lignin) found in
pretreated SG samples were determined by powder X-ray diffraction (pXRD). Fig. 4A (a)
shows the diffraction patterns of untreated SG and pretreated SG at 5 and 10 wt%
biomass loading. Three similar peaks are observed in the diffraction patterns for all of the
samples: the main peak position at 21.7° is indicative of the distance between hydrogen-
bonded sheets in cellulose I; the broad peak at ∼16° is known to be a composite of two
peaks from Iβ, Iα, or both;31 and the third small peak at 34.5°corresponds to 1/4 of the
6

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length of one cellobiose unit and arises from ordering along the fiber direction.32 SG
pretreated by [EOA][OAc] at 5 and 10 wt% biomass loading still retain primarily
cellulose I structure as similar with raw feedstock (Fig. 4A, a), reductions in the cellulose
crystallinity index (CrI) were observed (0.49 and 0.5 vs. 0.68, respectively) (Fig. 4A, a).
To further confirm cellulose structural changes during pretreatment with [EOA][OAc],
Avicel was pretreated under the same conditions and the XRD spectra is also plotted
(Fig. 4A, b). The reduction in the CrI of Avicel was also observed after pretreatment
(0.80 vs. 0.84), corroborating the SG pretreatment data (Fig. 4A, a). It has been shown
that anions of ILs play a critical role in dissolving cellulose, and those that accept
hydrogen bonds from cellulose hydroxyl protons can effectively disrupt the inter- and
intra-molecular hydrogen bonding in cellulose.33 However, the acetate anion with
different cations demonstrated the strong dependence of the performance on the cation.
The replacement of [C2C1Im] by [EOA] cation lead to a negligible effect on the
crystalline structure change of cellulose, which is possibly due to the stronger inter-
hydrogen bonds in the PIL than the intra-hydrogen bonds between PIL and cellulose.
Second, the pretreatment performance of the studied ILs in terms of lignin removal
during pretreatment were conducted by using compositional analysis method. As known,
lignin limits the rate of enzymatic hydrolysis by acting as a physical barrier, preventing
the digestible parts of the substrate to be hydrolyzed.34 High lignin removal is also a key
factor for an effective pretreatment of lignocellulosic biomass.6 Higher lignin removal
was observed when pretreating biomass using an IL with higher net basicity (Fig. 4B),
indicating a good relationship between lignin removal and net basicity. Besides the net
23, 24
basicity correlates with an IL’s ability to dissolve cellulose,
it might also correlate
with an IL’s ability to remove lignin. Although ILs that have high lignin removal,
including [DEOA][OAc], [EOA]F, [DEOA]F, and [EOA][La] are good for pretreatment,
the one-pot data demonstrated in Fig. 3B indicates that there are other contributing
factors in their lower sugar yields. By measuring the pH of ILs (5 wt% in H2O), we found
that these ILs are more acidic than [EOA][OAc] (Fig. 4C), and might limit their use in an
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efficient one-pot process.
Size exclusion chromatography (SEC) analysis of the hydrodynamic volume of lignin
(Fig. 5A, and Fig. S12, ESI) further gives more insight on the impact of IL net basicity
on the lignin extractability. It was found that the lignin streams generated from
pretreatment with ILs with high net basicity have significantly lower hydrodynamic
volume (or size) as compare to enzymatically mild acidolysis lignin (EMAL) extracted
from switchgrass, which is commonly believed as a representation of the ‘native’
switchgrass lignin.35, 36 The reduction in lignin size observed indicate possible lignin
depolymerization during the pretreatment process. Using GC-MS, the possible
depolymerized products from lignin were analyzed for [EOA][OAc], [Ch][OAc] and
[C2C1Im][OAc] based pretreatment (Fig. 5B).
2.3 Influence of process parameters on the production of sugars
Pretreatment conditions (e.g. biomass loading, temperature and time) and
saccharification conditions (e.g. IL and enzyme loading) can greatly affect sugar
production from IL-pretreated biomass, and thus the downstream fermentation yields.
Consequently, we conducted initial parameter optimization to increase process efficiency.
Although increasing biomass loading during pretreatment leads to a decrease in sugar
yield, glucose yields of 75% could still be obtained at biomass loadings varying from 5 to
20 wt% (Fig. 6A) indicating [EOA][OAc] possess an excellent capacity for high loading
biomass pretreatment.
We then determined the impact of pretreatment temperatures on sugar yields from 140
to 160 °C over specific time intervals. The resultant sugars appear to be largely affected
by the variation in pretreatment temperature. The increase in digestibility of SG was over
40% (60% vs. 85%) when temperature was increased by 20 °C (Fig. 6B). At 10% SG
loading, a satisfactory glucose yield (~85%) could be detected for the 160 °C pretreated
sample. Fig. 6B also depicted the effect of pretreatment time on the sugar yield.
Pretreatment at 160 ℃ for 15 min could release 45% glucose from SG. Increasing the
pretreatment time from 0.5 to 3 hrs slightly increased the yields of glucose and xylose
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from 85 to 87%, and 35 to 42%, respectively, indicating that this range of pretreatment
time has minimal impact on the enzymatic saccharification. The study has therefore
demonstrated that complete or near complete enzymatic glucan digestion of pretreated
SG can be achieved with [EOA][OAc] at a relatively shorter reaction time (30 min) than
[C2C1Im][OAc] (3 hrs).13 The compositional analysis of untreated, and pretreated SG
with [EOA][OAc] at different temperatures and time are summarized in Table 1. Three of
the major plant cell wall components of SG, such as glucan, xylan, and acid insoluble
lignin were monitored before and after pretreatment. Solid recovery refers to the mass
percentage of biomass (dry weight) recovered from the original biomass load. After
pretreatment, 55-81 wt% of the SG was recovered depended on the desired pretreatment
temperature and time. Pretreatment at higher temperatures results in lower solid recovery.
The glucan loading in the residual pretreated SG generally increased along with higher
temperature and/or longer time pretreatment. The lignin content of pretreated material
generally decreased as compared to the original biomass. This trend was most obvious
after pretreatment under higher temperature where lignin content was reduced by over
75% at 160 ℃ (untreated: 22% vs. pretreated: 4.7%). These results obtained with
[EOA][OAc] IL are consistent with our previous report by using ILs such as [Ch][Lys]
and [C2C1Im][Lys].14
It has been reported imidazolium-based ILs, such as 1-n-butyl-3-methylimidazolium
chloride ([C4C1Im][Cl]), and [C2C1Im][OAc] are known to deactivate cellulases at fairly
low concentrations (~5 wt%).13, 37, 38 Consistent with the previous reports,39, 40 the results
presented in Fig. 6C show that the commercial enzyme cocktails, Novozymes Cellic®
CTec2 and HTec2 (9:1, v/v), is not inactivated in the presence of 5 wt% PIL and can
tolerant 10 wt% loading of PIL by generating comparable sugars yields with those in the
absence of the IL. With further increasing IL loading to above 10 wt%, noticeable drop in
the sugar yields was observed which might be ascribed to a partial inactivation of the
cocktails.
Fig. 6D shows the effect of commercial enzyme cocktails loading on sugar yields.
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Glucose yields were improved from 35% to 87% when enzyme loading was increased
from 2.5 to 20 mg/g starting biomass. Further increase in enzyme loading resulted in
almost no improvement for sugar yields.
2.4 One-pot integrated process for cellulosic ethanol production
Simultaneous saccharification and fermentation (SSF) after pretreatment is a frequent
practice for cellulosic ethanol production recently. The presence of yeast together with
the cellulolytic enzyme cocktail reduces the accumulation of sugars-therefore increasing
yield and saccharification rate compared with separate saccharification and
fermentation.41 Our previous studies have demonstrated successful high ethanol
production from cellulosic biomass using SSF in the presence of [Ch][Lys].9, 10 However,
the basicity of IL necessitated pH adjustment for that case, due to which IL generation is
still a challenge. The utilization of [EOA][OAc] in the current process creates an
opportunity to eliminate pH adjustment.
SSF requires compatible fermentation and saccharification conditions, with a similar
pH, temperature and optimum substrate concentration. It is necessary to identify the
optimal IL concentration that enables fermentation using wild type strains. For toxicity
screening, a wild type S. cerevisiae BY4741 strain was technically used and its growth
curves were measured in various IL concentrations from 2.5 to 15 wt%. As shown in Fig.
7A, the IL concentration plays a significant effect on the yeast strain growth. Improved
growth occurs at IL loadings below 10 wt%. Since 5 wt% IL concentration could
improve yeast growth, it was chosen for the process intensification of the PIL based SSF.
Fig. 7B shows that 40 wt% starting biomass loading (i.e. 60 wt% IL) for pretreatment that
was diluted to a 5 wt% IL concentration in saccharification could generate a comparative
glucose yield with 20 wt% starting biomass loading.
Based on the above results, performance of yeast during SSF was then investigated
with different yeast inoculation varying from 0.01 to 0.5 wt%. Fig. 8A suggests that there
is no significant difference in ethanol yield when the yeast loading increased from 0.01 to
0.21 wt%. At very low yeast loading of 0.1 wt% (i.e. 1 g/L), ~70% of theoretical ethanol
yield was obtained. The weight ratio of biomass amount to yeast is as high as around
33.3:1 indicating that the one-pot process developed in this work could be more efficient
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with high biomass loading. Fig. 8B shows the dynamics of sugar consumption and
ethanol production under these conditions. After two days, the ethanol yield almost
reaches the top value as 70% of theoretical.
Fig. 9 presents the glucan balances for the [EOA][OAc] based integrated one-pot
process. By increasing the initial biomass loading, the one-pot process results in minimum
IL usage as low as 1.5 kg/kg of biomass. In addition, this process realizes yeast loading as
low as 0.03 kg/kg of biomass. The glucan/glucose balance suggests over 90% of glucose
from saccharification has been converted to ethanol, yielding an overall conversion of
70 % in one-pot. As a result, 117 g ethanol was produced from the glucan present in 1 kg
of SG. In the future, the utilization the xylose in the hydrolysates could generate a more
cost efficient process. For example, a microorganism that is capable of converting both
glucose and xylose could utilize this concentrated sugar stream for improved biofuel yield.
2.5 Ionic liquid cost estimation and process technoeconomic analysis
Ethanolamine and glacial acetic acid are easily available chemicals manufactured on
industrial scale with prices around $800-1500/MT (MT, metric ton) and $300-700/MT,
respectively (values depending on the supplier, quality and order quantity, see
www.alibaba.com). The cost of [EOA][OAc] is estimated using the equation reported by
Hallett et al.,42 where M1 and M2 are the molecular weights of the starting materials
while P1 and P2 are their prices.
ꢇꢈꢉꢈ + ꢇꢊꢉꢊ
ꢀꢁ ꢂꢃꢄꢅꢆ =
× ꢈ. ꢊꢋ
ꢇꢈ + ꢇꢊ
Therefore, the prices of [EOA][OAc] could be around $0.7-1.4/kg
lower than most of the commonly used ILs.
,
that is significantly
From the experimental demonstrations discussed in the above sections, it is clear that
the PIL enabled processes can eliminate important cost bottlenecks (i.e., IL cost, pH
adjustment and water-wash prior to hydrolysis) that otherwise would negatively impact
the economics of any IL based biorefinery processes. To further understand the economic
potential of PIL enabled biorefinery process in general and identify further advances
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needed from an economic perspective, a preliminary technoeconomic analysis (TEA) was
conducted based on an integrated biorefinery model (Fig. S13, ESI).
Multiple studies in the past have already indicated that the need for high ethanol yields
while co-utilizing both glucose and xylose for any cellulosic biofuel production process to
be cost competitive.43, 44 Therefore, to identify other key cost-drivers specific to the PIL
process in this study, a mature process with high yield is assumed in all the scenarios (S0-
S4) studied in this TEA. Effectively, the yield in these scenarios (S0-S4) corresponds to
90% conversion of glucan and xylan to fermentable sugars, followed by 90% conversion
of glucose and xylose to ethanol using an engineered microbe that is capable of co-
utilizing both glucose and xylose. Key process and cost specifications for all the scenarios
are provided in Table S2 (ESI).
The resulting estimated MESPs are provided in Fig. 10. Firstly, to elucidate quantify
the economic benefit of the PIL process that doesn’t require pH adjustment prior to
hydrolysis, the first two scenarios (S0 and S1) are modeled with and without pH
adjustment while other process specifications remain the same. Compared to S0, it was
found that the elimination of pH adjustment in S1 could potentially reduce the estimated
minimum ethanol selling price (MESP) in S1 by more than 40% (from $16.8/gal to
$9.5/gal, respectively). Due to the pH adjustment in S0, the IL recovery/recycle section
required an additional regeneration step leading to higher IL losses in S0 compared S1
(i.e., corresponds to 85% and 99% recovery, respectively) thereby negatively impacting
the economics of S0. This emphasizes the need for processes that would facilitate high IL
recoveries even in the context of relatively cheap ILs. Therefore, it is highly desirable to
minimize the number of steps/operations needed in IL recovery/recycle section by
eliminating regeneration steps that are required due to upstream pH adjustment. In
addition, it was further identified that the high MESP in S1 ($9.5/gal) is primarily due to
the energy intensity of the product recovery section that in turn is due to the need for
dilution prior to SSF. For instance, prior to SSF, the pretreated biomass slurry was needed
to be diluted to 5% IL to ensure high enzymatic and microbial activity during SSF.
Subsequently, the effective solids loading during SSF is <5 wt%, which resulted in low
ethanol titers and increased energy related costs for ethanol and IL recovery in the
downstream.
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To understand the economic impact of further advancements in relation to dilution
prior to SSF, two more projected scenarios (S2 and S3) are investigated. Scenario S2
assumes relatively higher IL loading (20 wt%) therefore reducing the amount of water
added during dilution prior to SSF. In practice, this scenario (S2) can be realized by
utilizing a PIL that is biocompatible at higher IL concentrations and/or engineered
enzymes/microbes that can tolerate higher IL concentration during SSF. Scenario S3 is
even more advanced and considers aqueous PIL (~25 wt% IL solution) for pretreatment
with effective solids loading of around 20% during pretreatment and SSF – therefore, this
scenario (S3) eliminated the need for dilution prior to (or during) SSF. Scenario S2 is able
to reduce MESP by more than 40% compared to S1 ($5.5/gal and $4.4/gal, respectively).
S3 is even more economical with an MESP of around $4.4/gal, as there is no intermediate
dilution and therefore the energy needs for the ethanol and IL recovery are minimal. In
addition, the current enzyme loading required is found to be another cost driver – for
instance, a three-fold reduction in enzyme loading (i.e., from 20 mg/g of biomass in
scenario S3 to 7 mg/g of biomass in scenario S4 could potentially reduce MESP further to
around $3.5/gal (S4). With these perceived advances, the PIL process is an attractive and
more economical route compared to conventional IL processes that involve water-wash
step that requires significant water and substantial amount of energy for subsequent
dehydration of IL – therefore, with the water-wash route, the MESP is unlikely to be lower
than $6/gal.44
Based on the discussions above, future research efforts should focus on identifying the
PILs that would minimize/avoid the need for intermediate dilution (i.e., improve solids
loading during SSF) and reduce enzyme loading at the same time. There is a need for
continued research to identify PILs that are more biocompatible and/or aqueous PILs that
are equally effective, compared to their dry counter parts, at relatively high solids loading
(~20 wt%) throughout pretreatment and SSF. With any such advancements, PILs would
represent a very promising class of ILs as they can enable a cost-competitive integrated
biorefinery processes while avoiding the need for intermediate pH adjustment and water-
wash steps. As the PIL technology evolves, there is a continued need to do more rigorous
TEA to further optimize the process. Nonetheless, the current TEA is successful in
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identifying initial cost drivers. This TEA has also highlights the importance of integrated
process evaluation to enable the design of cost-competitive biorefineries.
Conclusions
An integrated, one-pot process for the production of cellulosic ethanol from
switchgrass is demonstrated. This innovative conversion technology is enabled by low-
cost, biocompatible protic ionic liquid (PIL), ethanolamine acetate [EOA][OAc]. This IL
contains ions derived from cheap and commercially available compounds of
ethanolamine and acetic acid. Commercial enzyme mixtures and wild type yeast strain
are utilized without pH adjustment after pretreatment and require no water-wash
throughout the process. Under optimized conditions, an ethanol yield of 70% of
theoretical yield was achieved.. The high delignification effect of [EOA][OAc] is the key
reason for its high pretreatment efficiency. The process establishes an approach that
simplifies the operation and reduces costs to enable an affordable and scalable biomass
conversion process using efficient and low cost ILs under optimal processing conditions.
A preliminary technoeconomic analysis (TEA) revealed that an integrated biorefinery
concept based on the proposed PIL processes could potentially reduce the MESP by more
than 40% compared to a counterpart scenario requiring pH adjustment. Furthermore,
TEA identified additional improvements that would be required (including improved
yield, increased solids loading and reduced enzyme loading during SSF) to enable a cost-
competitive production of cellulosic biofuels. Given the potential of PILs, it is therefore
anticipated that there will be strong growth in the field of PILs, which will lead to
continued advances related to biomass pretreatment and, subsequently, their use in many
more applications. This study demonstrates the impact of a biocompatible IL in terms of
process optimization and conversion efficiency, and opens up avenues for an efficient
integrated biomass conversion technology that is based on the use of ILs.
Experimental Section
Materials
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Switchgrass (SG) (Panicum virgatum) was provided by Dr. Daniel Putnam, University of
California at Davis. Switchgrass was ground by a Wiley Mill through a 2 mm screen and
separated by a vibratory sieve system (Endecotts, Ponte Vedra, FL). Microcrystalline
cellulose (MCC, trademark name: Avicel) was purchased from Sigma-Aldrich (St. Louis,
MO). The commercial enzyme products cellulase (Cellic® CTec2, Batch#VCN10001)
and hemicellulase (Cellic® HTec2, Batch#VHN00001) were gifts from Novozymes,
North America (Franklinton, NC).
Compositional analysis
Compositional analysis of SG before and after pretreatment was performed using NREL
acidolysis protocols (LAP) LAP-002 and LAP-005, which was described in our previous
work.13 In a typical process, 200 mg of biomass and 2 mL of 72wt% H2SO4 were
incubated at 30 °C while shaking at 300 rpm for 1 h. The solution was diluted to 4wt%
H2SO4 with 56 mL of DI water and autoclaved for 1 h at 121 °C. The reaction was
quenched into an ice bath before removing the biomass by filtration. Carbohydrate
concentrations were determined from the filtrate using an Agilent HPLC 1200 Series
equipped with a Bio-Rad Aminex HPX-87H column and a Refractive Index detector. An
aqueous solution of H2SO4 (4 mM) was used as the mobile phase (0.6 mL min−1, column
temperature 50 °C). The injection volume was 20 ꢀL with a run time of 20 min. Acid
insoluble lignin was quantified gravimetrically from the solid after heating overnight at
105 °C (the weight of acid-insoluble lignin + ash) and then at 575 °C for at least 6 h (the
weight of ash).
One-pot pretreatment and saccharification
In a typical procedure, SG (100 mg) was mixed with ([EOA][OAc]) at a 10% biomass
loading in a 15 mL capped glass pressure tube and pretreated in a convection oven at
160 °C for 0.5 h. Untreated raw SG (30-40 mesh) was used as a control. After
pretreatment, the pretreated slurry was diluted with water to obtain a final IL
concentration of 10 wt%. Enzymatic saccharification step was operated at 50 °C for 72
hrs with constant agitation on an Enviro Genie SI-1200 rotator platform (Scientific
Industries, Inc., Bohemia, NY). The loading of enzyme cocktails (9CTec2/HTec2, v/v) is
20 mg EP per g SG.
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One-pot pretreatment & SSF
In a typical procedure, SG (100 mg) was mixed with ([EOA][OAc]) at a 40 wt% biomass
loading in a 15 mL capped glass pressure tube and pretreated in a convection oven at
160 °C for 0.5 h. After pretreatment, the pretreated slurry was diluted with DI-water to
obtain a final IL concentration of 5 wt%, and pre-saccharificated for 24 hrs under the
same aforementioned conditions. Simultaneously, wide type yeast strain BY4741 was
prepared. After the addition of yeast strain, the SSF step was conducted for 72 hrs.
X-ray diffraction (XRD) and NMR spectroscopy
The raw and pretreated biomass were dried and characterized with powder X-ray
diffraction (PXRD). The XRD analysis were performed on a PANalytical Empyrean X-
ray diffractometer equipped with a PIXcel3D detector and operated at 45 kV and 40kA
using Cu Kα radiation (λ=1.5418 Å). The patterns are collected in the 2θ range from 5 to
60° with a step size of 0.039° and the exposure time of 300 seconds. A reflection-
transmission spinner was used as a sample holder and the spinning rate was set at 8 rpm
throughout the experiment.
NMR spectra were acquired at 298 K using a Bruker Avance-600 MHz instrument in
DMSO-d6 and calibrated with the corresponding DMSO peak (δH=2.50 ppm for 1H and
δC= 39.50 ppm for 13C).
Size exclusion chromatography (SEC):
SEC was performed on the lignin in liquid stream to understand changes in lignin
molecular weight distribution during the solubilization and depolymerization process. An
Agilent 1200 series binary LC system (G1312B) equipped with DA (G1315D) detector
was used. Separation was achieved with a Mixed-D column (5 mm particle size, 300 mm
x 7.5 mm i.d., linear molecular weight range of 200 to 400,000 u, Polymer Laboratories,
Amherst, MA) at 80°C using a mobile phase of 1-methyl-2-pyrrolidinone (NMP, HPLC
grade) at a flow rate of 0.5 ml per min as previously described.45 Absorbance of materials
eluting from the column was detected at 300 nm (UV-A).
Technoeconomic Analysis
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To facilitate TEA conducted in this study, a detailed biorefinery model (Fig. S17, ESI)
developed in SuperPro designer is used. The biorefinery modeled represents a mature
(i.e., Nth plant facility that operates 350 days/year) industrial scale facility capable of
processing 2000 dry MT/day biomass to produce ethanol. The model encompasses
multiple sections such as pretreatment, SSF, product recovery, wastewater treatment
(WWT) and an onsite co-generation facility. While the upstream sections (i.e.,
pretreatment and SSF) are modeled to represent the PIL process discussed in this work,
the WWT and co-generation sections and most of the economic data are taken from the
design report by National Renewable Energy Technology Laboratory (NREL),46 and our
previous studies.44, 47 The product recovery section configuration conceived specifically
for this study and consists of multiple unit operations including distillation to recover
ethanol. After ethanol recovery, the residue is subjected to a series of operations to
recover IL from the stillage. First, a solid-liquid separation is used to remove most of the
solids. The aqueous stream is then concentrated using a multi-effect evaporation system.
The concentrated IL and water mixture is further dehydrated using a pervaporation unit to
recover dry IL that can be recycled to the pretreatment section (in the case of pH
adjustment prior to hydrolysis, the IL recovery/recycle section required an additional
‘regeneration’ step prior to recycle and was assumed to be accomplished by an
electrodialysis system with an effective IL recovery efficiency of ~85%).42,48 The WWT
section treats the used water in the process using multiple steps that include primarily
anaerobic and aerobic digestion. Solids residue from the product recovery section, and
biogas and sludge from WWT section are utilized in the co-generation section to produce
steam for processing needs. Any excess steam is used to produce electricity and any
surplus electricity is assumed to exported to the grid. There is a provision to import
natural gas from external sources if the steam generated on-site is not sufficient to meet
the overall process heating demand. In line with NREL study,43 minimum ethanol selling
price (MESP) is used as the benchmark for economic performance and it is equivalent to
the selling price of ethanol that corresponds to 10% internal rate of return. Using this
model, multiple scenarios (Table S2) are conducted to evaluate economic potential as
well as to identify key cost drivers specific to the PIL processes.
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Acknowledgements
This work was supported by the U.S. Department of Energy, Office of Science, Office of
Biological and Environmental Research (DE-AC02-05CH11231), Office of Basic Energy
Sciences and Division of Chemical Sciences, Geosciences and Biosciences. NWChem
calculations were conducted at Environmental Molecular Sciences Laboratory (EMSL)
supercomputing center.
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Figures and Tables
Low
toxicity
Cheap IL
CTec2+HTec2
50 oC, 24 h
S. Cerevisiae
Biomass
(High loading)
Sugars
Biofuel
Slurry
160 oC, 0.5 h
37 oC, 48-72 h
pH match
pH match
Pretreatment
Simultaneous saccharification & fermentation (SSF)
Figure 1. One-pot integrated IL process for biofuel production.
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Cation
Anion
DEOA
C2C1Im
Ch
TEA
A
TEOA
EOA
OAc
F
Cl
HSO4
La
9
8
7
6
5
4
3
2
1
0
Net Basicity
Acidity
B
Basicity
Ionic liquids evaluated
Figure 2. ILs investigated in the present work. (A): The information of cation and anion;
and (B): Prediction of acidity, basicity and net basicity of ILs.
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A
Sugar yield/%
B
0
10
20
30
40
50
60
[EOA][OAc]
[C2C1Im][OAc]
[DEOA][OAc]
[TEOA][OAc]
[Ch][OAc]
[EOA][La]
Glucose
Xylose
[EOA]F
[DEOA]F
[TEOA]F
[TEA][HSO4]
[EOA]Cl
[EOA][HSO4]
Figure 3. ILs screening for one-pot process in terms of sugar yields. (A): Photos of the
synthesized ILs; and (B): Performances on sugar yields. Conditions: pretreatment, 10
wt% SG loading, 90 wt% IL, 140 , 1h; saccharification, 10 wt% IL (in the case of
[C2C1Im][OAc], the IL was removed after pretreatment), 10 mg protein per g
switchgrass, 50 , 72h.
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A
b)
a)
Avicel
Untreated
10% SG
5% SG
10% Avicel
CrI=0.68
CrI=0.50
CrI=0.80
CrI=0.84
CrI=0.49
10
5
15
20
25
30
35
40
5
10
15
20
25
30
35
40
2-Theta
2-Theta
Lignin removal/%
Net basicity
B
80.0
60.0
40.0
20.0
0.0
2
1.6
1.2
0.8
0.4
0
Ionic liquids
C
8.0
6.0
4.0
2.0
Ionic liquids
Figure 4. Structure-property relationship of IL in one-pot process. (A): XRD analysis of
cellulose crystallinity in switchgrass and Avicel before and after pretreatment; (B):
Relationship between lignin removal and net basicity of ILs; and (C) pH Values of ILs
with 5 wt% in H2O.
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A
[EOA][OAc]
[EOA][HSO4]
[EOA][F]
[EOA][La]
[EOA][Cl]
EMAL
23
7
9
11
13
15
17
19
21
25
Retention time (min)
[EOA][OAc]
[Ch][OAc]
B
[C2C1Im][OAc]
Unknown
monomers
Unknown
monomer
12
14
16
18
20
22
24
26
Time (min)
Figure 5. GPC (A) and GC/MS (B) results of the lignin stream after pretreatment.
Conditions: pretreatment, 10 wt% SG loading, 90 wt% IL, 140 , 1h.
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A
B
100
80
60
40
20
0
100
Glucose
Xylose
Glucose
Xylose
80
60
40
20
0
140/3 150/1
150/3 160/0.25 160/0.5 160/1 160/3
5
10
15
20
Pretreatment temperature/time (℃/h)
Biomass loading/wt%
C
D
100
80
60
40
20
0
100
Glucose
Xylose
Glucose
Xylose
80
60
40
20
0
0
5
10
20
2.5
5
10
15
20
25
IL concentration/wt%
Enzyme loading/wt%
Figure 6. Effects of some parameters on the sugar yield: (A) Biomass loading during
pretreatment, (B) Pretreatment temperature/time, (C) IL concentration in saccharification,
and (D) Enzyme loading. Conditions: pretreatment, 10 wt% SG loading, 90 wt% IL,
160 , 0.5 h; saccharification, 10 wt% IL, 20 mg protein/g SG, 50 , 72 h.
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A
B
80
70
60
50
40
30
20
10
0
1
Glucose /%
Xylose /%
15 wt%
10 wt%
5 wt%
2.5 wt%
0.1
0.01
50-0.5 50-3 45-0.5 45-3 40-0.5 40-3 30-0.5 30-3 20-0.5 20-3
0
1
2
2
3
4
5
5
6
7
8
8
9
10 11 11 12 13 14 14 15 16 17 17 18 19 20
Time/h
Biomass loading/time (wt%/h)
Figure 7. Screening of [EOA][OAc] concentration for the biocompatibility with
Saccharomyces cerevisiae strain BY4741 (A, 31 , 10 g/L glucose in each IL solution
(A) and effect of biomass loading/time on sugar yield (B). Conditions: pretreatment, 160
℃, 0.5 h; saccharification, 5 wt% IL, 20 mg protein/g SG, 50 ℃, 72 h.
28

Page 29 of 33
Green Chemistry
View Article Online
DOI: 10.1039/C7GC01179B
A
B
80
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
Ethanol
Glucose
Glucose
Ethanol
0
0
24
48
72
0.01
0.02
0.05
0.09
0.15
0.21
Yeast loading/wt%
Time/h
Figure 8. Process optimization of one-pot ethanol fermentation after [EOA][OAc]
pretreatment. (A) Effect of yeast loading on ethanol fermentation; (B) Illustration of the
glucose consumption and ethanol production during SSF.
29