Dimethyl sulfoxide assisted ionic liquid pretreatment of switchgrass for

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Dimethyl sulfoxide assisted ionic liquid pretreatment of switchgrass for isoprenol production Shizeng Wang, Wenwen Zhao, Taek Soon Lee, Steven William Singer, Blake A. Simmons, Seema Singh, Qipeng Yuan, and Gang Cheng ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04908 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 28, 2018

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ACS Sustainable Chemistry & Engineering

Isoprenol fermentation from switchgrass after DMSO-assisted ionic liquid pretreatment is presented, and DMSO is industrially produced from lignin. 114x72mm (300 x 300 DPI)

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Dimethyl sulfoxide assisted ionic liquid pretreatment of switchgrass for isoprenol production Shizeng Wanga,b,# , Wenwen Zhaoa,#, Taek Soon Leeb,c, Steven W. Singerb,c, Blake A. Simmonsb,c, Seema Singhb,d, Qipeng Yuana, Gang Chenga* a

College of Life Science and Technology, Beijing University of Chemical Technology, North 3rd Ring East, # 15, Beijing, 100029, China. b Joint BioEnergy Institute (JBEI), 5885 Hollis Street, Emeryville, CA 94608, USA. c Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, 94702, CA, USA d Biomass Science and Conversion Technology Department, Sandia National Laboratories, 7011 East Avenue, Livermore, CA 94551, USA

#

These authors contributed equally to this work. * Corresponding author, Gang Cheng, email: [email protected]; [email protected] Postal address: Science and Technology Building, College of Life Science and Technology, Beijing University of Chemical Technology, North 3rd Ring East, # 15, Beijing, 100029, China.

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Abstract The production cost and viscosity of certain ionic liquids (ILs) are among the major factors preventing the establishment of economically viable IL-based biomass pretreatment technologies. Recently, mixtures of an IL with an organic solvent have been proposed for cellulose processing and biomass pretreatment. DMSO is an inexpensive organic solvent that is industrially produced from lignin, a by-product of pulping process. We carry out a mechanistic study of dimethyl sulfoxide (DMSO)-assisted IL pretreatment of switchgrass. The physical structures of biomass samples are studied by x-ray diffraction (XRD), N2 adsorption analysis and small angle neutron scattering (SANS). Both dry and aqueous suspension of biomass samples are measured by SANS that provides unique information on biomass pretreatment. A mixture of 42 wt.% [C2C1Im][OAc] and 58 wt.% DMSO is proposed as the optimal pretreatment solution and the recycling and reuse of the mixture of solvents are also studied. The fermentability of the hydrolysates generated after pretreatment is evaluated using an E.coli strain engineered to produce isoprenol. This study suggests an avenue for developing more efficient and cost effective IL-based processes for the production of lignocellulosic biofuels and bioproducts.

Keywords: ionic liquid; dimethyl sulfoxide; fermentation; pretreatment; biomass

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Introduction Over the last decade, the use of certain ionic liquids (ILs) in biomass pretreatment followed by enzymatic hydrolysis has been convincingly demonstrated by a rapidly growing number of publications.1-4 With the possibility to design the components of the ILs, some of them provide the ability to dissolve lignin5, 6 and/or carbohydrates,7, 8 which forms the foundation of IL pretreatment. Although advances in IL synthesis,9-12 enzyme and metabolic engineering,13, 14 process intensification15, 16 have greatly expanded the tools available for chemical engineers, the establishment of an economically viable IL-based biofuel production technology remains challenging due to confounding factors such as the cost and recyclability of ILs, their biocompatibility with enzymes and microbial hosts, lignin recovery and application, etc.17-22 Combining biomass deconstruction and hydrolysis in one pot using inexpensive acidic ILs offers a potential solution of this problem.12, 23 The continued development of systematic strategies to assess and engineer different processes is necessary to address these challenges and improve the efficiency of IL pretreatment and conversion processes. One of the approaches to reduce the cost of IL pretreatment has been the use of mixtures of ILs with inexpensive solvents such as water, glycerol and organic solvents.6, 24-30 Mixtures of an IL with an organic solvent, often referred to as “organic electrolyte solutions” (OESs),31 represent a novel class of mixture solvents that have been proposed for cellulose processing and biomass pretreatment.32-34 In 2011, Rinaldi discovered that some OESs containing only a small fraction of 1-butyl-3-methylimidazolium chloride can dissolve instantaneously large amounts of cellulose.31 Later, Ohira et al reported similar results with an amino acid IL, N,N-diethyl-N-(2-methoxyethyl)-N-methylammonium alanine, in 3


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DMSO.35 The organic solvents are usually inexpensive and possess lower viscosities. The cost of OESs for the dissolution of 1 kg cellulose has been shown to decrease significantly compared to that using pure ILs.33 DMSO is a unique solvent that is manufactured as a high quality product from black liquor, an aqueous lignin solution obtained as a by-product of the Kraft process for producing paper (Fig. S1).36 Although DMSO does not solubilize cellulose, recent studies have indicated that the addition of certain amounts of DMSO to some ILs improves cellulose solubility.37, 38 It is hypothesized that the DMSO molecules diminish the interaction strength between cations and anions of the ILs, thus facilitating cellulose dissolution as the IL ions are more likely to perturb the hydrogen bonds of cellulose.37-40 However, other investigations conclude that DMSO simply plays the role of a “viscosity reducer” and enables faster mass transport of the system without interacting with the ILs themselves.41, 42 In either case, DMSO can play an important role in IL-based cellulose solubilization and biomass pretreatment. There are only a few studies that report the use of mixtures of DMSO with ILs to pretreat biomass that result in improved enzymatic hydrolysis rates or yields.10,

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Literature

studies of biomass pretreatment using mixtures of ILs and organic solvents have been summarized in our previous paper.29 These studies suggest that it is possible to reduce the amount of ILs used for biomass pretreatment, up to a certain limit above which sugar yields decrease. In these studies, the mass ratio of biomass to the DMSO/IL mixtures is fixed while the ratio of DMSO to IL is varied. As the DMSO: IL ratio is increased, the biomass: IL ratio is decreased, and therefore the observed changes in sugar yields may result from two effects:1) dilution of biomass with IL; and 2) addition of DMSO. To this end, we recently examined the effect of addition of DMSO to [C2C1Im][OAc] on the sugar yields of 4



pretreated biomass by keeping the biomass loading in [C2C1Im][OAc] fixed.29 A synergistic effect between DMSO and IL was observed that leads to a maximum sugar yield from pine and white poplar samples pretreated using a 42 wt.% [C2C1Im][OAc] and 58 wt% DMSO solution.29 In the following, this mixture is named as the 58 wt% DMSO solution. The current work is an extension of the previous study. A deeper mechanistic understanding of biomass pretreatment is obtained by a combined study using XRD, N2 adsorption analysis and SANS. The effect of SG loading on pretreatment efficacy, as well as recycling and reuse of pretreatment solvents, are investigated and presented. Finally, a DMSO-enabled biofuel and chemical production platform is proposed where the carbohydrates are converted to isoprenol. Switchgrass (SG) is pretreated using a DMSO/IL mixture, and the sugars liberated by enzymatic hydrolysis of pretreated SG are converted to isoprenol via fermentation using an engineered strain of E. coli. Isoprenol (or isopentenol, l, 3-methyl-3-buten-1-ol) is a promising biofuel and a precursor for industrial chemicals such as isoprene. An engineered E. coli strain KG1R10 containing a heterologous mevalonatebased isoprenol pathway was used for isoprene production.43 Materials and methods Experimental procedures and materials characterization are provided in the supporting information.

Results and discussion Biomass pretreatment and structural changes

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Table 1 presents the biomass composition and solid recovery after pretreatment. Untreated SG has an ash content of 2.7 wt.%, measured on a dry matter basis, which is similar to one literature report.44 The solid recovery of the DMSO-pretreated sample is 76.6 ± 3.1 wt.%, much lower than expected. To understand this low solid recovery, untreated SG samples were extracted with water and ethanol using a Soxhlet apparatus. The solid recovery after the extraction was ~86 wt.%. Therefore it is believed that DMSO dissolved most of the soluble components during the pretreatment process. The mass loss of the samples after pretreatment thus includes the extractable components, which is about 14 wt.%. From an industrial point of view, it is not feasible to remove the extractives prior to IL pretreatment. The losses of cellulose, xylan and lignin during pretreatment were not calculated since it requires removal of nonstructural extractives prior to pretreatment.7 It is noticed that the solid recoveries of non-extracted SG after [C2C1Im][OAc] pretreatment at 150 C -160 oC ranged from 50 wt.% to 60 wt.% in prior studies.45-47 Table 1 Biomass Compositions and solid recovery DMSO Biomass Cellulose Xylan concentration concentration (wt. %) (wt. %) in IL (wt. %) in IL (wt.%) Untreated 100 95 58 0

5 5 5 5

32.4±1.5 28.8±1.7 30.8±2.5 36.8±3.6 37.1±2.4

16.1±2.0 19.2±0.3 17.7±2.0 19.7±4.1 19.8±3.9

Lignin (wt. %)

Solid recovery (wt. %)

17.3±1.2 17.2±1.4 14.4±2.0 13.5±0.2 13.4±0.8

-76.6±3.1 66.1±2.6 67.0±2.3 67.0±1.5

The impact of DMSO on IL pretreatment was studied by progressively diluting [C2C1Im][OAc] with DMSO while keeping the mass ratio of SG to [C2C1Im][OAc] fixed at 5 wt.%. In the prior work, different concentrations of DMSO in [C2C1Im][OAc] were used

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to pretreat pine and eucalyptus samples: 0, 14, 32, 58, 72, 90, 95 and 100 wt %.29 The highest sugar conversion is observed for the samples pretreated in the 58 wt% DMSO solution.29 It is expected that the 58 wt% solution represents one of the optimal pretreatment solvent mixture. Therefore, only four DMSO solutions were used in this work: 100, 95, 58 and 0 wt %. The sugar conversion as a function of DMSO concentrations (100, 95, 58 and 0 wt %) in [C2C1Im][OAc] is presented in Fig.1a. There is not a significant difference between 24 h and 72h sugar conversion for all the samples studied; suggesting efficient enzyme mixtures were used in this work. DMSO-pretreated SG shows slightly higher sugar conversion (10.2%) than that of untreated SG (6.4 %). Meanwhile, [C2C1Im][OAc]-pretreated SG exhibits about 10 fold increase in sugar conversion (66.5%) in comparison to untreated SG. The sample pretreated using the 58 wt% DMSO solution shows a higher sugar conversion (72.5%) than that using pure [C2C1Im][OAc]. A similar result has been observed in the prior work with white poplar and pine 29. Although [C2C1Im][OAc] is diluted with DMSO, the effective interactions between biomass and [C2C1Im][OAc] are not affected at this dilution; and more importantly, the reduced viscosity of the system may facilitate the interactions between biomass and [C2C1Im][OAc].29 In one study, the viscosity of DMSO and 1-butyl-3-methylimidazolium chloride solutions was shown to decrease exponentially with increasing mole fraction of DMSO.48 It may also benefit from improved cellulose solubility in the mixture solvent as some studies suggest.33, 37 The sample pretreated using the 95 wt% DMSO solution exhibits a lower sugar conversion than that pretreated by the 58 wt% DMSO solution. Noting that the mass ratio of SG to [C2C1Im][OAc] was kept

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constant, this decrease is due to prevailing DMSO dilution effect that decreases the probability of contacts between biomass and [C2C1Im][OAc].

Fig. 1. (a) Total sugar conversion as a function of DMSO concentration; (b) XRD of untreated and pretreated samples; (c) pore size distribution and (d) pore volume and surface area of untreated and pretreated samples.

Structural changes in SG during pretreatment were investigated using XRD, N2 adsorption analysis and SANS. Fig. 1b indicates that native crystalline cellulose was transformed into amorphous cellulose after pretreatment using the 58 wt.% DMSO solution, which is consistent with its higher sugar yield. [C2C1Im][OAc]-pretreated SG and other samples still contain recalcitrant cellulose I structures, suggested by the broad peak around 15.6.7 This indicates that addition of certain amount of DMSO to the SG/[C2C1Im][OAc] promotes

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effective interactions between SG and [C2C1Im][OAc] which eventually led to transformation of cellulose I to amorphous cellulose.49 Biomass crystallinity of cellulose I was rapidly evaluated by comparing the XRD intensity ratio at 18.5 and 22.2.50 Untreated SG has a biomass crystallinity of 0.54 while 100 wt.% DMSO-, 95 wt.% DMSO- and 100 wt.% [C2C1Im][OAc]- pretreated SG samples have biomass crystallinity of 0.51, 0.50 and 0.22, respectively. Although DMSO- and 95 wt.% DMSO-pretreated SG samples exhibit similar biomass crystallinity, the sugar conversion of 95 wt.% DMSO-pretreated SG is higher. This will be explained in the following paragraph. Pore size distribution for pores with diameters larger than 5nm, which corresponds to the lower limit that typical cellulases can penetrate,51, 52 is shown in Fig. 1c. The corresponding porosity expressed as cm3/g biomass and overall specific surface area are presented in 1d. Fig.1c shows that untreated SG contains small pores concentrated around 10nm. DMSO pretreatment increased porosity with slight changes in pore size distribution. Pretreatments using [C2C1Im][OAc] and the [C2C1Im][OAc]/DMSO altered pore size distribution and porosity, with pores concentrated towards larger sizes. Pretreatment increased both the specific surface area and porosity. The higher sugar yield from 95 wt.% DMSO-pretreated SG than that of 100 wt.% DMSO-pretreated one is explained by its higher specific surface area and porosity. However, a continuous increase in porosity does not always lead to improved sugar conversion, as shown in Fig. 1a. This is determined by the relative contributions of biomass porosity and biomass crystallinity to sugar conversion.53

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Figure 2 (a) SANS data of dry biomass samples; (b) SANS data of wet biomass samples. The solid lines on curves are a fit to corresponding scattering functions as described in the text. After biomass pretreatment, wet samples are often dried for subsequent quantitative analysis since accurate weight of the samples is required. For enzymatic hydrolysis, certain amount of dry samples is measured and added to the buffer solution. Thus, the effect of swelling with water on the morphology and integrity of biomass samples needs to be considered as well. Wet samples are close to the environment in which the enzymatic hydrolysis is performed. Here both dry and wet samples are studied using SANS.54, 55 Due to space limit, the background information of SANS on biomass and other porous system will not be introduced here. Interested readers may consult relevant references such as ref. 53 as well as ref 4 in the supporting information. The SANS data obtained from dry and wet samples are shown in Figure 2. The scattering curves of the four dry samples, i.e., untreated, 100 wt.% DMSO-, 58 wt.% DMSO- and 100 wt.% [C2C1Im][OAc]-pretreated samples, are described by a power-law function (Eq.1 in supplementary information) at q = 0.004 to 0.1Å-1. For polydisperse porous media, an appropriate relationship between pore radius (R) and the q is derived: R≈ 2.5/q.56, 57 Therefore, SANS data of dry samples cover pores with radii in a range from ~ 2 to 60 nm. The scattering at q = 0.1 to 0.4Å-1 is 10



attributed to incoherent background mainly due to hydrogen atoms in the biomass.58 The exponent of the power law function (n) reflects the fractal behavior of the biomass samples.59 If the value of the exponent lies between 3 and 4, it suggests scattering from a surface fractal with the fractal dimension of Ds = 6 − n.60 A higher value of the Ds indicates a rougher surface with 2 being from the perfect surface. If the exponent is less than 3, a mass fractal behavior from can be expected where Dm = n. A lower value of the Dm indicates the mass distribution becomes less compact in the object.59 The values of obtained exponents are 3.3, 3.4, 3.2 and 3.2 for untreated, 100 wt. % DMSO-, 58 wt.% DMSO- and 100 wt.% [C2C1Im][OAc]-pretreated samples, respectively. The corresponding surface fractal dimensions of the pore/matrix interfaces are 2.7, 2.6, 2.8 and 2.8, respectively. After pretreatments with 100 wt.% DMSO, the surface roughness decreases. This suggests that DMSO mainly washes surfaces of particulates and pore/matrix interfaces without causing much erosion to the surfaces. As shown in Figure 1d, the porosity and specific surface area increases after DMSO pretreatment, indicating that this increase is caused by removing materials that were previously blocking pores. The increased porosity and specific surface area may also increase chances of contacts between biomass and [C2C1Im][OAc]. This provides a new understanding of the role of DMSO in biomass pretreatment using DMSO/IL mixtures. After pretreatment with the 58 wt. % DMSO and 100 wt.% IL, the surface roughness of SG particulates increases, which is likely due to deconstruction of biomass particulates. SG samples were also measured as they were dispersed in D2O. Different scattering curves were observed due to swelling or disintegration of biomass particulates. Untreated and DMSO-pretreated samples exhibit excess scattering in the q = 0.1 to 0.3Å-1. The scattering 11


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data within this q range have been interpreted either as from small pores54,

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or from

cellulose microfibrils62 in biomass. It is believed it is due to scattering from small pores in this work since a fit to the form factor of a sphere is better than that of a cylinder and the obtained radius of the cylinder is around 0.7nm which is about half of typical values of cellulose microfibrils.55 In comparison to the dry samples, the presence of this excess scattering is due to increased neutron scattering contrast of the small pores as well as lower incoherent background of D2O.54 The SANS data is well described by a model comprised of a power law term and a term describing a single population of spheres (Eq. 2 in supplementary information). The model suggests that the small pores in the sample are rather scarce and they do not form a mass fractal as do in a previous work.61 The small pores have rather uniform radii of 1.5 nm. It is noted that the N2 adsorption analysis produced pores with radii centered on 5 nm in dry samples. They were not detected by SANS from wet samples which may be explained by two aspects: 1) SANS detects both open and closed pores while N2 adsorption analysis measures open pores; 2) swelling in water changes structure of pores including sizes and its distribution. This change in pore structure during hydration is understandable when considering collapse of pores during drying biomass samples.63 There is no evidence that suggests a correlation between pores with radii centered on 1.5nm and those around 5nm. The obtained exponents for untreated and 100 wt.% DMSO pretreated samples are 2.9 and 2.7, respectively. The values of the exponents indicate mass fractals. Thus, a transition from surface fractals to mass fractals within the same q range is observed upon suspending the samples in water. This indicates disintegration of biomass particulates due to swelling in 12



water. Fractal surfaces or interfaces are broken down and form smaller particles that led to a transition from surface fractals to mass fractals. For the 58% DMSO- and 100% [C2C1Im][OAc]-pretreated samples, the excess scatterings are largely gone and SANS curves are again described by the power-law function. This suggests that most of the small pores in this range are destroyed by pretreatments. It is noted that the power-law function cannot fit the data well in the q=0.1 to 0.2Å-1. This is likely due to the scattering from residual small pores.56 The mass fractal dimension derived from the power-law function analysis is around 2.1 for both samples. A lower mass fractal dimension suggests that the subunits constituting the fractals are loosely associated with each other. In compared to the mass fractal dimension of untreated and 100 wt.% DMSO-pretreated samples, the pretreatment using the 58 wt.% DMSO solution and 100 wt.% [C2C1Im][OAc] led to biomass particulates with loose internal structures. This is beneficial for enzymatic hydrolysis since it increased specific surface area. Recycle and reuse of [C2C1Im][OAc]/DMSO One recent study shows that the costs of a mixed solvent system for processing cellulose significantly decrease for ratios of DMSO to IL between 25 wt.% - 50 wt.%.33 Thus, the 58 wt.% solution represents one of the optimal mixtures based on the cost and its pretreatment efficiency. Therefore, this solution was used as the pretreatment solvent for further studies carried out in this work. Recycle and reuse of pretreatment solvents was tested using the 58 wt.% DMSO solution and a SG loading of 5 wt.%. The 5 wt.% biomass loading was used a starting point to gain some insights into this recycle and reuse process. A distillation apparatus was used to remove water from the pretreatment liquor while performing the pretreatment.7 The 13


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recovered amount of water by distillation from the pretreatment liquor during each cycle is presented in Table 2. It was observed that the temperature needed to be increased to 130 C in order for the water to be distilled, and therefore the pretreatment temperature was kept at 130 C during recycle and reuse experiments. This process was repeated 4 times. Fig.3 presents the sugar conversion of pretreated SG using recycled liquors. It shows that the sugar conversion decreases using recycled liquor, however it still reaches around 50 % after 4th reuse. During the pretreatment process, the IL concentration increased due to the evaporation of water. The faster the water is removed from the pretreatment liquor, the better the pretreatment efficiency. Due to the presence of DMSO, it takes a longer time to remove water from the solution than without DMSO, as shown in our previous work.7

Table 2 Recycle and reuse of pretreatment liquor Biomass Mass of used for recycled pretreatment pretreatment liquor (g) (g) Neat IL 0.2 3.8, neat IL Recycle/reuse, 0.2 22.0 1st Recycle/reuse, 0.2 21.7 2nd Recycle/reuse, 0.2 21.9 3rd Recycle/reuse, 0.2 21.7 4th Recycle, 5th 21.4

Antisolvent water (g)

Water collected by Distillation (g)

Liquor recovery (wt %)

15.2 15.2

13.5

90.7

15.2

13.5

91.6

15.2

13.7

94.4

15.2

13.6

92.7

14


91.8


Fig. 3. Impact of recycle and reuse of the pretreatment liquor on sugar conversion Increasing the amount of water in the pretreatment liquor from 4 fold to 16 fold significantly decreases sugar conversion, as shown in Fig. 3. To improve the sugar conversion, one needs longer pretreatment time, higher pretreatment temperatures and better design of the reactor. A balance between sugar conversion and pretreatment cost needs to be investigated in future work. The accumulated lignin in the recycled pretreatment liquor may affect the pretreatment efficiency, as previously discussed.7 The role of dissolved lignin on the pretreatment efficacy is still under investigation. The IL concentration in the pretreatment liquor is difficult to estimate, however it is expected the [C2C1Im][OAc] concentration decreases in the pretreatment liquor with increasing the number of recycling and reuse.7 This requires more efficient washing of the pretreated biomass. A short summary of literature studies on IL recyclability was presented in our most recent work and will not be repeated here.7 High biomass loading

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To be economically viable, higher biomass loading levels during pretreatment is preferred. Fig.4 shows the effect of biomass loading on sugar conversion after pretreatments using the 58 wt. % DMSO solution. With increasing biomass loading from 5 wt.% to 17 wt. %, sugar conversion modestly decreases from 73 % to 63 %.

Fig. 4. Impact of biomass loading on sugar conversion from pretreated SG using the 58 wt.% DMSO solution. Isoprenol production by SHF In order to study the fermentability of the pretreated SG, we performed SHF of pretreated SG using an engineered strain of E. coli DH1 capable of producing isoprenol from glucose. SG was pretreated in the 58 wt.% DMSO solution at 110C for 3 h with a biomass loading of 15 wt.% and saccharification was performed at a solid loading of 10% (w/v). The glucose concentration is 34 g/L and xylose concentration is 12 g/L in the hydrolysate. The titer of isoprenol using EZ-rich medium reached 1.23±0.09 g/L with glucose and xylose from enzymatic hydrolysate as carbon source. In a prior work, the titer of isoprenol was 1.85 g/L using10 g/L glucose as carbon source in EZ-rich medium.43 Known inhibitors

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in the hydrolysates including residual [C2C1Im][OAc]64 and by-products from pretreatment could contribute to the decreased titer of isoprenol. To reduce the operation cost, saccharification and fermentation were also carried out in one-pot configuration. For isoprenol production, we added concentrated EZ-rich medium and inoculants directly into enzymatic slurry without sterilization. The final isoprenol titer reached 0.72 ± 0.05 g/L, which was lower than that of SHF. This lower yield could be due to the cell absorption by biomass, the low fluidity of slurry, residual [C2C1Im][OAc] and possible contamination. The yield of isoprenol reached 24.5 g/kg SG.

Conclusions A comparative study of dry and wet SG samples by SANS revealed disintegration of biomass particulates upon hydration which is enhanced by pretreatment with IL solutions. Pure DMSO caused minimal degree of deconstruction to biomass; it however may have enhanced interactions between IL and biomass by increasing porosity of biomass samples and possibly improving cellulose solubility. The interplay between porosity and cellulose crystallinity determines total sugar release where an increase in porosity is not always leading to improved sugar conversion. Although the 58 wt.% DMSO solution is studied here, it is noted that the optimal composition of pretreatment solution will depend on many variable such as pretreatment temperature and time, biomass loading, etc. The recycle and reuse of the pretreatment liquor is a significant challenge that remains to be optimized. Hydolysates generated by this process are readily converted to isoprenol using SHF and SHF in one-pot configuration, reaching a titer of 1.23 and 0.72 g/L, respectively. The

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results of this study provide a foundation for developing a potential economically viable IL based pretreatment technology.

Supporting Information Materials, strains and medium, biomass pretreatment, x-ray diffraction, nitrogen (N2) adsorption, SANS, compositional analysis and enzymatic hydrolysis, isoprenol production in shake flask by separate hydrolysis and fermentation (SHF) and one-pot method, [C2C1Im][OAc]/DMSO recycle and reuse for pretreatment. Notes The authors declare no competing financial interest. Author Information Corresponding Authors Email: [email protected]; [email protected] Acknowledgements We thank Dr. Boualem Hammouda (NCNR, NIST) for his discretionary neutron beam time and Mr. Cedric Gannon (NCNR, NIST) for the help with the SANS experiment. Gang Cheng acknowledges support for this research by the National Natural Science Foundation of China (U1432109) and China Scholarship Council (201606885004). The work carried out at the DOE Joint BioEnergy Institute (http://www.jbei.org) was supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy. Access to NGB 30m SANS was provided by the Center for High Resolution Neutron Scattering, a partnership between the National Institute 18



of Standards and Technology and the National Science Foundation under Agreement No. DMR-1508249. We acknowledge the support of the National Institute of Standards and Technology, U.S. Department of Commerce, in providing the neutron research facilities used in this work. References 1. Brandt, A.; Grasvik, J.; Hallett, J. P.; Welton, T., Deconstruction of lignocellulosic biomass with ionic liquids. Green Chem. 2013, 15, (3), 550-583, DOI: 10.1039/C2GC36364J. 2. Elgharbawy, A. A.; Alam, M. Z.; Moniruzzaman, M.; Goto, M., Ionic liquid pretreatment as emerging approaches for enhanced enzymatic hydrolysis of lignocellulosic biomass. Biochem. Eng. J. 2016, 109, 252267, DOI: 10.1016/j.bej.2016.01.021. 3. Zhang, Q.; Hu, J.; Lee, D.-J., Pretreatment of biomass using ionic liquids: Research updates. Renewable Energy 2017, 111, (Supplement C), 77-84, DOI: https://doi.org/10.1016/j.renene.2017.03.093. 4. Vancov, T.; Alston, A.-S.; Brown, T.; McIntosh, S., Use of ionic liquids in converting lignocellulosic material to biofuels. Renewable Energy 2012, 45, (Supplement C), 1-6, DOI: https://doi.org/10.1016/j.renene.2012.02.033. 5. Ren, H.; Zong, M.-H.; Wu, H.; Li, N., Efficient Pretreatment of Wheat Straw Using Novel Renewable Cholinium Ionic Liquids To Improve Enzymatic Saccharification. Ind. Eng. Chem. Res. 2016, 55, (6), 1788-1795, DOI: 10.1021/acs.iecr.5b03729. 6. Parthasarathi, R.; Sun, J.; Dutta, T.; Sun, N.; Pattathil, S.; Konda, N. V. S. N. M.; Peralta, A. G.; Simmons, B. A.; Singh, S., Activation of lignocellulosic biomass for higher sugar yields using aqueous ionic liquid at low severity process conditions. Biotechnol. Biofuels 2016, 9, 160, DOI: 10.1186/s13068-016-0561-7. 7. Yuan, X.; Singh, S.; Simmons, B. A.; Cheng, G., Biomass Pretreatment Using Dilute Aqueous Ionic Liquid (IL) Solutions with Dynamically Varying IL Concentration and Its Impact on IL Recycling. ACS Sustain.Chem.Eng. 2017, 5, (5), 4408-4413, DOI: 10.1021/acssuschemeng.7b00480. 8. Cao, Y.; Zhang, R.; Cheng, T.; Guo, J.; Xian, M.; Liu, H., Imidazolium-based ionic liquids for cellulose pretreatment: recent progresses and future perspectives. Appl. Microbiol. Biotechnol. 2017, 101, (2), 521532, DOI: 10.1007/s00253-016-8057-8. 9. Gschwend, F. J. V.; Brandt, A.; Chambon, C. L.; Tu, W.-C.; Weigand, L.; Hallett, J. P., Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids. J Vis Exp. 2016, 114, (114), e54246, DOI: 10.3791/54246. 10. Tao, J.; Kishimoto, T.; Hamada, M.; Nakajima, N., Novel cellulose pretreatment solvent: phosphonium-based amino acid ionic liquid/cosolvent for enhanced enzymatic hydrolysis. Holzforschung 2016, 70, (10), 911-917, DOI: 10.1515/hf-2016-0017. 11. George, A.; Brandt, A.; Tran, K.; Zahari, S. M. S. N. S.; Klein-Marcuschamer, D.; Sun, N.; Sathitsuksanoh, N.; Shi, J.; Stavila, V.; Parthasarathi, R.; Singh, S.; Holmes, B. M.; Welton, T.; Simmons, B. A.; Hallett, J. P., Design of low-cost ionic liquids for lignocellulosic biomass pretreatment. Green Chem. 2015, 17, (3), 1728-1734, DOI: 10.1039/C4GC01208A. 12. Chen, L.; Sharifzadeh, M.; Mac Dowell, N.; Welton, T.; Shah, N.; Hallett, J. P., Inexpensive ionic liquids: [HSO4]--based solvent production at bulk scale. Green Chem. 2014, 16, (6), 3098-3106, DOI: 10.1039/C4GC00016A. 13. Frederix, M.; Mingardon, F.; Hu, M.; Sun, N.; Pray, T.; Singh, S.; Simmons, B. A.; Keasling, J. D.; Mukhopadhyay, A., Development of an E. coli strain for one-pot biofuel production from ionic liquid pretreated cellulose and switchgrass. Green Chem. 2016, 18, (15), 4189-4197, DOI: 10.1039/c6gc00642f. 14. Xu, J.; Wang, X.; Liu, X.; Xia, J.; Zhang, T.; Xiong, P., Enzymatic in situ saccharification of

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For Table of Contents Use Only Synopsis: Isoprenol fermentation from switchgrass after DMSO-assisted ionic liquid pretreatment is presented, and DMSO is industrially produced from lignin.

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Dimethyl sulfoxide assisted ionic liquid pretreatment of switchgrass for

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