Santiago Rodríguez-Valderrama

Carlos Escamilla-Alvarado

Pasiano Rivas-García

Jean-Pierre Magnin

Mónica Alcalá- Rodríguez

Refugio Bernardo

ScDr. Carlos Escamilla Alvarado

Biorefinery concept comprising acid hydrolysis, dark fermentation and anaerobic digestion for co-processing of fruit and vegetable wastes and corn stover

Keywords: Biohydrogen, Furfural, Hydrochloric acid, Lignocellulosic waste, Methane, Multisubstrate, Phenolic compounds

A new biorefinery conceptual process is proposed for biohydrogen and biomethane production from a combination of fruits and vegetables wastes (FVW) and corn stover (CS). The objective of this work was to perform the acid hydrolysis (HCl 0.5 % v/v, 120 °C, 2 h) of the FVW and CS at 3:1 dry basis ratio, and to process its main physical phases, liquid hydrolyzates (LH) and hydrolyzed solids (HS), by mesophilic dark fermentation (DF) and anaerobic digestion (AD), respectively. In DF of LH as carbon source, hydrogen was produced at maximum rate of 2.6 mL H2/(gglucose h) and maximum accumulation of 223.8 mL H2/gglucose, equivalent to 2 mol H2/molglucose, in a butyric-pathway driven fermentation. HS were digested to methane production assessing inoculum to substrate ratios in the range 2 -4 ginoculum/gVS. The main results in AD were, 14 mmol CH4/gvs. The biorefinery demonstrated the feasibility to integrate the acid hydrolysis as pretreatment and subsequently use the LH for hydrogen production, and the HS for methane production, with an energy yield recovery of 9.7 kJ/gvs, being the energy contribution from anaerobic digestion 8-fold higher than of dark fermentation.

Introduction

Mexico is committed to reducing its greenhouse gases (GHG) by the year 2030 to 22%, whereas emissions of short-lived climate pollutants (black carbon) are aimed to 51% reduction (Gobierno de México 2015).

The main proposals for obtaining energy from renewable sources are bioenergy, solar energy, geothermal energy, hydroelectric energy, wind energy, and tidal energy. The biomass as a source for bioenergies, i.e. biohydrogen, biomethane, bioethanol, and biobutanol [START_REF] Mahlia | Palm oil and its wastes as bioenergy sources: a comprehensive review[END_REF], is most promising in the energetic share it could reach; according to some estimates, in Mexico it could supply ca. 46% of the annual energy, yet currently, only 5% is supplied (International Energy Agency 2016).

Residual biomass can be classified by its origin in forestry residues, agricultural residues, agroindustrial wastes and organic fraction of municipal solid waste [START_REF] Li | A review of methane production from agricultural residues in China[END_REF]. Residual biomass exhaustive use has led to the development of the biorefinery of residues.

The biorefinery concept is associated with the intensive conversion of organic matter into added-value products [START_REF] Escamilla-Alvarado | Bioenergy and bioproducts from municipal organic waste as alternative to landfilling: a comparative life cycle assessment with prospective application to Mexico[END_REF][START_REF] Moncada-Botero | Design strategies for sustainable biorefineries[END_REF]. Biorefineries are outlined by the following sections: i) pretreatment section to fractionate biomass, ii) section of bioprocesses (e.g. fermentation, digestion), iii) purification of bioproducts to discard impurities and pollutants, and iv) energy conversion section [START_REF] Giuliano | Process optimization of a multi-product biorefinery: The effect of biomass seasonality[END_REF][START_REF] Hernández-Flores | Bioelectricity generation from wastewater and actual landfill leachates: A multivariate analysis using principal component analysis[END_REF]).

The pretreatment with the highest efficiency of fermentable sugars release is diluted acid hydrolysis [START_REF] Gonzales | Optimization of substrate concentration of dilute acid hydrolyzate of lignocellulosic biomass in batch hydrogen production[END_REF][START_REF] Joglekar | Process of fruit peel waste biorefinery: a case study of citrus waste biorefinery, its environmental impacts and recommendations[END_REF]. It employs high temperatures and acids in low concentrations to hydrolyze macromolecules such as cellulose and hemicellulose into monomeric sugars such as glucose, xylose, arabinose, and others [START_REF] Kumar | Kinetic studies of two-stage sulphuric acid hydrolysis of sugarcane bagasse[END_REF]. Such soluble hydrolyzate is prone to any kind of fermentation, yet the insoluble fraction remaining from the pretreatment (hydrolyzed solids) is also susceptible for downstream processing. In the biorefinery perspective, the use of both hydrolyzates would be very attractive to increase the productivities of the installation.

Fermentable sugars in liquid hydrolyzates (HL) can be used as a carbon source to produce hydrogen through dark fermentation (DF) bioprocess [START_REF] Roy | Biohythane production from organic wastes: present state of art[END_REF]. On the other hand, the insoluble biomass, or hydrolyzed solids (HS), may be converted into a bioenergetic such as methane through anaerobic digestion (AD) [START_REF] Oliwit | Comparative evaluation of biochemical methane potential of various types of Ugandan agricultural biomass following soaking aqueous ammonia pretreatment[END_REF]. Actually, only few works have dealt with the either DF or AD of hydrolyzates with interesting results. For instance, [START_REF] Datar | Hydrogen production from the fermentation of corn stover biomass pretreated with a steam-explosion process[END_REF] applied steam explosion hydrolysis followed by the separated fermentation of the liquid hydrolyzates and the solid hydrolyzates for hydrogen production. Curiously, the dark fermentation of solid hydrolyzates did not produce hydrogen during the first 21 d, which was attained only after 20 h since the addition of cellulases. On the other hand, Tapia-Rodríguez et al. ( 2019) evaluated the parallel production of hydrogen and methane from enzymatic hydrolyzates of agave bagasse, however the HS were not included in the biorefining process.

The sources of biomass for biorefineries are numerous and in order to not compromise the food supply, biowastes should be mainly used as its substrates [START_REF] Romero-Cedillo | A review of the potential of pretreated solids to improve gas biofuels production in the context of an OFMSW biorefinery[END_REF]. Considering that Mexico was the 11 th agriculture producer worldwide with an annual production of 210 million tons in 2018 (SADER 2019), its wastes generation along the food supply chains (production, postharvest, processing, distribution, consumption) are also considerable. Indeed, as corn is the second most important crop in Mexico, the wastes associated to the over 26x10 6 metric tons production (SADER 2019) are corn stover (CS) and corn cobs. Since close to 82% of the CS generated is used for animal feed, the remaining 18% does not have a defined use (ca. 4.7x10 6 metric tons in dry base) and is therefore underused [START_REF] Hernández | Wheat straw, corn stover, sugarcane, and Agave biomasses: chemical properties, availability, and cellulosicbioethanol production potential in Mexico[END_REF]. Currently, CS is the most promising lignocellulosic waste for biofuels production due to its high cellulose (23-40%) and hemicellulose (12-32%) contents, and its low-cost abundant biomass [START_REF] Kim | Integration in a depot-based decentralized biorefinery system: Corn stoverbased cellulosic biofuel[END_REF][START_REF] Tan | Efficient fractionation of corn stover by bisulfite pretreatment for the production of bioethanol and high value products[END_REF].

Another underused biowaste in Mexico are the fruits and vegetable wastes (FVW), which compose ca. 44% of municipal solid wastes and that are disposed in landfills and open dumps [START_REF] Taboada-González | Waste characterization and waste management perception in rural communities in Mexico: A case study[END_REF][START_REF] Díaz | Treatment of supermarket vegetable wastes to be used as alternative substrates in bioprocesses[END_REF][START_REF] Gavilán | Municipal solid waste management[END_REF].

Even though worldwide distribution of biowaste has raised attractiveness on biorefineries, some traits of these substrates such as its recalcitrant nature and seasonal availability are some of the most important challenges for the development of biorefineries at large scale (Sultana and Kumar 2011;[START_REF] Giuliano | Process optimization of a multi-product biorefinery: The effect of biomass seasonality[END_REF]. It has been proposed that the combination of multiple lignocellulosic biomass would be a feasible solution as it also improves biofuels yields, complements the nutritional balance and reduces delivery costs (Sultana and Kumar 2011;[START_REF] Wang | Biohydrogen and methane production by co-digestion of cassava stillage and excess sludge under thermophilic condition[END_REF]. Therefore, this work aimed to evaluate a biorefinery concept for biohydrogen and biomethane production, as well as the resulting energy potential, from a combination of fruits and vegetables wastes (FVW) and corn stover (CS). The biorefinery concept (h-H-M) integrated the diluted acid hydrolysis (h) as pretreatment and the parallel production of hydrogen by DF (H) from liquid hydrolyzates and methane (M) from hydrolyzed solids.

Materials and methods

Biorefinery h-H-M set up

The FVW and CS were used as substrates to test the h-H-M biorefinery concept as shown in Fig 1 . The co-substrates were combined in dry basis (db) at the ratio 3:1 FVW:CS according to [START_REF] Rodríguez-Valderrama | Enfoque de Biorrefienría para la Producción de Hidrógeno y Metano a partir de residuos Orgánicos. Universidad Autónoma de Nuevo[END_REF]. The co-substrates were subjected to hydrolysis pretreatment (h-stage) using HCl as the catalyst acid. After the dilute acid hydrolysis, the liquid and solid phases were separated by centrifugation. Liquid hydrolysates (LH) were limed to decrease the concentration of inhibitory compounds, and used as a carbon source in an H2-producing dark fermentation stage (H-stage) at mesophilic temperature. The hydrolyzed solids (HS) were washed to remove the catalyst acid excess and used as carbon source in a methane producing anaerobic digestion bioprocess (M-stage). Each of the three stages will be described in detail in the following sections.

INSERT FIG. 1

Co-substrates

Corn stover (collected from Cuencamé, Durango, Mexico) and FVW (collected from a local cafeteria Chemical Sciences Faculty, Universidad Autónoma de Nuevo León, Nuevo León, Mexico) were separately dried in an oven at 85 °C for 24 h, grinded to 180 µm particle size of using a manual mill, and stored in distinct tightly closed plastic bags at room temperature. FVW and CS were physico-chemically characterized (Table 1). The empirical molecular formulas and the heat power value were CH4.31O0.68N0.01, 3606 cal/gdb, and CH0.8O0.62N0.04, 2712 cal/gdb for CS and FVW, respectively.

INSERT TABLE 1

Diluted acid hydrolysis and overliming

The acid hydrolysis was carried out using dilute HCl (0.5% v/v) in 0.5 L Schott bottles. The reaction volume was 0.3 L and the solid content was 6.6% of reaction volume. The co-substrates ratio was 3:1 FVW:CS (g:g). The hydrolysis reaction was performed in an autoclave (121 °C) for 120 min [START_REF] Yan | Dilute sulfuric acid cycle spray flow-through pretreatment of corn stover for enhancement of sugar recovery[END_REF][START_REF] Kumar | Kinetic studies of two-stage sulphuric acid hydrolysis of sugarcane bagasse[END_REF]. After the hydrolysis, liquid hydrolyzates (LH) were separated by centrifugation (10 000 g, 10 min) and characterized in terms of reducing and monomeric sugars and the inhibitory compounds, i.e. acetic acid, formic acid, furfural, 5-hydroxymethyl-furfural (HMF), and total phenolic compounds (TPC).

Overliming treatment was used applied to the LH in a two-step approach [START_REF] Chang | Hydrogen production by the anaerobic fermentation from acid hydrolyzed rice straw hydrolysate[END_REF]. Firstly, the pH of LH was adjusted to 10 by adding powder Ca(OH)2 in continuous stirring for one hour. Secondly, the pH was reduced to to 7 by 6 M HCl addition, following a centrifugation (10 000 g, 15 min) and the separation of liming precipitates and supernatant. Sample were retrieved from the LH to analyze the removal of inhibitory compounds and sugars.

Dark fermentation

DF was carried out by duplicate in 0.5 L Schott bottles with 0.4 L of reaction volume. The methanogenic anaerobic sludge was heat-treated to inhibit methane-producing microflora in water bath at 96° C for 2 h.

The initial reducing sugars (RS) were adjusted to 13 g/L and the amount of substrate and inoculum were added according to the inoculum to substrate ratio (ISR) of 1.2 (VS basis). The fermentation volume was supplemented with 4 mL of 200 fold mineral medium previously reported by [START_REF] Rodríguez-Valderrama | Enfoque de Biorrefienría para la Producción de Hidrógeno y Metano a partir de residuos Orgánicos. Universidad Autónoma de Nuevo[END_REF]. The medium was supplemented with nitrogen source (1 g NH4Cl/L) and buffer medium (3 g K2HPO4/L, 1.5 g KH2PO4/L). The experimental units were stirred at 150 rpm in a multiple magnetic stirrer inside an incubator at 35 °C.

Anaerobic digestion

HS from the separation of LH were washed twice with distilled water (0.03 L of water per 10 gwb of HS), vortexed and sedimented for 10 min, to remove the residual acid catalyst. Afterwards, HS were recovered by centrifugation (15 min at 10 000 g), dried at 80 °C and characterized (Table 1). HS empirical molecular formula based on elemental composition was CH2.30O0.42N0.03.

AD was carried out in 0.120 L serum bottles with 0.08 L of work volume. The inoculum was anaerobic sludge from a semi-continuous digester fed with FVW at 30 d of hydraulic retention time. The inoculum was degassed during 3 d and then used for HS anaerobic digestion. The inoculum had the following characteristics: 7.81 pH, 93.64%wb moisture, 6.36%wb TS, 51.46%db VS, 48.54%db ashes. The alkalinity and total VFA were 12 300 mg CaCO3/L and 8 040 mg VFA/L, respectively. Its empirical molecular formula based on elemental composition was CH1.34O2.02N0.10.

Inoculum to substrate ratios (ISR) were assayed in batch mode: 2, 2.5, 3, 3.5 and 4 gvs inoculum/gvs HS. A control was run with fresh FVW at ISR 3. A blank was loaded only with inoculum and water to to determine the methane production by the organic matter present in the inoculum. The methane production (2.16 mmol CH4) from blanks was subtracted from the methane produced by HS. All the experimental units were kept constant in its inoculum content at 2.5 gvs, the substrate amounts were adjusted according to each ISR. The anoxic environment in each bottle was promoted by flushing N2 during 3 min.

Afterwards, the bottles were sealed with a rubber stopper and aluminum rings. The operation temperature and stirred velocity were 35 ± 1 °C and 150 rpm, respectively. All the experiments were carried out by duplicate.

Analytical methods

The pH was determined according to the procedure described by NMX-AA-25-1984[START_REF] Anderson | Determination of bicarbonate and total volatile acid concentration in anaerobic digesters using a simple titration[END_REF]. Solids profile was measured according to standard methods (APHA/AWWA/WEF 2005). Cellulose and acid-insoluble lignin contents were determined by the gravimetric method base on AOAC methods (AOAC 1992).

Hemicellulose amount was determined subtracting the cellulose content from holocellulose content after by lignin oxidation by NaClO (Escamilla-Alvarado et al. 2015). The extractives in CS and FVW were determined by differential weight after extraction in a water bath at 60 °C 24 h [START_REF] Sluiter | Optimal configuration and combination of multiple lignocellulosic biomass feedstocks delivery to a biorefinery[END_REF]). The elemental characterization (C, H, O, N) was determined by an elemental analyzer (Thermo Scientific Flash 2000, U.S.A), O2 was used as combustion gas and He was used as carrier gas.

The reducing sugars (RS) in hydrolyzate were determined by the 3,5-dinitrosalicylicacid method (DNS) using dextrose for the calibration curve [START_REF] Miller | Use of dinitrosalicylic acid reagent for determination of reducing sugar[END_REF]. Glucose, xylose, and arabinose were quantified by high performance liquid chromatograph (LDC Analytical, U.S.A.) equipped with a Rezex RHM-Monosaccharide (300mm X 7.8 mm) column and a refractive index detector (Varian Prostar, U.S.A.). The column temperature was 65 °C, whereas the mobile phase (H2O) flow rate was maintained at 0.6 mL/min. The total amount of biogas produced in DF and AD was determined by the acid-brine displacement method [START_REF] Escamilla-Alvarado | A multivariable evaluation of biohydrogen production by solid substrate fermentation of organic municipal wastes in semi-continuous and batch operation[END_REF]. Hydrogen and methane gas contents were determined in a gas chromatograph (Thermo Scientific Trace 1310, U.S.A.); the gas chromatograph was equipped with a thermal conductivity detector and a molecular sieve column (TG-BOND Msieve 5A, 30 m x 0.33 mm).

The operating temperatures were 100 °C, 150 °C, and 200°C for the oven, injector, and detector, respectively. Nitrogen gas was used as a carrier gas with a flow rate of 3 mL/min. The total phenolic compounds (TPC) were determined by the Folin-Ciocalteu method proposed by [START_REF] Blainski | Application and analysis of the Folin Ciocalteu method for the determination of the total phenolic content from Limonium Brasiliense L[END_REF] using tannic acid as the standard. Furfural, HMF, formic acid, acetic acid, propionic acid, succinic acid, and lactic acid were demined by gas chromatography (Varian CP 3380, U.S.A.) with a column ZB-FFAP (15m x 0.53 x 1 µm) and flame ionization detector. The injector and detector temperatures were 230 and 280 °C, respectively. The temperature program for the column initiated at 90 °C for 3 min, then increased to 200 °C at 20 °C/min rate and maintained for 3 min, and finally raised to 250 °C at 30 °C/min, which was maintained for 4 min.

Total volatile fatty acids (TVFA) and alkalinity concentration were determined by a titration method [START_REF] Anderson | Determination of bicarbonate and total volatile acid concentration in anaerobic digesters using a simple titration[END_REF]. Acetic acid, propionic acid, butyric acid, and ethanol were quantified using a gas chromatograph according to the method described in our previous work (Rodríguez-Valderrama et al.

2019).

Calculations

A set of response variables was calculated according to Table 2 to analyze the production of hydrogen, methane and energy potential. The cumulative specific hydrogen production H(t) (mL H2/gglucose) was used for describing the accumulated production of hydrogen in time and to obtain kinetic parameters from fitting the results by the Gompertz equation (Eq. 1). Thus, the maximum cumulative specific hydrogen production Hmax (mL H2/gglucose), the maximum specific hydrogen production rate rmax,H (mL H2/(gglucose h)) and the adaptation time 𝜆 (h) were determined.

The hydrogen molar pseudoyield, Y'H2 (mol H2/molglucose) in Eq. 2 was obtained from the Hmax (mL H2/gglucose) as a means to compare the system to the maximum theoretical hydrogen yield (2 and 4 mol H2/gglucose for butyrogenic and acetogenic pathways, respectively).

Other variables in the equations listed in Table 2 used in Eq. 1 or Eq. 2 are t is fermentation time (h), e is 2.718, CRS,0 and CRS,f (g/L) are the concentration of RS at the beginning and at the end of DF, and MWglucose is the glucose molar weight (180.16 g/mol).

Regarding the AD equations and parameters (Table 2), the cumulative methane production B(t) (mmol CH4) and cumulative specific methane production b(t) (mmol CH4/gvs) were used to calculate the kinetic parameters by two methods: an adaptation of the Gompertz equation (Lo et al. 2010, Eq. 3 and Eq. 4), and the first-order model proposed by Hashimoto (1989, Eq. 5 and Eq. 6). Through Eq. 3 and Eq. 5 the maximum cumulative methane production Bmax is obtained, whereas through Eq. 4 and 6 the maximum cumulative specific methane production bmax is estimated. The other parameters estimated through these equations are the maximum methane production rate Rmax,M (mmol CH4/ d), the maximum specific methane production rate rmax,M (mmol CH4/(gvs d)), 𝜆, and the methane production rate k (1/d).

The specific gross energy potential Êp (kJ/gvs) was used to compare our h-H-M biorefinery against other biorefinery models in the literature, either serial where DF is followed by AD (Eq. 7), or in parallel DF and AD systems (Eq. 8). In these equation, the hydrogen high heating value HHVH2 is 282.8 kJ/mol, the methane high heating value HHVCH4 is 889.9 kJ/mol, VM is the molar volume of an ideal gas at standard conditions (22.4 L/mol), 1000 is the mL to L conversion factor, ηDF is the quotient of VS consumed and VS fed in the DF units, and ηAH is the quotient of glucose released and VS fed in acid hydrolysis experiments.

INSERT TABLE 2

Results and Discussion

Diluted acid hydrolysis and overliming

The RS concentration after co-substrates hydrolysis was 23.49 g/L, containing high amounts of glucose (10.36 g/L) followed by xylose (8.61 g/L) and arabinose (0.39 g/L). The RS production yield was 48.54% (calculated as the amount of RS produced divided by the sum of volatile solids added) for the 3:1 FVW:CS combination. This yield is comparable to those reported in the literature for acid hydrolysis of either FVW or CS. For instance, [START_REF] Datar | Hydrogen production from the fermentation of corn stover biomass pretreated with a steam-explosion process[END_REF] obtained a hydrolysis yield of 47% in the steam-explosion treatment of acid impregnated CS. [START_REF] Cao | Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16[END_REF] managed to extract the 35.20% of sugars in the CS acid hydrolysis. On the other hand, [START_REF] Díaz | Treatment of supermarket vegetable wastes to be used as alternative substrates in bioprocesses[END_REF] extracted 35.9% of the reducing sugars available in tomato wastes through acid hydrolysis. Additionally, one of the main benefits of the co-substrates acid hydrolysis is the improvement of the monomeric sugars distribution. For instance, in our 3:1 FVW:CS experiment the monosaccharide distribution in the liquid hydrolyzates was 53.5% glucose, 44.5% xylose and 2% arabinose (Table 1). In contrast, the main monomeric sugars distribution from hydrolysis of only CS were 9.08% of glucose, 83.08% of xylose and 7.84% of arabinose [START_REF] Datar | Hydrogen production from the fermentation of corn stover biomass pretreated with a steam-explosion process[END_REF]), whereas FVW were only composed of 100% hexoses (Del [START_REF] Campo | Diluted acid hydrolysis pretreatment of agrifood wastes for bioethanol production[END_REF].

The overliming treatment of acid hydrolyzates successfully reduced the inhibitory compounds as TPC in 33.86%, although 10.05% RS were also lost (Table 3). RS loss is commonly expected in such treatments [START_REF] Saha | Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol[END_REF], for instance, [START_REF] Chang | Hydrogen production by the anaerobic fermentation from acid hydrolyzed rice straw hydrolysate[END_REF] reported 9% RS loss after the overliming of rice husk hydrolyzates, and Purwadi et al. ( 2004) had 8.42% RS loss from detoxification by Ca(OH)2 of Swedish forestry residues hydrolyzates. The concentration of TPC, HMF, furfural, and acetic acid, did not surpass the concentrations known to inhibit hydrogen production, which are in the following ranges: 0.8-2.28 g/L for TPC, 0.86-1.89 g/L for HMF, 0.8-3.41 g/L for furfural and 0.6-7.80g/L for acetic acid [START_REF] Ren | Dark fermentation of xylose and glucose mix using isolated Thermoanaerobacterium ihermosaccharolyticum W16[END_REF][START_REF] Gonzales | Optimization of substrate concentration of dilute acid hydrolyzate of lignocellulosic biomass in batch hydrogen production[END_REF].

INSERT TABLE 3

Dark fermentation

After overliming pretreatment, the LH were used for hydrogen production at initial pH of 7 and 35 °C.

After 150 h of fermentation, maximum experimental cumulative biogas and maximum cumulative specific hydrogen production were 2717 mL and 223.8 mL H2/gglucose, respectively (Fig. 2). The hydrogen average content in biogas was 50.89%, whereas methane was not detected. According to the Gompertz parameters (Table 4), the production of hydrogen from the acid hydrolyzates of the co-substrates mixtures (3:1 FVW:CS) showed a rmax,H of 2.60 (mL H2/(gglucose h)) and 19.25 h of adaptation time.

rmax,H is comparable to other studies presented in literature where hydrogen production from acidic hydrolyzates of organic waste has been studied. [START_REF] Zhang | Fermentative hydrogen production from corn stover hydrolyzate by two typical seed sludges : Effect of temperature[END_REF] reported a rmax,H of 0.92 (mL H2/(gglucose h)) in a 100 mL serum vials with acid hydrolyzates (1.7% v/v H2SO4, 120 min, 120 °C) from CS produced by activated sludge. The reason for their low rmax,H compared to our study might be attributed to the low initial sugars concentration (5 g/L against 13 g/L, respectively) and the low initial ISR (0.19 against 1.2, respectively), as such parameters are directly related to hydrogenogenic performance [START_REF] Fan | Efficient conversion of wheat straw wastes into biohydrogen gas by cow dung compost[END_REF][START_REF] Ozmihci | Thermophilic dark fermentation of acid hydrolyzed waste ground wheat for hydrogen gas production[END_REF]). In addition, the hydrogen batch fermentation at ISR lower than 0.16 could have presented inhibition due to the high amounts of substrate [START_REF] Wong | A review of sustainable hydrogen production using seed sludge via dark fermentation[END_REF].

INSERT FIG. 2

The maximum productivity of hydrogen from LH was 2909 mL H2/Lreactor, which can be compared with other studies also using LH for hydrogen production. In the case of [START_REF] Datar | Hydrogen production from the fermentation of corn stover biomass pretreated with a steam-explosion process[END_REF], they reported hydrogen productivity of 3310 mL H2/Lreactor from CS hydrolyzates in a CSTR reactor using a pre-treated anaerobic sludge as an inoculum; the volumetric productivity reported in our work was 12.1% lower than theirs. However, the adaptation time reported by [START_REF] Datar | Hydrogen production from the fermentation of corn stover biomass pretreated with a steam-explosion process[END_REF] was 49.5% higher due to the initial concentrations of inhibitors present in their hydrolyzates.

INSERT TABLE 4

The molar pseudoyield obtained at 35°C was 2.02 mol H2/molglucose, comparable to those using pure and mixed cultures from acid hydrolyzates. Biohydrogen production from starch hydrolyzates led to a maximum YH2 of 1.28 and 0.85 mol H2/molglucose by either Clostridium pasteurianum CH5 or by consortium from dark fermentation sludge, respectively [START_REF] Chen | Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species[END_REF]. Both experiments were carried out at 37 °C and initial sugars concentration of 26.7 g/L. Using acid hydrolyzates from wheat starch (121 °C, 15 min, pH 2.5, H2SO4), [START_REF] Cakr | Comparison of bio-hydrogen production from hydrolyzed wheat starch by mesophilic and thermophilic dark fermentation[END_REF] obtained a Y'H2 of 2.4 mol H2/molglucose at 55 °C and initial sugars concentration of 18.5 g/L using heat-treated anaerobic sludge. This value was 1.2-fold higher than ours, however, the use of thermophilic temperatures may decrease the energy gains from biofuels production [START_REF] Rodríguez-Valderrama | Enfoque de Biorrefienría para la Producción de Hidrógeno y Metano a partir de residuos Orgánicos. Universidad Autónoma de Nuevo[END_REF].

The final pH in hydrogen production was 5.34 (Table 4), an adequate value for hydrogen production as reported in literature [START_REF] Braguglia | Anaerobic bioconversion of food waste into energy: A critical review[END_REF]. In our experiments, the pH0 was adjusted to 7 as commonly done in free fermentation experiments. For instance, [START_REF] Liu | Biohydrogen production evaluation from rice straw hydrolysate by concentrated acid pre-treatment in both batch and continuous systems[END_REF] observed a considerable hydrogen yield increase from 0.02 mol H2/molsugars at pH0 5, to 0.44 mol H2/molsugars at pH0 7 when using rice straw acid hydrolyzates.

The main metabolites in DF were butyric acid (6.13 g/L), acetic acid (1.11 g/L), propionic acid (0.36 g/L) and a small amount of ethanol (0.07 g/L), which indicates that the fermentation was carried out in greatly by butyric acid metabolic pathway [START_REF] Chen | Stoichiometric analysis of biological hydrogen production by fermentative bacteria[END_REF][START_REF] Wang | Kinetic models for fermentative hydrogen production: A review[END_REF]. These results demonstrated the good performance of dark fermentation from acid hydrolyzates obtained from organic substrates mixtures by anaerobic sludge.

Anaerobic digestion

The cumulative methane production and specific methane production as a function of time for different ISR are shown in Fig 3 . The best results for each parameter were obtained at ISR 2.5 and 4, respectively. Indeed, all the experiments evaluated with HS presented methane productions and specific methane productions higher than the experiments with FVW (control) during the first five days due to the readily biodegradable organic matter in the HS. Still, at the end the control produced as good results as most of them due to the RIS used and the good biodegradability of FVW at high digestion times [START_REF] Li | Solid-state anaerobic digestion for methane production from organic waste[END_REF].

The cumulative methane production (Bmax) at the different studied ISR (2-4) was in the range 6.37 to 8.78 mmol CH4. The best results were obtained in the order ISR 2.5>4>3>2>3.5 (Table 5,Fig 3A). On the other hand, the cumulative specific methane production (bmax) was in the range 5.84-13.64 mmol CH4/gvs, obtained from highest to lowest in the order ISR 4>3>3.5>2.5>2. This was in agreement to reports in literature that have ascribed this phenomenon to the fact that a greater inoculum to substrate ratio allows a more exhaustive conversion of biomass into methane [START_REF] Hashimoto | Effect of inoculum/substrate ratio and pretreatments on methane yield and production rate from straw[END_REF]).

The predicted maximum cumulative methane production (Bmax, 8.90 mmol CH4) and the lowest deviation in respect to the experimental value (1.30%) were presented with the first-order kinetic model for ISR=2.5, whereas Gompertz prediction had a deviation of 3.36% respect to the measured production.

The coefficient of determination for this experiment (ISR = 2.5) was 0.99, slightly higher than the obtained by the Gompertz model (0.99). The maximum methane rates (k) for each ISR evaluated were in the range of 0.19 to 0.28 (1/d). The maximum k (0.28 1/d) corresponded to ISR of 2.5, which could have reflected a positive interaction of a rapid substrate biodegradability due to pretreatment and an adequate inoculum load. Similarly, at 2.5 ISR [START_REF] Moset | The impact of inoculum source, inoculum to substrate ratio and sample preservation on methane potential from different substrates[END_REF] registered a k of 0.16 (1/d) in the anaerobic digestion of corn stover silage (1 year), and 0.08 (1/d) for wheat straw without any previous treatment.

The predicted specific methane productions (bmax) by the first-order kinetic and Gompertz models were 14 mmol CH4/gvs and 13.2 mmol CH4/gvs, respectively (Table 6). However, the first-order kinetic model presented the best fit to the experimental data since its error (2.62%) was lower than the respective of the Gompertz model (3.07%). These errors are usual when comparing the fits of mathematical models.

As reported by [START_REF] Zhang | Batch anaerobic co-digestion of pig manure with dewatered sewage sludge under mesophilic conditions[END_REF] who obtained deviations from 1.5% for the first-order kinetic model and 3.7% for the Gompertz model when they evaluated the anaerobic digestion of dewatered sewage sludge at ISR of 1 and 37 °C of fermentation temperature.

INSERT FIG. 3 INSERT TABLE 5 The maximum predicted bmax (14 mmol CH4/gvs or 342.3 L CH4/kgvs) is comparable to the maximum methane specific productions in literature, only 8% lower than the value of 372.4 L CH4 /kgvs, from alkaline pretreated (5% NaOH) CS on solid-state [START_REF] Zhu | AD, anaerobic digestion; bmax, Maximum cumulative specific methane production; DF, dark fermentation; CM, cattle manure NA, not applicable; FVW, fruit and vegetable waste; Hmax[END_REF]. This difference may be mainly due to the positive traits of alkaline treatment and its less severity compared to acid treatments. Besides lignin solubilization, alkaline pretreatments also provide neutralization of the different acids in lignocellulosic compounds degradation. However, the maximum methane production rate (9.3 L CH4/(kgvs d)) found by [START_REF] Zhu | AD, anaerobic digestion; bmax, Maximum cumulative specific methane production; DF, dark fermentation; CM, cattle manure NA, not applicable; FVW, fruit and vegetable waste; Hmax[END_REF] after 40 d, was 6.2 fold lower than that obtained from HS during 15 d (2.35 mmol CH4/(gvs d); 57.4 L CH4/(kgvs d)), which indicates the rapid substrate degradation due to the acid pretreatment process.

The bmax and the rmax,M obtained in the control experiment with fresh untreated FVW were 10.01 mmol CH4/gvs (244.5 L CH4/ kgvs) and 1.29 mmol CH4/ (gvs d) (31.6 L CH4/(kgvs d)), respectively. This bmax was 28.63% lower than that obtained with HS (342.3 L CH4/kgvs), because when pretreating a substrate, either by alkaline pretreatment or acidic pretreatment, the methane production is raised, since soluble organic matter was increased and then easily used by microorganisms [START_REF] Abudi | Batch anaerobic co-digestion of OFMSW (organic fraction of municipal solid waste), TWAS (thickened waste activated sludge) and RS (rice straw): Influence of TWAS and RS pretreatment and mixing ratio[END_REF]. The increase in methane production (28.63%) due to substrate pretreatment was also observed in the literature. For instance, [START_REF] Abudi | Batch anaerobic co-digestion of OFMSW (organic fraction of municipal solid waste), TWAS (thickened waste activated sludge) and RS (rice straw): Influence of TWAS and RS pretreatment and mixing ratio[END_REF] registered the increment of 6.69% in the methane production from rice straw without pretreatment and 45.15% from alkali rice straw organic fraction of municipal solid wastes (OFMSW). On the other hand, both substrates (rice straw and pretreated rice straw) showed higher methane production when compared to OFMSW digestion, but this phenomenon seems to be related to the nature of the substrate rather than to the pretreatment applied.

INSERT TABLE 6

Energy evaluation of the h-H-M concept biorefinery

The energy yield from h-H-M biorefinery model and its comparison with two-stage systems from different biomasses as substrates are shown in Table 7. The energy yield for h-H-M biorefinery based on gaseous biofuels production was 9.7 kJ/gvs. The stage with the greatest contribution to the energy yield was the methane production (M) stage with a contribution near to 89.4% (8.7 kJ/gvs), whereas the contribution of hydrogen produced in dark fermentation (H) was the remaining 10.6% (1.0 kJ/gvs). [START_REF] Ghimire | Biohydrogen production from food waste by and residue post-treatment to anaerobic digestion : A synergy for energy recovery[END_REF] calculated a maximum energy yield of 4.4 kJ/gvs for hydrogen and methane production from food waste.

Energy contribution of DF was 29.6% (1.3 kJ/gvs) when the food wastes were fermented in a 1.5 L semicontinuous reactor at 55°C, whereas the maximum energy yield contribution was presented by AD (70.4%, 3.1 kJ/gvs). The relatively low energy contribution of DF is ascribed to the inherent energy loss in the form of metabolites in the effluents [START_REF] Xia | Comparison in dark hydrogen fermentation followed by photo hydrogen fermentation and methanogenesis between protein and carbohydrate compositions in Nannochloropsis oceanica biomass[END_REF].

INSERT TABLE 7

Conclusions

This work demonstrated that a new biorefinery approach coined as h-H-M, was technically feasible for treating a preparation of fruits and vegetables wastes and corn stover through acid hydrolysis, dark fermentation and anaerobic digestion. Acid hydrolysis of the preparation 3:1 fruit and vegetable wastes to corn stover produced hydrolyzates containing up to 10.4 g/L of glucose, 8.61 g/L of xylose and 0.39 g/L of arabinose.

Dark fermentation from liquid hydrolyzates performed competitively at inoculum to substrate ratio 1.2, reaching productivity of 2909.7 mL H2/Lreactor and pseudoyield 2.02 mol H2/molglucose. The hydrolyzed solids were adequately used as carbon source for anaerobic digestion, promoting higher initial methane production when compared to compared to fresh substrate. The maximum cumulative methane production, 8.9 mmol CH4, and the highest methane specific production, 13.6 mmol CH4/gvs, were obtained at the inoculum to substrate ratios of 2.5 and 4, respectively.

The total energy potential from the h-H-M biorefinery concept in the form of hydrogen and methane reached 9.7 kJ/gvs, of which hydrogen contributed to 10.6 %.

Using a combination of co-substrates such as FVW and CS opens up the possibility of interesting configurations for real biorefineries, as this approach may provide an alternative to mono-substrate drawbacks.

RS

         =  +           (1)
Hydrogen molar pseudoyield

( ) ( ) 2 ,0 cos ,0 , ( ) max ' 1000 RS glu e H RS RS f M H C MW Y C C V   = -   (2)
Cumulative methane production (Gompertz-modified model)

( ) max 0 max, ( ) exp -exp - 1 R e M B t B t B           =  +           (3)
Cumulative specific methane production (Gompertz-modified model)

( ) max 0 max, ( ) exp -exp - 1 r e M b t b t b           =  +           (4)
Cumulative methane production (first-order kinetic model)

( ) max ( ) 1 exp kt B t B - =  - (5)
Cumulative specific methane production (first-order kinetic model)

( ) max ( ) 1 exp kt b t b - =  - (6)
Specific gross energy potential for serial hydrogen and methane production (1 ) 1000

P AH H AH CH M E H HHV b HHV V   =    + -    (8) 660

List of Figures

Fig. The

  Fig. The h-H-M biorefinery concept

Fig. 2 Fig. 3

 23 Fig. The h-H-M biorefinery concept

Table 1

 1 Characteristic of co-substrates (FVW and CS) and HS

		Reducing sugars
	T	Operational temperature
	t	Time
	TPC	Total phenolic compounds
	TS	Total solids
	TVFA	Total volatile fatty acids
	VM	Molar volume at standard conditions (22.4 L/mol H2 or CH4)
	VO	Operational volume
	VS	Volatile solids
	Y'H2	Hydrogen molar pseudoyield (mol H2/molglucose)
	Subindices
	db	dry basis
	wb	wet basis
	Greek characters
		adaptation time (h or d)
	ηAH	Acid hydrolysis efficiency (gglucose/gvs)
	ηDF	Dark fermentation efficiency (gvs consumed/gvs added)

Abbreviations: CS, corn stover; db, dry base; FVW, fruits and vegetables wastes; ND, not determined; TS, total solids; VFA, volatile fatty acids; VS, volatiles solids; wb, wet base.

Table 2

 2 Equations used to describe the H and M stages and the biorefinery performance

	Response variable	Equation

Table 3

 3 Main sugar production and secondary products from acid hydrolysis pretreatment of co-substrates (3:1 FVW:CS) Gompertz coefficients and performance parameters from dark fermentation of LH

	Parameter	LH before overliming	LH after overliming
	RS (g/L)	23.49	21.13
	Glucose (g/L)	10.36	9.65
	Xylose (g/L)	8.61	8.36
	Arabinose (g/L)	0.39	0.07
	HMF (g/L)	0.65	ND
	Furfural (g/L)	0.14	ND
	TPC (g/L)	1.14	0.76
	Formic acid (g/L)	4.02	ND
	Acetic acid (g/L)	0.53	ND
	Abbreviations: HMF, 5-hydroxymethylfurfural; ND, not detected; LH, liquid hydrolyzates; RS, reducing sugars; TPC, total
	phenolic compounds.

Abbreviations: Hmax, maximum cumulative specific hydrogen production; Rmax,H, maximum specific hydrogen production 670 rate; R 2 , coefficient of determination ; Y'H2, hydrogen molar pseudoyield; λ, adaptation time.

Table 6

 6 Kinetic parameters comparison between the first-order model and Gompertz model on the maximum cumulative specific methane production (bmax,) during anaerobic digestion of HS and FVW

	Model	ISR-substrate
		2-HS 2.5-HS	3-HS	3.5-HS	4-HS	3-FVW
	Experimental bmax (mmol CH4/gVS)	5.84	8.78	9.63	9.10	13.64	9.67
	First-order
	bmax (mmol CH4/ gVS)	6.04	8.89	10.12	9.77	14.00	NA
	k (1/d)	0.23	0.28	0.23	0.19	0.25	NA
	R 2	0.99	0.99	0.99	0.99	0.99	NA
	Error (%)	3.47	1.29	5.09	7.36	2.62	NA
	Gompertz
	bmax (mmol CH4/ gVS)	5.68	8.49	9.62	9.02	13.22	10.01
	rmax,M (mmol CH4/ (gVS d))	0.90	1.67	1.43	1.18	2.35	1.29
	λ	0.00	0.00	0.00	0.00	0.00	1.46
	R 2	0.99	0.99	0.99	0.99	0.99	0.99
	Error (%)	2.61	3.36	0.11	0.87	3.07	1.46

Abbreviations: bmax, maximum cumulative specific methane production; k, methane production rate; HS, hydrolyzed solids; FVW, fruits and vegetables wastes; NA, not applicable; rmax,M, maximum specific methane production rate; R 2 , coefficient of determination; λ, adaptation time.

Note: range of final pH values 7.53-7.72.

Acknowledgments

The authors express their gratitude to the Directive Board of the Chemical Sciences Faculty of the Universidad Autónoma de Nuevo León for supporting the project (02-106534-PST-15/123). Santiago Rodríguez-Valderrama thanks Consejo Nacional de Ciencia y Tecnología (Conacyt) for the Scholarship No. 714579.

Abbreviations

Coefficient of determination

List of Tables


Yangyang Mao

Mengchen Zhang

Long Cheng

Jianwei Yuan

Gongping Liu
email: gpliu@njtech.edu.cn

Libo Huang

Mihail Barboiu

Wanqin Jin
email: wqjin@njtech.edu.cn

Bola-amphiphile-imidazole -GO membrane with enhanced solvent dehydration properties

Keywords: artificial water channel, imidazole, graphene oxide membrane, solvent dehydration, pervaporation

Inspired by fast and selective water transport in natural Aquaporin (AQP), an water binding bola-amphiphile-imidazole compound (IU) was embedded with the hydrophilic and laminated graphene oxide (GO) to prepare IU-GO membrane.

H-NMR, TG, XRD, SEM, AFM and FTIR were conducted to characterize physicochemical properties of IU and IU-GO membranes. It was demonstrated that IU could well interact with GO to generate a robust membrane structure. The resulting IU-GO membranes were applied for pervaporation dehydration of n-butanol-water.

The total flux and the separation factor of the optimized IU-GO membrane were 3506 g•m -2 •h -1 and 4454 respectively when the membrane was measured at 343 K with butanol content of 80 wt%. Furthermore, the membrane exhibited good stability and separation performance during 120 hours continuous operation test, showing great potential in application of solvent dehydration.

Introduction

In recent years, pervaporation (PV) membrane separation technology is widely applied in solvent dehydration and organic separation due to its convenient operation, energy-efficiency, and environmental friendliness [START_REF] Cheng | Hybrid membranes for pervaporation separations[END_REF][START_REF] Zhao | Facilitated water-selective permeation via PEGylation of graphene oxide membrane[END_REF] . An ideal dehydration PV membrane allows for rapid transport of water, while simultaneously rejecting organic solvent [START_REF] Huang | Vapor transport in graphene oxide laminates and their application in pervaporation[END_REF] . Poly(vinyl alcohol) [START_REF] Xia | In-situ crosslinked PVA/organosilica hybrid membranes for pervaporation separations[END_REF] and chitosan [START_REF] Qian | Chitosan/graphene oxide mixed matrix membrane with enhanced water permeability for high-salinity water desalination by pervaporation[END_REF] are traditional polymer membrane materials for solvent dehydration due to the good hydrophilicity while suffering from an undesirable trade-off between permeability and selectivity which limits their further application [START_REF] Liu | Ultrathin two-dimensional MXene membrane for pervaporation desalination[END_REF] .

In nature, aquaporin (AQP) [START_REF] Siwy | Improving on aquaporins[END_REF] , as a trans-membrane protein, possesses both superior water permeability (~10 8 -10 9 water molecules/s/channel) and high selectivity (filter out molecules and charged hydrated ions larger than water molecule). From this point of view, the selectivity water transport observed with AQP could provide valuable guidance in membrane separation, especially in PV dehydration of organic solvent. Typical behaviors of AQP are the hourglass-type structure, hydrophobicity and charge repulsion in the aromatic arginine (ar/R) constriction with diameter of 2.8 Å and hydrophilicity on both side of the pore [START_REF] Shen | Biomimetic membranes: A review[END_REF] . This unique structural property of AQP provides important insights for designing synthetic membrane materials with rapid water transport. The basic approach for mimicking this channel is to extract the AQP proteins and reconstitute them into vesicles or synthetic bilayers otherwise they will lose biological functions [START_REF] Shen | Biomimetic membranes: A review[END_REF][START_REF] Zhong | Aquaporin-embedded biomimetic membranes for nanofiltration[END_REF] . Wang et al. [START_REF] Li | Nature gives the best solution for desalination: Aquaporin-based hollow fiber composite membrane with superior performance[END_REF] reported on a highly permeable and selective biomimetic composite membrane with a high AQP covering density exhibited water permeance of 4 L•m -2 •h -1 •bar -1 or 8 L•m -2 •h -1 •bar -1 for flat sheet or for hollow fiber polyamide thin layer RO membranes, respectively with a salt rejection of >97.5%. In the application of AQP biological membranes, main challenges might include: i) difficulty in controlling stable structure and scaling-up because of insufficient stability of AQP proteins in high saline and operational pressure; ii) fragile lipid bilayers show poor compatibility with the practical applications; iii) high fabrication cost compared with synthetic counterparts [START_REF] Kim | Biomimetic Selective Ion Transport through Graphene Oxide Membranes Functionalized with Ion Recognizing Peptides[END_REF][START_REF] Lee | Facilitated Water Transport through Graphene Oxide Membranes Functionalized with Aquaporin-Mimicking Peptides[END_REF] . Therefore, researchers attempt to mimic the structure of AQP [START_REF] Kocsis | Artificial water channels-deconvolution of natural Aquaporins through synthetic design[END_REF] and synthesize artificial simpler molecules with similar structural and thus water transport properties, namely artificial water channels, first reported by Barboiu et al. [START_REF] Le Duc | Imidazole-Quartet Water and Proton Dipolar Channels[END_REF] Common artificial water channels such as carbon nanotubes [START_REF] Geng | Stochastic transport through carbon nanotubes in lipid bilayers and live cell membranes[END_REF][START_REF] Kim | Osmotically-Driven Transport in Carbon Nanotube Porins[END_REF][START_REF] Tunuguntla | Enhanced water permeability and tunable ion selectivity in subnanometer carbon nanotube porins[END_REF] (CNTs), imidazole-quartet water channel [START_REF] Le Duc | Imidazole-Quartet Water and Proton Dipolar Channels[END_REF][START_REF] Barboiu | From Natural to Bioassisted and Biomimetic Artificial Water Channel Systems[END_REF][START_REF] Licsandru | Salt-Excluding Artificial Water Channels Exhibiting Enhanced Dipolar Water and Proton Translocation[END_REF] (IQWC) and peptide-appended pillar [5]arene [START_REF] Kumar | Highly permeable polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z[END_REF][START_REF] Hu | Single-Molecular Artificial Transmembrane Water Channels[END_REF][START_REF] Shen | Highly permeable artificial water channels that can self-assemble into two-dimensional arrays[END_REF] (PAP) have been explored during the last decades. Noy et al. [START_REF] Tunuguntla | Enhanced water permeability and tunable ion selectivity in subnanometer carbon nanotube porins[END_REF] indicated that water permeability in 0.8-nanometer-diameter CNTs, which confined water down to a single-file chain, exceeded that AQP. Hou et al. [START_REF] Hu | Single-Molecular Artificial Transmembrane Water Channels[END_REF] , Kumar et al. [START_REF] Shen | Achieving high permeability and enhanced selectivity for Angstrom-scale separations using artificial water channel membranes[END_REF] et al. and Wang et al. [START_REF] Li | Hyperfast Water Transport through Biomimetic Nanochannels from Peptide-Attached (pR)-pillar 5 arene[END_REF] chronologically investigated the molecular transport of water through PAP channels embedded in in bilayer membranes and amphiphilic block copolymers [START_REF] Discher | Polymer vesicles[END_REF][START_REF] Mai | Self-assembly of block copolymers[END_REF] . The PAP channel based on block copolymer membranes showed sharp selectivity for a molecular weight cutoff of ~ 500 Da and achieving large enhancement in permeability reaching about 65 L•m -2 •h -1 •bar -1 . Alternatively, imidazole-quartet water channel artificial water channels [START_REF] Barboiu | From Natural to Bioassisted and Biomimetic Artificial Water Channel Systems[END_REF] are excellent candidates to construct membranes with fast and selective water transport properties, while have not been further realized due to the lack of suitable embedding matrix and membrane fabrication approach. Two-dimensional graphene oxide (GO) nanosheets, can be easily assembled into bilayer structure, and show better mechanical and chemical stability [START_REF] Huang | High-Efficiency Water-Transport Channels using the Synergistic Effect of a Hydrophilic Polymer and Graphene Oxide Laminates[END_REF] than phospholipid bilayers, together with rich functional groups [START_REF] Guan | Incorporating Graphene Oxide into Alginate Polymer with a Cationic Intermediate To Strengthen Membrane Dehydration Performance[END_REF] , making it an ideal platform for incorporating water channel molecules. Recently, Park et al. [START_REF] Kim | Biomimetic Selective Ion Transport through Graphene Oxide Membranes Functionalized with Ion Recognizing Peptides[END_REF] demonstrated that GO membranes functionalized with a peptide motif can realize selective recognition and transport between Co 2+ and Cu 2+ . State-of-art design and fabrication of GO membranes with a surface-tethered peptide RF8 to mimic the water-selective filter of natural AQP were also explored [START_REF] Lee | Facilitated Water Transport through Graphene Oxide Membranes Functionalized with Aquaporin-Mimicking Peptides[END_REF] .

In this work, we selected bola-amphiphile-imidazole compound (IU) [START_REF] Le Duc | Imidazole-Quartet Water and Proton Dipolar Channels[END_REF][START_REF] Licsandru | Salt-Excluding Artificial Water Channels Exhibiting Enhanced Dipolar Water and Proton Translocation[END_REF] embedded into GO interlayer to fabricate biomimetic IU-GO membrane (Fig. 1). The main purpose of this work is to study water transport behavior through the interlayer space resulted from IU-GO membrane. The chemical structure of as-synthesized IU, morphologies and structure of resulting IU-GO membranes were systematically characterized. The water transport properties of the IU-GO membranes were evaluated by dehydration of butanol-water mixtures via pervaporation process. The solvent dehydration performance was further studied by varying the feed concentration, temperature and operating time. with a molar ratio of 1:2 (Fig. 2) using tetrahydrofuran (THF, Shanghai No. 4 Reagent & H.v Chemical Co., Ltd., China) as a solvent [START_REF] Le Duc | Imidazole-Quartet Water and Proton Dipolar Channels[END_REF] . The resulting mixture was refluxed heating at 70 °C for 10 h, and then washed with THF for three times, followed by centrifugation at room temperature. Finally, the product was dried in vacuum at 70 °C for 48 h to receive white powder of bola-amphiphile-imidazole compound: N,N'-hexane-1,6-diylbis(1H-imidazol-4-ethylcarboxamide), further used as molecular scaffold to construct IU-GO membranes.

Membrane Fabrication

Dried IU powder was solubilized in methanol with the concentration of 0.2 mg•ml -1 . Graphene oxide (GO, Nanjing XFNANO Tech Co., Ltd, China) nanosheets with lateral dimension of 2~5 μm and thickness of ~1 nm were dispersed into deionized water to get 0.25 mg•ml -1 suspension by ultra-sonication and centrifugation.

IU and GO were mixed with ice bath as well as stirred for 0.5 h. The resultant mixture was vacuum-assisted filtered by polyacrylonitrile (PAN, Shandong Megavision Membrane Technology & Engineering Co., Ltd, China) [START_REF] Guan | Precisely Controlling Nanochannels of Graphene Oxide Membranes through Lignin-Based Cation Decoration for Dehydration of Biofuels[END_REF] to obtain the pristine GO and IU embedded GO (IU-GO) membranes. According to the O element content analyzed by XPS, the actual IU mass fraction in IU-GO membranes were calculated as 2.4 wt %, 3.1 wt %, 3.8 wt %, 5.2 wt %, 6.7 wt %, 9.0 wt % and 9.3 wt % respectively. 3-6 membrane samples were prepared for GO or each IU-GO membrane.

The deviation of membrane thickness (average error is 7.5 %) was controlled by using the same concentration of suspension for filtration and same filtration time for the fabrication of IU-GO membranes.

Characterizations

Molecular structure and thermal stability of imidazole-ureido were determined by using Nuclear Magnetic Resonance Spectrometry (NMR, AVANCE II 400, Germany) and thermogravimetric (TG, NETZSCH 209F1, Germany) from 30°C to 200 °C with a rate of 10 °C•min -1 under N2. Surface morphologies and thicknesses of the membranes were characterized by scanning electron microscopy (SEM, Hitachi Limited S-4800, Japan). Roughness of the membrane surface was obtained from the atomic force microscope (AFM, Bruker Dimension Icon, Korea). A contact angle drop-meter was used to test membrane surface water contact angle (WCA, A100P, MAIST Vision Inspection & Measurement Co., Ltd.). Interlayer spacing of the membrane was examined by X-ray diffraction (XRD, Rigaku Miniflex 600, Japan) at the range of 5°≤ 2θ ≤ 40°. We utilized X-ray photoelectron spectroscopy (XPS, Thermo ESCALAB 250, USA) and Fourier transform infrared (FTIR, Thermo AVATAR-FT-IR-360, USA) spectra with the range of 400-4000 cm -1 to analyze the elements and functional groups of the samples.

Pervaporation Measurement

Pervaporation measurement was conducted by a homemade module. All membranes were supported by a stainless-steel construction. Flow rate of feed solution was fixed with 100 ml•min -1 by a peristaltic pump and the temperature needed to be maintained steady for some time before testing. The pressure of permeate side was below 160 Pa which was controlled by vacuum pump. The permeate vapor was collected at low temperature provided by liquid nitrogen. The separation performance of each sample was tested for at least three times. The deviation of separation performance (average error is 8.7 %) was controlled by applying the same operating conditions such as feed temperature and concentration, downstream pressure, as well as the conditions for measuring the membrane area, permeate weight and concentration. Flux (J, g•m -2 •h -1 ) and separation factor (β) are often used to describe the separation performance of the membrane [START_REF] Guan | Incorporating Graphene Oxide into Alginate Polymer with a Cationic Intermediate To Strengthen Membrane Dehydration Performance[END_REF] . We definite water flux and separation factor as the following:

𝐽 = 𝑀 𝐴•𝑡 (1) 𝛽 = 𝑌 𝑊 𝑌 𝑂 ⁄ 𝑋 𝑊 𝑋 𝑂 ⁄ (2)
M, A and t represent the mass of permeates (g), effective separation area (m 2 ) and collecting time (h) respectively. 𝑌 𝑊 , 𝑋 𝑊 mean the mass fraction of water on the permeate side and feed side. Similarly, 𝑌 𝑂 , 𝑋 𝑂 mean the mass fraction of organic component on the permeate side and feed side.

Calculations of activation energy by the Arrhenius Equation [START_REF] Zhao | Facilitated water-selective permeation via PEGylation of graphene oxide membrane[END_REF] is used to explain the relationship between feed temperature and flux:

𝐽 𝑖 = 𝐽 0 • 𝑒 -𝐸 𝑎 𝑅𝑇 ⁄ (3) While 𝐽 𝑖 (g•m -2 •h -1 ) is the flux of component i; 𝐽 0 (g•m -2 •h -1
) is the pre-exponential factor; 𝐸 𝑎 (kJ•mol -1 ) is the permeation-process-associated activation energy;

T (K) is the operation temperature; R (kJ•mol -1 •K -1 ) is the gas constant, R=8.314 × 10 -3 kJ•mol -1 •K -1 .
We take the log of both side of above equation to get simple calculation formula:

𝑙𝑛(𝐽 𝑖 ) = 𝑙𝑛(𝐽 0 ) - 𝐸 𝑎 8.314 • 1000 𝑇 (4)
And the value of 𝐸 𝑎 can be calculated by the slope of 𝑙𝑛(𝐽 𝑖 ) vs 1000/T curve.

Main factors to influence separation performance include driving force (temperature and concentration) and membrane structure (membrane thickness) [START_REF] Zhao | Facilitated water-selective permeation via PEGylation of graphene oxide membrane[END_REF] . To explore the influence of feed concentration and temperature, the driving force-normalized permeance and selectivity of IU-GO membrane were calculated.

When considering the effect of membrane thickness on the pervaporation performance, permeability Pi is the key parameter to evaluate. The permeance ((𝑃/𝑙), GPU), permeability (Pi, Barrer) and selectivity (α) are obtained by:

(𝑃/𝑙) 𝑖 = 𝐽 𝑖 𝑝 𝑖0 -𝑝 𝑖𝑙 = 𝐽 𝑖 𝑥 𝑖0 𝑦 𝑖0 𝑝 𝑖𝑜 𝑠𝑎𝑡 -𝑝 𝑖𝑙 (5)
𝑃 𝑖 = 𝐽 𝑖 •𝑙 𝑝 𝑖0 -𝑝 𝑖𝑙 = 𝐽 𝑖 •𝑙 𝑥 𝑖0 𝑦 𝑖0 𝑝 𝑖𝑜 𝑠𝑎𝑡 -𝑝 𝑖𝑙 (6

Results and Discussion

Characterization of Imidazole-Ureido

1 H NMR spectroscopy was used to confirm the molecular structure of IU. In this work, 1 H NMR spectra of IU was recorded in d6-DMSO, by using the residual solvent peak as reference to examine the molecule structure of IU. and the observed peaks were in accord with the previously reported results and proposed formula [START_REF] Le Duc | Imidazole-Quartet Water and Proton Dipolar Channels[END_REF] : δ = 1.22 (s, 2H), 1.31 (t, 2H), 2.56 (t, 2H), 2.94 (q, 2H), 3.20 (q, 2H), 5.7 (t, 1H), 5.85 (t, 1H), 6.75 (s, 1H), 7.65 (s, 1H), 11.80 (s,1H) (Fig. 3a) It confirms that IU compound was successfully synthesized in this work.

TG and DTG were also investigated to make sure the synthesized IU can remain stable under certainly high temperature conditions used during application (Fig. 3b). It shows a slight mass loss occurring over a temperature range of 30-100 °C owing to a strong water binding, as previously reported [START_REF] Le Duc | Imidazole-Quartet Water and Proton Dipolar Channels[END_REF] . The mass of compound IU starts to decompose at 180 °C and occurs secondary decomposition at 410 °C, indicating that the IU can keep its self-assembled form below 180 °C and thus can meet the requirements of solvent dehydration process.

Characterization of Membrane

Morphology

Fig. 4 a,d display the surface and cross-section morphology of the pristine GO membrane, displaying a typical wrinkles morphology [START_REF] Liu | Graphene oxide membrane for molecular separation: challenges and opportunities[END_REF] and a membrane thickness of 104±12 nm. embedding bola-amphiphile IU into GO interlayer, the thicknesses of IU-GO membrane was increased 130±10 nm (3.1 wt %) and 153±24 nm (9.3 wt %) respectively, while the laminar structure was well preserved [START_REF] Liu | Graphene oxide membrane for molecular separation: challenges and opportunities[END_REF][START_REF] Zhang | Nanoparticles@rGO Membrane Enabling Highly Enhanced Water Permeability and Structural Stability with Preserved Selectivity[END_REF] . When the mass fraction of IU of membrane was increased from 0 wt % to 9.3 wt %, the membrane surface showed less wrinkles and an increase of local roughness with the addition of bola-amphiphile IU. Besides that, we analyzed 3D AFM figures of pristine GO membrane and IU-GO membrane with different mass fraction of IU (3.1 wt % and 9.3 wt %). Both of the pristine GO membrane and 3.1 wt % IU-GO membrane surface showed typical wrinkled morphology. Nano-bumps were observed on the 9.3 wt % IU-GO membrane surface. The result indicates that IU in the membrane gradually agglomerated to form nanoparticles when the solvent was evaporated. AFM also gave the surface roughness Rq (10.0 nm, 14.7 nm and 15.1 nm) of IU-GO membranes (0 wt %, 3.1 wt % and 9.3 wt %) respectively. According to the WCA test, the hydrophilicity of IU-GO membranes showed a slight decrease compared with pristine GO membrane. Both chemical structure and surface roughness of the membrane surface determine the hydrophilicity [START_REF] Shen | High-performance thin-film composite polyamide membranes developed with green ultrasound-assisted interfacial polymerization[END_REF][START_REF] Xu | Novel thermally cross-linked polyimide membranes for ethanol dehydration via pervaporation[END_REF] . For a hydrophilic material, the hydrophilicity increased with its roughness. Here, a reduced hydrophilicity (slightly higher WCA) was found in the GO membrane with a rougher surface. It suggests that the chemical structure of the membrane surface (long hydrophobic chain alkanes) dominates the membrane hydrophilicity in our case. It would affect the water permeation of IU-GO membrane which will be discussed later.

Chemical and Physical Structures

The chemical structure of GO and IU were investigated by FTIR (Fig. 6) and XPS (Fig. 7), which revealed the molecular interaction between GO and IU. Fig. 6 demonstrates the FTIR results of the pristine GO membrane (red curve), IU-GO membrane (blue curve) and the PAN substrate (black curve). We could clearly figure out peaks of hydroxyl group (3252 cm -1 , -OH), carboxyl group (1736 cm -1 , C=O, and and aromatic ring (C-C, 284.8 eV, accounts for 42.13%). For XPS C1s spectra of IU-GO membrane, we found that carboxyl group could even hardly be detected but carbon-nitrogen bond (C-N, 285.7 eV, 9.99%) was observed. By comparing the XPS N1s spectra of IU with that of IU-GO, we found that the ratio of functional group -NH-CH=N-decreased (from 72.43% to 47.00 %) and the ratio of functional group -NH-C=O-increased (from 27.57% to 53.00%). Therefore, in addition to hydrogen bonding between GO and IU, there are dehydration condensation of the carboxyl group and ring opening reaction of the epoxy group on GO with the pyrrole nitrogen on the imidazole ring of the IU. The physical properties and d-spacing of IU-GO membranes with different IU mass fraction were characterized by XRD (Fig. 8a,b). Three typical peaks represent PAN substrate where 2θ values are equal to 17.60, 22.75 and 25.90, respectively. The d-spacing of GO membrane were calculated from Bragg's Law [START_REF] Kacher | Bragg's Law diffraction simulations for electron backscatter diffraction analysis[END_REF] . It shows that d-spacing of the pristine GO membrane is 0.85 nm, which agrees with reported value [35, 36] . The typical peak gradually shifts towards lower 2θ value with the embedding of the IU into GO interlayer, indicating an enlarged interlayer space. When the IU accounts for 3.1 wt %, d-spacing of the IU-GO membrane is 0.87 nm. Considering the graphene thickness of 0.34 nm, the empty interlayer space of IU-GO membrane for molecular transport is calculated as 0.53 nm. As further increasing the embedding IU content to 9.3 wt %, the d-spacing was reached 1.07 nm. Thus, an empty interlayer space of 0.73 nm was obtained, which is larger than the molecular diameter of most small organic solvents such as ethanol and butanol. which is able to accommodate components larger than its averaged interlayer spacing.

This phenomenon has been reported by many papers studying nanomaterials-intercalated GO membranes [START_REF] Zhao | Facilitated water-selective permeation via PEGylation of graphene oxide membrane[END_REF][START_REF] Huang | Vapor transport in graphene oxide laminates and their application in pervaporation[END_REF][START_REF] Guan | Precisely Controlling Nanochannels of Graphene Oxide Membranes through Lignin-Based Cation Decoration for Dehydration of Biofuels[END_REF][START_REF] Guan | 3D nanoporous crystals enabled 2D channels in graphene membrane with enhanced water purification performance[END_REF] . The dimension of assembled imidazole-quartet water channel (IQWC) are bigger than interlayer spacing:

1.07x0.97 nm, excluding the diameter of C-atoms. We further measured the XPRD data on samples containing constant amount of 20 mg IU and variable amounts of GO to cover the concentration domain of the prepared IU-GO membranes. They are not reminiscent with the formation of lamellar quartet phases observed in crystal structure of the IU, including the imidazole-quartet water channels (IQWC). Based on x-ray single structure analysis they are probably accommodating partial assembled structures of IU-ribbon, which probably can be accommodated by pairs, with the dimensions of 0.65±0.2 nm within the GO interlayer space. They are binding water at their extremities, generating the clustering of water within the membrane Further studies would be carried out to confirm the self-assembly of IU at supramolecular level maintained in the presence of GO nanosheets. distances observed in the x-ray crystal structure of IU (center) [START_REF] Le Duc | Imidazole-Quartet Water and Proton Dipolar Channels[END_REF] and possible models for the embedding of the IU in IU-GO layers (right).

Pervaporation Performance

Effect of IU Mass Fraction

Water transport properties of IU-GO membranes with different IU mass fractions were investigated by measuring pervaporation dehydration performance. The total flux and separation factor are shown in Fig. 9. To normalize the effect of driving force and membrane thickness on the separation performance, permeance, permeability and selectivity are calculated in Table 1. All the membrane samples in Fig. 9 are tested under the same pervaporation conditions (90 wt % butanol-water solution at 313 K) to keep an identical driving force. Meanwhile the membrane thickness was controlled at ~100-150 nm for IU-GO membranes with different IU mass fractions. Thus, the permeability and selectivity follow similar variation trends for flux and separation factor, respectively.

The embedded bola-amphiphile IU into GO membranes would affect the water transport behavior in three possible ways: i) presumed water binding by bola-amphiphile IU to allow highly selective water transport through membrane; ii) enlarged GO d-spacing (XRD result in Fig. 8) to promote the water diffusion rate while reduce the diffusion selectivity; iii) decreased hydrophilicity (WCA result in Fig. 5 g-i) to suppress the water adsorption. The separation factor and selectivity of IU-GO membranes were enhanced by one order of magnitude with introducing IU up to 3.1 wt%, confirming the highly selective transport properties of IU towards water. However, excessive IU (>3.1 wt%) reduced the separation factor and selectivity, due to decrease of diffusion selectivity caused by the enlarged d-spacing, as well as suppressed water preferential adsorption resulted from the lowered hydrophilicity.

The flux and permeance (or permeability) were co-determined by above mentioned IU, d-spacing and hydrophilicity with a complicated way. The flux and permeance would be increased because of the embedded IU and enlarged d-spacing while reduced due to the lowered hydrophilicity with the addition of IU. As increasing the IU mass fraction to 3.1 wt%, ideal water clusters might be gradually formed, together with the enlarged d-spacing, achieving the highest water flux and permeance (or permeability). Additionally, the hydrophobic alkane chains of IU would suppress the water transport simultaneously, leading to the variable flux and permeance (or permeability) for GO membranes embedded with different IU mass fractions.

Effect of Feed Concentration

Pervaporation dehydration performance of organic solvents would largely depend on water concentration in the feed [START_REF] Guan | Incorporating Graphene Oxide into Alginate Polymer with a Cationic Intermediate To Strengthen Membrane Dehydration Performance[END_REF] . Here, we studied the influence of 5 wt %, 10 wt %, 15 wt % and 20 wt % water in n-butanol water mixture at 343 K (Fig. 10) respectively. When the water concentration ranged between 5 wt % ~20 wt %, flux of IU-GO membrane increased from 575 to 3506 g•m -2 •h -1 and separation factor changed from 830 to 4454 at 343 K. It could be found that increasing feed water content significantly promoted both the flux and separation factor of IU-GO membrane. The enhanced water transport can be attributed to enhancement of driving force and membrane swelling in the butanol-water solution with higher water concentration. In order to further understand the effect of feed water concentration on the transport properties, the flux and separation factor was normalized by the driving force as permeance and selectivity, respectively. As shown in Table 2, (P/l) Water and P Water was enhanced with increasing the feed water concentration owing to the swelled membrane structure caused by water molecules adsorbed in the membrane. The properly loosened membrane structure lowers the transport resistance for water molecules, meanwhile is insufficient to facilitate butanol permeation due to the larger molecular size of butanol. In addition, the facilitated penetration of water molecules occupies more water clusters in membrane, thus inhibiting the diffusion of butanol molecules [START_REF] Zhao | Facilitated water-selective permeation via PEGylation of graphene oxide membrane[END_REF] . Hence, the butanol permeance shows a decreased tendency with the increasing feed water concentration and resulted in enhanced selectivity of IU-GO membrane. In polymeric membranes, swelling often happens with increase of feed water content, resulting in a decrease in selectivity [START_REF] Vane | Review: membrane materials for the removal of water from industrial solvents by pervaporation and vapor permeation[END_REF] . The suppressed swelling behavior enabled by the IU-GO structure would allow the membrane application in dehydration of solvent containing a wide range of water content. Generally, the relationship between feed temperature and total flux follows Arrhenius law [START_REF] Zhao | Facilitated water-selective permeation via PEGylation of graphene oxide membrane[END_REF][START_REF] Guan | Precisely Controlling Nanochannels of Graphene Oxide Membranes through Lignin-Based Cation Decoration for Dehydration of Biofuels[END_REF] . Fig. 11 b displays Arrhenius plots of water and butanol permeation through the IU-GO membrane in dealing with 90 wt % butanol-water feed solutions.

Effect of Feed Temperature

According to the calculation, we obtained that permeation activation energies for water and butanol are 16.81 kJ/mol (endothermic) and -27.21 kJ/mol (exothermic) respectively. Water flux was increased when the temperature raised due to the flux was consistent with incremental temperature driving force [START_REF] Liu | Elevating the selectivity of layer-by-layer membranes by in situ bioinspired mineralization[END_REF] . In the same way, the attenuation of butanol flux also relies on the permeation activation energy of butanol, which means the butanol permeation decreased with the temperature. Namely, water was much easier to pass through IU-GO membrane than butanol if the membrane was operated at higher temperature. It explained why both flux and separation factor were improved by increasing the feed temperature. The water selectivity over butanol for IU-GO membrane kept increasing with operation temperature, which indicated butanol encountered higher transport resistance under high temperatures than water.

Like the effect of feed concentration discussed above, the feed temperature affects the PV performance also in two aspects: driving force and membrane structure.

The driving-force-normalized permeance and selectivity were calculated in Table 3 to study the influence of temperature on the intrinsic transport properties of the IU-GO membrane. Both (P/l)Water and (P/l)Butanol were decreased with rising the feed temperature while the selectivity increased. According to the solution-diffusion model (b) (a) [39] , the mass transfer of PV process is governed by sorption and diffusion. At higher temperature, the sorption would be inhibited while the diffusion would be promoted.

The lowered permeance reveals that adsorption process plays a dominant role during mass transfer. Moreover, the IU-GO membrane might show higher adsorption selectivity for water towards butanol which resulted in higher selectivity.

Effect of Operation Time

Continuous pervaporation on the 3.1 wt % IU-GO membrane was investigated in 90 wt% butanol-water solution at 333 K to evaluate the stability of the membrane (Fig. 12). It was noted that prior to long-term performance test, this membrane had been working for over 120 h. During additional 120 h continuous pervaporation separation, the membrane selectivity kept stable and the water concentration in permeate was around 99.5%. According to the XPS analysis, strong interaction was confirmed between GO and IU, which is favorable for achieving this stable separation performance. The stability can be also related to the insolubility of the resulted H bonding aggregates or particles of IU in the membrane. Flux demonstrates a minor decline, from ~1147 g•m -2 •h -1 to ~1000 g•m -2 •h -1 . This kind of decline might result from the metastable GO [START_REF] Kim | Room-temperature metastability of multilayer graphene oxide films[END_REF][START_REF] Kumar | Scalable enhancement of graphene oxide properties by thermally driven phase transformation[END_REF] . The O/C ratio of GO will decrease to fixed value of 0.38 within the relaxation time and final reaches a quasi-equilibrium state [START_REF] Zhao | Facilitated water-selective permeation via PEGylation of graphene oxide membrane[END_REF] . High temperature accelerates the reduction of GO compared to room temperature and leads to the minor decline of flux. While after that, IU-GO membrane showed stable separation performance. and GO-based membranes [START_REF] Huang | High-Efficiency Water-Transport Channels using the Synergistic Effect of a Hydrophilic Polymer and Graphene Oxide Laminates[END_REF][START_REF] Tsou | Effect of microstructure of graphene oxide fabricated through different self-assembly techniques on 1-butanol dehydration[END_REF][START_REF] Chen | Fabrication of graphene oxide composite membranes and their application for pervaporation dehydration of butanol[END_REF] for butanol dehydration performance reported in literature [START_REF] Vane | Review: membrane materials for the removal of water from industrial solvents by pervaporation and vapor permeation[END_REF][START_REF] Liu | Pervaporation Membranes for Biobutanol Production[END_REF] . Operation conditions of each membrane reported in Fig. 13 are listed in Table 4. In general, compared with polymeric membranes, IU-GO membrane displays higher flux and separation factor, while shows lower separation performance compared with inorganic membranes. As discussed above, the assembly manner and hydrophilicity of IU significantly affect the separation performance, which requires further optimization in future study.

Fig. 1

 1 Fig.1 Schematic diagram of designed IU-GO membrane structure

Fig. 2

 2 Fig.2 Synthesis of bola-amphiphile-imidazole compound (IU)

  𝑃/𝑙) 𝑖 is the permeance of component i; ( (𝑃/𝑙) 𝑊 and (𝑃/𝑙) 𝐵 represent permeance of water and butanol respectively); 𝑃 𝑊 and 𝑃 𝐵 represent the permeability of water and butanol respectively, l is the membrane thickness (μm); pi0-pil is the total driving force for transport of component i; pi0 and pil are the vapor pressure of component i on the feed and permeate side, respectively; pil is considered as 0 due to the low pressure of the permeate side (close to the vacuum, ~160 Pa); xio and yio represent the activity coefficient and mole fraction of component i of the feed respectively, and 𝑝 𝑖𝑜 𝑠𝑎𝑡 is the saturated vapor pressure (Pa) of the pure component i at a certain temperature.

Fig. 3 (

 3 Fig.3 (a) 1 H-NMR spectrum and (b) TG-DTG curves of the synthesized IU

  Fig.4 b,e and Fig.4 c,f represent the SEM images of IU-GO membranes with different mass fraction of IU (3.1 wt % and 9.3 wt % respectively). After

Fig. 4

 4 Fig.4 SEM images of (a),(d) GO membrane (b),(e) IU-GO membrane with 3.1 wt % IU and (c),(f) IU-GO membrane with 9.3 wt % IU;

Fig. 5

 5 Fig.5 AFM and WCA images of (a), (d), (g) GO membrane (b), (e), (h) IU-GO membrane with 3.1 wt % IU and (c), (f), (i) IU-GO membrane with 9.3 wt % IU

1359 cm - 1 ,Fig. 6 FTIR

 16 Fig.6 FTIR spectra of IU-GO membrane, GO membrane and PAN substrate As revealed by XPS spectra in Fig.7 a-d, C and N elements were analyzed to further study the chemical structure of GO and IU. Typical peaks of GO were listed as carboxyl group (-O-C=O, 289.4 eV, accounts for 2.39%), carbonyl group (C=O, 288.4 eV, accounts for 7.08%), epoxy group (C-O-C, 286.9 eV, accounts for 48.4%)

Fig. 7

 7 Fig.7 XPS spectra for C1s of (a) GO membrane and (b) IU-GO membrane; N1s of (c) synthesized IU and (d) IU-GO membrane

  From the XRD, we can obtain an averaged interlayer spacing (< 1 nm) of the GO-based membrane. While the flexible GO nanosheets can easily generate local bulges (typical wrinkles morphology of GO membrane surface shown in Fig 4a),

Fig. 8 (

 8 Fig.8 (a) XRD patterns and (b) d-spacings of GO membrane and IU-GO membrane with different mass fraction of IU (c) XRD patterns of IU-GO powders (left) the measured main

Fig. 9

 9 Fig.9 Butanol dehydration performance of GO membrane and IU-GO membranes fabricated in different mass fraction of IU (90 wt % butanol-water at 313 K)Table 1 Permeance, permeability and selectivity of IU-GO membrane fabricated in different mass fraction of IU in 90 wt % butanol-water solution

Fig. 10

 10 Fig.10 Effect of feed water concentration on the butanol dehydration performance at 343 K

Fig. 11 aFig. 11

 1111 Fig.11a shows the influence of feed temperature (313 K, 323 K, 333 K, 343 K) on the dehydration performance of IU-GO membrane with IU mass fraction of 3.1 wt %. Both flux and separation factor were increased as the feed operation temperature raised. Specifically, when the temperature of feed solution was 343 K, the flux and separation factor rose up to 1468 g•m -2 •h -1 and 4601 respectively. This phenomenon is favorable for the application of IU-GO membrane since its separation efficiency can be enhanced by simply increasing the feed temperature.

Fig. 12

 12 Fig.12Effect of operation time on butanol dehydration performance of IU-GO membrane in dealing with 90wt % butanol-water feed solutions at 333 K Fig.13concludes common polymeric membranes (polyimides, cellulose, poly(vinyl alcohol) and polyacrylonitrile), inorganic membranes (zeolite and silica)

  Fig.13PV performance of membranes for butanol dehydration reported in literature

4 Conclusions

 4 In summary, inspired by natural fast water transport channel AQP, we introduced the molecular bola-amphiphile imidazole compound (IU) into the layered structure of assembled GO laminate to fabricate a new type of IU-GO membrane. IU-GO membrane demonstrated high water transport selectivity over butanol due to the introduction of IQWC. The total flux and separation factor of the optimized IU-GO membrane were 3506 g•m -2 •h -1 and 4454 respectively for pervaporation dehydration of 80 wt% butanol-water solution at 343 K. The IU-GO membrane exhibited a simultaneous enhancement in flux and separation factor with increasing feed water concentration and temperature, together with a stable performance during continuous operation, showing great potential in solvent dehydration. The explored molecular transport behavior through IU-GO membrane would also inspire design and fabrication of GO-based membranes for organic separation in future.

Table 2

 2 Permeance, permeability and selectivity of IU-GO membrane at 343K under different

		feed water concentration
	Water concentration	(P/l) Water	(P/l) Butanol	P Water	P Butanol
						𝛼
	/ wt%	/ GPU	/ GPU	/ Barrer	/ Barrer
	5	2507	2.21	326	0.29	1134
	10	2742	1.76	356	0.23	1558
	15	4451	1.70	579	0.22	2618

Table 3

 3 Permeance, permeability and selectivity of IU-GO membrane under different feed

		temperatures in 90 wt % butanol-water feed solution
	Temperature	(P/l) Water	(P/l) Butanol	P Water	P Butanol
						𝛼
	/ K	/ GPU	/ GPU	/ Barrer	/ Barrer
	313	8971	5.01	1166	0.65	1791
	323	7226	1.92	939	0.25	3764
	333	5179	0.78	673	0.10	6640
	343	3726	0.54	484	0.07	6900

Table 4

 4 Operation conditions of each membrane demonstrated in Fig.13

		Membrane	T /°C	Butanol /wt %	Flux /g•m -2 •h -1	Separation factor	Reference
		PAI/PEI	60	85	846	1174	[44]
	Polymer membranes	PI-ZrO 2 CS PVA	40 30 60	90 95 90	140 210 950	109 2657 140	[45] [46] [47]
		HPAN	30	70	2030	99	[48]
		NaA zeolite	70	90	2700	8000	[49]
	Inorganic membranes	A-type zeolite T-type zeolite Hybrid silica	75 75 95	90 90 95	1370 1700 1800	90000 18000 1100	[50] [50] [51]
		Tubular silica	70	95	4500	600	[52]
	GO based membranes	GO PASA CS@GO GO/AAO	30 70 50	90 90 90	2540 10000 3100	2241 1523 230	[39] [27] [42]
		IU-GO	40	80	1948	1817	This work
	IU-GO	IU-GO	60	90	1318	4352	This work
		IU-GO	70	80	3506	4454	This work

* PAI means polyamide-imide; PEI means polyetherimide; PI-ZrO2 represents mixed-matrix membranes based on polyimides with ZrO2 nanostars; CS represents chitosan membrane; PVA represents means poly(vinyl alcohol) membrane; HPAN represents hydrolyzed polyacrylonitrile membrane.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (21776125, 21490585, 51861135203, 21922805), the Innovative Research Team Program by the Ministry of Education of China (grant No. IRT_17R54) and the Topnotch Academic Programs Project of Jiangsu Higher Education Institutions (TAPP).


Maëlle Cartel

Christine Didier
email: christine.didier@inserm.fr

Regulation of CHK1 by the Ubiquitin-Proteasome System

Keywords: CHK1, Ubiquitin Proteasome System ATR, ataxia telangiectasia and Rad3-related; CHFR, checkpoint

The checkpoint kinase 1 (CHK1) is a master regulator of genome integrity in vertebrate cells. Despite its important cell cycle functions, its regulation is still incompletely understood. Cassidy et al. provide novel insights on the regulation of the CHK1 abundance by the HECT E3 ligase HUWE1 during unperturbed cell cycle as well as in response to replicative stress. These results may help us to apprehend the underlying mechanism of tumorigenesis.

Introduction/Main text/Conclusion-Discussion

Maintenance of genome integrity is crucial for the development, homeostasis, and survival of all organisms, and essential for restricting tumorigenesis. In response to DNA damage, a complex network of proteins sense and mediate repair of damaged DNA or apoptosis. Serine threonine checkpoint kinase 1 (CHK1) is a central protein involved in the DNA damage response (DDR). Its phosphorylation by the upstream kinase ataxia telangiectasia and Rad3related (ATR) induces its activation in response to DNA damage, and its subsequent phosphorylation of cell cycle substrates leads to DNA repair, cell cycle arrest and cell death in the case of excessive DNA damage [START_REF] Guo | Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts[END_REF]. Moreover, DDR unrelated functions of CHK1 during DNA replication and mitosis have also recently been described [START_REF] Petermann | Chk1 promotes replication fork progression by controlling replication initiation[END_REF][START_REF] Zachos | Chk1 is required for spindle checkpoint function[END_REF]. This confirms the dual functions of CHK1 and reinforces the attractive notion that CHK1 is a "molecular switch" from normal cell cycle progression to checkpoint arrest. Significant progress has recently been made in our understanding of CHK1 regulation and its implications in cancer etiology and therapy (4). However, areas of darkness remain regarding the regulation of the expression and activation of CHK1, despite its important cell cycle functions. In depth studies are needed to establish a more integrated view of CHK1 regulation modes depending on the cellular context, normal cell proliferation or under various types of damage and stress. The work of Cassidy et al. is part of this research and describes for the first time the participation of HUWE1, a HECT domain-containing ubiquitin E3 ligase, in the regulation of the stability of CHK1 in the unperturbed cell cycle and in response to replication stress. It is well known that CHK1 protein levels are tightly regulated by the ubiquitin-proteasome system and any deregulation can have important consequences in terms of proliferation, tumor development and resistance to therapies. To date, two Cullin RING Ligase (CRL) complexes E3s have been described to control CHK1 abundance including the SKP1-Cullin1(Cul1)-Fbx6 complex, preferentially ubiquitylating CHK1 in the nucleus [START_REF] Zhang | The F box protein Fbx6 regulates Chk1 stability and cellular sensitivity to replication stress[END_REF], and the other forming the CDT2-Cullin4A(Cul4A)-DDB1 complex preferentially targeting CHK1 in the cytoplasm (6) (referee to Figure 1). However, questions still remain as to the subcellular localization, and the cell cycle phases during which these various E3 ligase complexes intervene. To this purpose, it would be interesting to investigate whether the stability of CHK1 protein is co-ordinately regulated by these multiple E3 ligase complexes and whether crosstalks exist between HUWE1 and other E3 ligases involved in CHK1 regulation, which have probably not yet been identified. Furthermore, although these E3 ligases are mainly involved in the addition of K48-linked polyubiquitination, HUWE1 is a multifaceted HECT domain-containing ubiquitin E3 ligase, which catalyses both mono-ubiquitination and K6-, K48-and K-63-linked poly-ubiquitination of its substrates. It would be interesting to specify the context and the conditions that favour the addition of K48 versus K9 or K63 linkages on CHK1 protein, as the latter linkage types create specific poly-ubiquitin topographies involved in a wider variety of regulations, including signal transduction, protein localization, DNA repair, endocytosis, and protein-protein interaction [START_REF] Kao | Ubiquitination by HUWE1 in tumorigenesis and beyond[END_REF]. Furthermore, these poly-ubiquitination linkage topographies may depend on the phosphorylation status of the CHK1 protein. On this specific aspect, Cassidy et al. demonstrate that HUWE1 preferentially targets active CHK1 (S296 auto-phosphorylated form, referee to Figure 1B and7) as the poly-ubiquitinated lysines identified are mainly located in regions of the CHK1 protein that are known to be inaccessible in its closed-inactive conformation (referee to Figure 5C and Table 1). Interestingly, these poly-ubiquitination modifications on CHK1 by HUWE1 E3 ligase are preferentially K48-linked poly-ubiquitin chains, targeting the kinase for proteasome degradation, K6 and K63 ubiquitin linkages have also be detected, consistent with previous analyses [START_REF] Heidelberger | Proteomic profiling of VCP substrates links VCP to K6-linked ubiquitylation and c-Myc function[END_REF][START_REF] Michel | Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling[END_REF]. This opens the way for exciting and potential new mechanisms of CHK1 regulation. Moreover, whether other post-transcriptional modifications, partners or adaptor proteins, such as claspin are required for this ubiquitinylation should be investigated in the near future, as suggested by observations from Cassidy et al. (referee to Figure 7). Ubiquitination modification is a tightly regulated dynamic process, and is antagonized by deubiquitylating enzymes (DUBs). These enzymes can interact with E3 ligases and their substrates and thus regulate their stability and/or their addressee to the proteasome. At least 3 Ubiquitin Specific Proteases (USP), USP1, 3 and 7, have been reported to be able to cleave the ubiquitin chain on CHK1 and promote its stabilization [START_REF] Guervilly | USP1 deubiquitinase maintains phosphorylated CHK1 by limiting its DDB1-dependent degradation[END_REF][START_REF] Cheng | Deubiquitinating enzyme USP3 controls CHK1 chromatin association and activation[END_REF][START_REF] Vega | USP7 controls CHK1 protein stability by direct deubiquitination[END_REF], referee to Figure 1). It would be interesting (I) to understand the regulatory mechanisms involved that dictate protein fate; (II) to identify whether a specific deubiquitylating enzyme interacts and works together with HUWE1 E3 ligase, as it has been shown for USP7 and the E3 ubiquitin ligase, Mouse Double Minute 2 homolog, MDM2, and the mitotic E3 ubiquitin ligase, checkpoint with forkhead and Ring-finger, CHFR [START_REF] Li | A dynamic role of HAUSP in the p53-Mdm2 pathway[END_REF][START_REF] Giovinazzi | USP7 and Daxx regulate mitosis progression and taxane sensitivity by affecting stability of Aurora-A kinase[END_REF]. Furthermore, the work of Cassidy et al. provides interesting data on the modes of regulation of CHK1 protein stability. Authors have shown that CHK1 itself could control its stability by phosphorylation of the last 60 amino acids of the HECT domain of the HUWE1 E3 ubiquitin ligase (referee to Table 3). It would be important to establish whether CHK1 is able to prime and regulate its own ubiquitinylation in specific contexts, such as unperturbed conditions or in response to a replicative stress. Finally, CHK1 has been found to be overexpressed in a variety of human tumors, and plays an essential role in cellular proliferation and resistance to replicative stress [START_REF] López-Contreras | An extra allele of Chk1 limits oncogene-induced replicative stress and promotes transformation[END_REF]. On the other hand, HUWE1 is frequently overexpressed in solid tumors, but can be downregulated in brain tumors [START_REF] Kao | Ubiquitination by HUWE1 in tumorigenesis and beyond[END_REF]. Depending on the substrate and the context, HUWE1 is implicated in controlling proliferation and differentiation, apoptosis, DNA repair, and responses to stress. Understanding the cell-specific functions of HUWE1 and its relationship with the CHK1 deserves to be investigated particularly in response to DNA damage conditions. In agreement with this point, Cassidy et al. reported that the interaction between CHK1 and the HECT domain of the HUWE1 E3 ligase is strengthened in response to replicative stress (Figure 7B). Altogether, Cassidy et al., provide interesting new reports and information on the regulation of the CHK1 protein by the HUWE1 E3 ligase and highlight for the first time an auto-regulatory mechanism controlled by CHK1 itself that may help us to understand the underlying mechanism of tumorigenesis.

Figure Legends

Regulation of CHK1 by the Ubiquitin Proteasome System and cellular fate. Several transcriptional and post-transcriptional pathways that regulate CHK1 protein have been described, including mechanisms that positively or negatively affect its stability, activity, localization and protein-protein interactions. Deregulation of the ubiquitination and deubiquitination process may underlie the heterogeneity of CHK1 expression observed in tumors. CHK1 is degraded by the Ubiquitin-Proteasome System. Ubiquitin E3 ligases involved in CHK1 ubiquitination and degradation during normal cell cycle progression or in response to DNA damage include two Cullin RING Ligase (CRL) complexes (CDT2-CUL4-DDB1 and SKP1-CUL1-Fbx6) and the HECT E3 ligase HUWE1. Inversely, stabilization of CHK1 by the Ubiquitin Specific Proteases (USP), USP1, USP3 and USP7 has also been described.

Acknowledgements

The authors would like to thank fellow group members, and Dr Stéphane Manenti for ongoing support and stimulating insights.

Authors Contributions

MC and CD wrote the manuscript.

Conflict of interest

The authors declare no conflict of interest.