Biomass waste dissolution with imidazole ionic liquids for biohydrogen production

Cellulosic biomass waste from municipal solid and agricultural biomass residue are Second Generation energy source, mainly contain glucose and xylose monomers, were`extensively studied in present research for fermentable sugar hydrolysate in biohydrogen production. Scrap paper cup (SPC), bamboo stem wall (BSW), sugar cane bagasse (SCB) and oil palm empty fruit bunch (OPEFB) were dissolved in laboratory prepared imidazole ionic liquids; 1-allyl-3-methylimidazolium chloride ([Amim]Cl) and 1-butyl-3-methylimidazolium chloride ([Bmim]Cl). A comparative study on biomass composition was presented by Van Soest and thermalgravimetric analysis (TGA) method. TGA was proved as comparative, cheaper and faster method in measuring the lignoellulose composition. Experimental result show that the ionic liquids were completely dissolved the SPC, BSW, SCB and OPEFB with high cellulose recovery; 96.00%, 91.34% 87.16% and 99.51% respectively. The used ionic liquids were highly recovered from the mixture at 94% to 99% recovery rate and FTIR analysis proofed that the recycled ionic liquid is principally consistent with the original. The regenerated cellulose was undergo acid hydrolysis to reducing sugars (glucose/xylose) hydrolysate to be used as feedstock fermentation for biohydrogen production. Acid hydrolysis of the recovered cellulose resulted up to 96% sugar conversion. IL-SPC hydrolysate reported higher total sugar conversion compare to SPC (control) due to higher surface area and disintegration of the cellulosic bril structure resulted from the dissolution process. IL-SPC, IL-BSW, IL-SCB and IL-OPEFB hydrolysate contained higher total sugar compared to SPC hydrolysate even though their cellulose recovery are lower that the SPC (control). Biohydrogen fermentability test of this hydrolysate was carried out using biohydrogen producing bacterium Clostridia sp. Almost 85% of biomass waste hydrolysate substrate was utilized by the bacteria. Up to 196 ml H 2 / 100 ml cumulative bioydrogen production was collected for fermentation using the biomass hydrolysate while 174.91 ml H 2 / 100 ml was produced from the control.


Introduction
Circular economy is an international trend, and how to utilize waste to high-value products is a big-pro le issue. Bio-hydrogen is one of attractive future energy carrier compared to other biofuel due to its high energy density, low energy input and higher conversion e ciency to usable power including its nonpolluting nature (Dutra et al., 2017). Scrap paper cup (SPC) was widely used all over the world and become a threat to the environment. It composed of 90% high strength paper with 5% thin coating of polyethylene to make the paper containers waterproof but make it very complicated to recycle (Arumugam et al., 2017). Agriculture waste which is plentiful and readily available, is categorized as lignocellulosic biomass. It has complex molecular structure with tangled chain of cellulose, hemicellulose and lignin (Kim, 2018). Hydrolysis of the biomass and SPC can be used as source of fermentable sugar for biohydrogen production (Harun et al., 2013, Kim, 2018. However, the complex lignocellulose biomass structure with intra and intermolecular hydrogen bonds make the hydrolysis of the biomass and SPC become a major challenge (Xing et al., 2014). Therefore, this paper exploring an environment friendly preprocessing step of the SPC and agricultural biomass, to provide e cient process of cellulose conversion to fermentable sugar hydrolysate for biohydrogen production.
The difference composition of chemical components in biomass waste is directly in uence their chemical reactivity. This is why determination of the total amount of each lignocellulose components is crucial to foresee the e ciency of a biomass conversion process (Carrier et al., 2011). There are several established composition analysis standard methods (Ioelovich, 2015) that applied at lignocellulose based research area such as National Renewable Energy Laboratory (Sluiter et al., 2008), Technical Association of the Pulp and Paper Industry, Scandinavian Pulp, Paper and Board (Scandinavian, 2009), Van Soest method (Van Soest et al., 1991) and Thermogravimetry analysis (TGA) that primarily introduced by American Society for Testing and Materials (Earnest, 1988). Since different methods may give different results, this study presented comparison of lignocellulose biomass composition determined by using Van Soest and TGA method. Van Soest also known as detergent ber analysis where the lignocellulose ber fractioned into neutral detergent ber (NDF), acid detergent ber (ADF) and acid detergent lignin (ADL). In this method, NDF is the raw materials removed protein, fat and other extracts, which mainly includes cellulose, hemicellulose, lignin and ash. ADF mainly containing cellulose, lignin and ash while ADL mainly consists of lignin and ash. By difference, hemicellulose (NDF-ADF), cellulose (ADF-ADL) and lignin (ADL-Ash) are calculated (Hindrichsen et al., 2006). Compare to wet chemical method by Van Seost, TGA is an analytical technique, monitoring fraction material weight degradation that occurs as sample is heated at constant rate. TGA can quantitatively measure the components of complex mixtures because of ability to determine characteristic thermal decomposition each one of them. The composition of cellulose, hemicellulose, lignin and others from the TGA were calculated as the literature on pyrolysis of lignocellulose components as reported by Yang et al. The research report on pyrolysis of individual and mixtures components of cellulose, hemicellulose and lignin. Simplex-lattice approach was used for analysis of both approach and linear relationship occurred between the weight losses of each lignocellulose components during pyrolysis. From the study, rst weight loss of biomass occurred at room temperature to 100 °C, indicate water content, hemicellulose started the weight loss at temperature 220 to 315 °C, follow by cellulose that degraded at temperature from 315 to 400 °C, while lignin have widest temperature range of degradation which at 100 °C to 900 °C (Yang et al., 2006 Ionic liquids have received extensive attention as a promising solvent in dissolving cellulose under unpressurised condition. Ionic liquids are organic salt composed of anions and cations. They usually melt below 100 °C and also recognized as "green" solvent. Application of ionic liquids have several advantages such as low vapor pressure, high polarity, good dissolving and extracting ability, wide liquid range, good thermal stability and excellent design ability (Xia et al., 2018). Ionic liquids provide excellent characteristics in chemical processes which able to dissolve polar and non-polar organic, inorganic and polymeric compounds. The presence of anion in the ionic liquid can extensively disrupt the hydrogen bonding interactions in the three dimensional network of lignocellulose, leading to dissolution of biomass components. Besides that, the ionic liquids can be recovered after the dissolution process (Singh et al., 2018). Formerly, several literatures were reported utilization of variety ionic liquids for the dissolution of However, dissolution of scrap paper cup by ionic liquids for preparation of fermentable sugar hydrolysate is a novel, presented in this paper.
There were many types of ionic liquid that was studied especially in cellulose dissolution. Imidazole cations ionic liquid has reported could dissolve lignocellulose biomass (Singh et al., 2018;Xia et al., 2018). Therefore, in this study two imidazole based ionic liquids were prepared in laboratory; 1-allyl-3methylimidazolium chloride ([Amim]Cl) and 1-butyl-3-methylimidazolium chloride ([Bmim]Cl). The difference between the two ionic liquids are [Amim]Cl contained C = C structure on allyl chain of the imidazole ring while [Bmim]Cl consist of C-C butyl chain (Xing et al., 2014). The ionic liquids are analyzed by FTIR to con rm the target ionic liquid has synthesized. The ability and e ciency of prepared [Amim]Cl and [Bmim]Cl are determined by monitoring the regenerated cellulose/hemicellulose recovery and ionic liquid recycled recovery.
Most organisms cannot directly consume cellulose/hemicellulose because of its complex structure and high molecular weight. In order to convert cellulose/hemicellulose into a carbon source that can be utilized by organisms, hydrolysis is an indispensable step in bioenergy. Acid hydrolysis is one of promising method for degradation of cellulose/hemicellulose into monosaccharides or destroy the ber structure by cut the glycosidic bond composed of cellulose/hemicellulose (Xiang et al., 2003). The biohydrogen production was carried out by dark fermentation, and used the mixed consortium of Clostridia sp. The experiments show that green processes is a feasible way to make the biomass waste to energy.

Experimental Method
The experiments were design by several steps to work out a green process of recovering cellulose/hemicellulosse from municipal solid waste SPC; and plant biomass: BSW, SCB, and OPEFB, to be used as sugar feedstock in biohydrogen production as shown at Fig. 1. I. A control step was established to distinguish the effect of ionic liquids on the acid hydrolysis and biohydrogen production by directly hydrolyzed the biomass waste without ionic liquid treatment. SPC was hydrolysed with 10M sulfuric acid at 65 °C for 50 min, then the obtained hydrolysate was used as the feedstock for hydrogen production. Biomass composition analysis by Van Soest and TGA method Biomass composition was analyzed by analytical method develop by Van Soest et al. (1991) and compared with TGA method. Two different biomass were used in the study; BSW and SCB. In this study, the biomass were analyzed by Van Soest standard method as reported at Van Soest et al., (1991). Analysis of the cellulose, hemicellulose, and lignin contents of the biomass were determined synchronously. It involved preparation of natural detergent ber (NDF), acid detergent ber (ADF), acid detergent lignin (ADL) and ash content determination. NDF is the raw materials removed protein, fat and other extracts, which mainly contained cellulose, hemicellulose, lignin and ash. ADF is the acid detergent ber that mainly containing cellulose, lignin and ash. Thus, hemicellulose content can be determined by minus the NDF and ADF while the cellulose composition calculate as ADF -ADL. As ADL is mainly consists of lignin and ash, lignin composition can be found by minus the ash amount from the ADL.

Synthesis of ionic liquids
The preparation of ionic liquids are a modi ed method of Xing et al., (2014). For the synthesis of [Amim]Cl, 1-methylimidazole and allyl chloride were added to a three-necked ask, the temperature was controlled at 55 °C for 8 hours, and then the excess allyl chloride and impurities were distilled off to give pure [Amim]Cl.
[Bmim]Cl was synthesized with combination of 1-methylimidazole and 1-chlorobutane in three-necked ask and kept the temperature at 85 °C for 24 h. After that, it was cooled to room temperature before wash with ethyl acetate and concentrated. Then, the pure nal product was obtained by lyophilization. Schematic diagram of preparation of the ionic liquid was presented at Fig. 2.

Dissolution of biomass with ionic liquids
The ionic liquids were heated to 120 °C while stirring, and 10 wt% of biomass waste SPC, BSW, SCB and OPEFB were added gradually into the ionic liquids individually.

Cellulose recovery and ionic liquids recycle
After completed the dissolution process, water was added to the mixture to precipitate the cellulose/hemicellulose. Two apparent layer was formed and the upper layer was separated from the mixture for further lignin and ionic liquid recovery. The precipitated cellulose/hemicellulose was further washing with deionized water on pre-weighed stainless steel lter paper by suction ltration. While washing, the precipitate cellulose/hemicellulose turned to clear solution. The obtained cellulose/hemicellulose was further washed with hot water to completely remove the ionic liquid. The cellulose/hemicellulose recovery was calculated as Equation

Analysis
Composition of the SPC and agricultural biomass were analyzed by thermogravimetric analysis (TGA) brand TA Instrument TGA2950. The physical morphology of biomass waste, before and after dissolution by ionic liquid were gured out by scanning electron microscope (SEM) Hitachi S-3400H analysis. The synthesized ionic liquid [Amim]Cl and [Bmim]Cl were analysed by fourier transform infrared spectroscopy (FTIR) (JASCO FTIR 460 Plus) to con rm the target ionic liquids were produced. After the acid hydrolysis, the composition of hydrolysate was measured by high performance liquid chromatography (HPLC) Brand Hitachi with ICESep Coregel 87H3 column to determine the amount of primary monosaccharides (glucose, xylose, arabinose, sucrose), the metabolites (butyric acid and acetic acid) and other fermentation inhibitor (formic acid, propionic acid,and furfural). Biogas constituents were analysed using gas chromatography (GC) with thermal conductivity detector (SHIMADZU GC-14B). Phenol-sulfuri acid method by UV/VIS spectrophotometer (HITACHI U-5100).

Results And Discussion
Characterization of ionic liquid The laboratory synthesized ionic liquids were analyzed with FTIR to characterize the target products. [Bmim]Cl spectrum at Fig. 3(a)  The functional groups and their wavelengths of [Amim]Cl were showed in Fig. 3(b). Imidazole ring skeletons vibration absorption peaks indicated at peak 3150 cm − 1 (alkyl group), 1430 cm − 1 and 1570 cm − 1 (C = C stretching vibration) and 1165 cm − 1 (HCC). 1639 cm − 1 was the peak for 1 C = C, allyl group while peak at 951 cm − 1 was the R-CH = CH-H. According to the analysis results, the speci c functional groups are consistent with the results analyzed by and the results proved that the target product was successfully synthesized.

Comparison of biomass composition by Van Soest and TGA method
The biomass composition analysis by Van Soest and TGA were compared and showed at  Biomass waste composition by TGA method All the biomass waste's chemical compositions were analyzed with TGA method. According to the literature on pyrolysis lignocellulose components, hemicellulose started the weight loss at temperature 220-315 °C, follow by cellulose that degraded at temperature from 315 to 400 °C, while lignin have widest temperature range of degradation which at 100 °C to 900 °C. Figure 4(a) shows the pyrolysis curve of scrap paper cups (SPC) in TGA analysis. The rst weight loss of SPC occurred at room temperature (RT) to 100 °C, it accounts for 7.52% of total weight, which indicate the water content in the SPC. From the pyrolysis curve, it was determined the hemicellulose and cellulose composition of SPC which are 58.50% and 17.17% respectively. There might be some weight of lignin together with the composition of cellulose and hemicellulose because of the wide degradation of lignin. Nonetheless, as the degradation of lignin is very low mass loss rate (< 0.14 wt%/°C) the amount can be negligible. The SPC pyrolysis curve has comparable study of Dikobe and Luyt (2010) which reported on the properties of PP/LLDPE/wood composites polymer. There were identi ed on the SPC pyrolysis curve, 11.46 wt% weight loss at 400-500 °C and this signal is visibly known as linear low-density polyethylene (LLDPE) material. Above 500 °C, some residual was decomposed and identi ed as ash. The composition of scrap paper cup, rice husk, sugar cane bagasse and oil palm empty fruit bunch were shown in Table 3 to dissolved completely the SPC, BSW, SCB and OPEFB biomass waste at temperature 120 °C. The complete dissolution of the biomass waste was con rmed though scanning electron micrograph (SEM) images as shown at can indirectly in uence the dissolving ability by impacting their physical properties, such as melting point, density and viscosity. The researcher also found that weak hydrogen bond present between the oxygen atom of cellulose and the acidic hydrogen ring ofcation. Small van der Waals interaction formation between of glucose and cations. Lignin has well-known properties as water impermeability, hydrophobicity and lm-forming ability. Once the lignocellulose biomass was dissolved, it can be fractionated to its principle component by adding anti-solvent. In this experiment, water was used as the anti-solvent. The dissolution of lignin is more tolerant to water compared to the dissolution of cellulose. High solubility of lignin can be achieved in the mixture of ionic liquids and water, while the presence of water has negative impact on the dissolution ability of cellulose in ionic liquid. They also found that addition of applicable amount of water to ionic liquid can signi cantly increase the lignin solubility. This is due to the increased interaction probability between lignin and free ions, which the amount and mobility were remarkably increased after the addition of water (Hou et al., 2017). Thus, addition of water in a complete dissolution of lignocellulose with ionic liquid is applicable for fractionation of cellulose, lignin and the ionic liquid recyclability. The lignin separated from lignocellulose biomass is not only a desirable for preparation of lignin-free renewable feedstock for the production of bioenergy, but the extracted lignin also can be a promising starting material for production of novel materials. Therefore, the extraction of lignin from biomass has greatly improve the biore nery productivity and pro tability.
Further washing of cellulose solution resulted a smooth and transparent layer of regenerated cellulose as shown at Fig. 5 (a). Theoretically, the regeneration of cellulose was started when water is added in the dissolve cellulose with ionic liquid. This is because, the hydrogen bonds that formed during dissolution process, between the hydroxyl group of cellulose and Cl − anions are dismissed by adding more H 2 O. The regenerated cellulose was converted to cellulose II from cellulose I in original raw biomass. Addition of water was promoted formation of hydrogen bond of water with anions Cl − and resulted the hydrogen bonds between cellulose are connected again leading to precipitation (Gupta & Jiang, 2015). It was observed that the surface morphology and cellulose structure of the cellulose was change signi cantly.
Subsequently, relative homogeneous textures were displayed as shown in SEM images (Fig. 5b, d, f, h). As much as ionic liquid cost is important, the ionic liquid recycling rate is a key variable for the process economics perspective. In this experiment, the recycled ionic liquids were highly recovered by vacuum distillation. The ionic liquid recovery rate after dissolution of SPC, BSW, SCB and OPEFB were 94.0%, 95.04%, 97.12% and 98.15% individually. The purity of the recycle ionic liquids were proof by FTIR analysis at Fig. 7. The result shows that the FTIR spectra of recycled ionic is principally consistent with the original. The SPC, IL-SPC, IL-BSW, IL-SCB and IL-OPEFB were hydrolyzed by 10 M sulfuric acid at temperature 65 °C to produce fermentable reducing sugar. In the acid hydrolysis process, glycosidic bonds between the cellulose molecules are break to form monosaccharides. Literatures reported that, compare to other method of hydrolysis, acid hydrolysis promising higher sugar yield and good reproducibility. However, the method will produce large amount of degradation products from the monosaccharids such as furfural generated from pentose and hydroxymethylfurfural produced from hexose which further degrade into formic acid. The composition of the biomass waste hydrolysate including the sugars and degradation products were shown at Table 5. From the experimental data, IL-SPC hydrolysate contained higher glucose compare to SPC. It is because, IL-SPC cellulose has undergo ionic liquid dissolution which resulted higher surface area and disintegration of the cellulosic bril structure then provide better accessibility for the hydrolysis process (Jeong et al., 2018;Loow et al., 2016). Almost negligible amount of xylose (0.29 g/L) was found and neither furfural nor HMF were detected in the hydrolysate of IL-SPC compare to SPC's hydrolysate. It shows that, the ionic liquid dissolution was removed most of hemicellulose components from the SPC which also contribute to high glucose conversion from IL-SPC (Kim, 2018). This result also supported by the result of biomass hydrolysis (IL-BSW, IL-SCB and IL-OPEFB) where their total sugar are higher that the SPC even though, their cellulose content are lower that the SPC.
Besides glucose, sucrose, arabinose and xylose, biomass waste hydrolysate contained other unknown soluble sugar. The unknown soluble sugar may consist of other monomers of hemicellulose such as galactose and mannose. Besides monomer, the soluble sugar can also be cello-oligomer and xylooligomers. Oligomer is a molecule that consists of a few monomers units which can be dimer, trimer, and tetramer, depends on the number of monomers, two, three or four. Dimer can be digest by the bacteria but trimer oligomers are cannot (Nivea et al., 2006).  Fermentation for biohydrogen production Biohydrogen producing bacterium Clostridia sp. was used to examine the suitability of sugar feedstock from acid hydrolysis of SPC, IL-SPC, IL-BSW and IL-SCB for biohydrogen production. Control set was established in the experiment which the fermentation was fed with pure glucose as carbon source. The biohydrogen production pro le are illustrated in Fig. 8. As shows at Thus for maximum biohydrogen yield, fermentation parameters must be manipulated to divert the metabolism pathway towards producing butyric acid and acetic acid as the nal fermentation product (Abdul et al., 2016). This report is the rst of a trial of biohydrogen production from SPC and IL-SPC hydrolysates. Optimisation of the fermentation process will bring higher yields of biohydrogen.  with high cellulose recovery and reusability of the ionic liquids. To prepare fermentable sugar that can be consumed by the biohydrogen producing bacteria, acid hydrolysis of the regenerated cellulose was hydrolysed by 10% sulfuric acid which resulted up to 96% sugar conversion. IL-SPC hydrolysate reported higher total sugar conversion compare to SPC (control) due to higher surface area and disintegration of the cellulosic bril structure resulted from the dissolution process. IL-SPC, IL-BSW, IL-SCB and IL-OPEFB hydrolysate contained higher total sugar compared to SPC hydrolysate even though their cellulose content are lower that the SPC (control). In the fermentation process, almost 85% of biomass waste hydrolysate substrate was utilized by the bacteria and producing up to 196 ml H 2 /100 ml cumulative bioydrogen. As compare to control fermentation, the bacteria utilized 94.81% substrate, but producing lower cumulative biohydrogen which is 174.91 ml H 2 /100 ml. The biohydrogen fermentability results show that the recovered cellulose from the biomass waste is compatible to be used as a source of sugar in producing bio-hydrogen. This method of recycling cellulose from municipal solid waste and agriculture biomass was a new alternative for renewable energy resources, and provide new solutions to environmental pollution problems. Not applicable. The study does not involve the use of any animal or human data or tissue.

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Not applicable. The manuscript does not contain any other individual person's data.

Availability of data and materials
All data generated or analyzed during this study are included in the main manuscript le.

Competing interests
The authors declare that they have no competing interests.  (a) transparent layer of regenerated cellulose after washing with water. SEM images of biomass waste before and after ionic liquid dissolution (b) raw SPC 50x (c) SPC regenerated cellulose/hemicellulose 200x optical microscope (d) raw BSW 500x, (e) BSW regenerated cellulose/hemicellulose 3000x, (f) raw SCB 500x (g) SCB regenerated cellulose/hemicellulose 3000x ,(h) raw OPEFB 500x (i)OPEFB regenerated cellulose/hemicellulose 10000x   Biohydrogen production curve of different matrix carbon sources

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