Efficient biohydrogen and advanced biofuel coproduction from municipal solid waste through a clean process

Journal Pre-proofs Efficient biohydrogen and advanced biofuel coproduction from municipal solid waste through a clean process Farinaz Ebrahimian, Keikhosro Karimi PII: DOI: Reference:

S0960-8524(19)31885-1 https://doi.org/10.1016/j.biortech.2019.122656 BITE 122656

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

17 October 2019 6 December 2019 10 December 2019

Please cite this article as: Ebrahimian, F., Karimi, K., Efficient biohydrogen and advanced biofuel coproduction from municipal solid waste through a clean process, Bioresource Technology (2019), doi: https://doi.org/10.1016/ j.biortech.2019.122656

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Efficient biohydrogen and advanced biofuel coproduction from municipal solid waste through a clean process

Farinaz Ebrahimiana and Keikhosro Karimia, b*

a

Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran b

Industrial Biotechnology Group, Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran

* Corresponding author: Tel: +983133915623 Fax: +983133912677 E-mail: [email protected]

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Abstract The cleanest form of energy, i.e., biohydrogen, and advanced biofuel, i.e., biobutanol, were produced from the organic fraction of municipal solid waste (OFMSW). Ethanol as a byproduct of this process was used for the pretreatment of this substrate, and this pretreatment was improved by other process byproducts, i.e., acetic acid and butyric acid. The pretreatment was conducted with 85% ethanol and 0-1% (w/w) acetic/butyric acid at 120 and 160 °C for 30 min. The pretreatment catalyzed by 1% (w/w) acetic acid at 120 °C resulted in a hydrolysate with 49.8 g/L total fermentable sugars, which was fermented to the highest overall yield of acetone, butanol, and ethanol (ABE) and hydrogen. Through this process, 114.1 g butanol, 43.8 g acetone,15.1 g ethanol, 97.5 L hydrogen were obtained from each kg of OFMSW, producing 270 g ABE and 151 L H2 from each kg of substrate, corresponding to 6000 kJ energy production.

Keywords: ABE fermentation, Butanol, C. acetobutylicum, Organosolv pretreatment, Acid catalyst

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1. Introduction The extensive utilization of fossil fuels is considered as one of the primary causes of greenhouse gases emission, global warming, and unexpected climate change. These environmental concerns along with the unclear future of fossil fuels have led to growing interest in the development of alternative, non-polluting, and renewable energy sources (Kumari & Singh, 2018; Tonini et al., 2016). On the other hand, the rapid growth of human population causes the generation of huge amounts of municipal solid waste (MSW), threatening human and ecosystem health (Karak et al., 2012). The quantity and composition of MSW depend on the cultures and social development, and it is expected that the amount of MSW will increase to 4 billion tons by 2100, making serious problems for human living worldwide (Hosseini et al., 2013; Pour et al., 2018). Landfilling and incinerating are the most typical strategies for MSW management; however, they negatively affect the global environment by releasing toxic leachates and toxic gases, respectively (Chen et al., 2016; Cucchiella et al., 2014). The main fraction of MSW consists of biodegradable organic materials, including kitchen waste, solid paper, and garden waste (Cesaro & Belgiorno, 2014). Food waste (mainly stale bread and waste rice) comprises a significant proportion of organic solid waste in most Iranian cities. For instance, the average quantity of stale bread generated in Tehran, Iran, is reported to be more than 42% (Damghani et al., 2008; Hosseini et al., 2013). Therefore, starch is the predominant component in the MSW of Iranian cities. As biodegradable materials are a dominant part of MSW and due to their abundance and zero cost, the best environmentally friendly policy to alleviate the huge amounts of organic waste is their bioconversion to renewable and clean fuels (Elsamadony & Tawfik, 2015). Besides providing a

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sustainable alternative to fossil fuels, the consumption of organic fraction of municipal solid waste (OFMSW) reduces the volume of MSW and renders it manageable. Hydrogen, the cleanest fuel, has the highest energy content (120 MJ/kg) compared to other gaseous fuel, i.e., methane (50 MJ/kg), gasoline (44 MJ/kg), and ethanol (26.8 MJ/kg) (Sharma & Melkania, 2017). Hydrogen can be produced from both fossil and non-fossil primary energy sources. The examples of these two methods as well as their advantages and disadvantages are given in Table 1. Biohydrogen production from dark fermentation is a more economically and environmentally feasible process among other biological processes. This is due to low energy consumption, no oxygen or light source limitations, and a high potential for minimizing unwanted wastes (O-Thong et al., 2008). However, the main obstacle for biohydrogen production by dark fermentation is its relatively high production cost as well as low production rate and inefficiency, which can be resolved by the utilization of low-value substrates or by coproduction of hydrogen with other sustainable biofuels (Liu et al., 2019; Ren et al., 2016). In this regard, acetone-butanol-ethanol (ABE) has gained great attention as an advanced drop-in biofuel. Butanol, the main product of ABE fermentation, offers superior characteristics in comparison with bioethanol, including higher energy density (50%), lower volatility (50.2% higher boiling point), lower corrosiveness (1.2% higher pKa), and its possible blending with gasoline or diesel up to 20% (Bankar et al., 2013). Clostridium species, e.g., Clostridium acetobutylicum, have the ability to ferment a wide range of organic wastes for the anaerobic coproduction of hydrogen and ABE. The low titer of hydrogen and ABE in the fermentation is one of the main bottlenecks of the process (Liu et al., 2019). Thus, compared with ABE or hydrogen fermentation, coproduction of hydrogen and ABE might be more competitive. C. acetobutylicum is an efficient microorganism meeting the requirements of the “biorefinery” concept, i.e., installations developed to produce a variety of value-added products and biofuels 4

by biomass conversion. Feedstock cost is one of the primary challenges for process optimization. OFMSW, containing appreciable amounts of starch and lignocellulosic materials, has been suggested as the substrate for cost-effective biorefinery development. Having high amylolytic activities, C. acetobutylicum can directly utilize unhydrolyzed starchy materials (Gutierrez et al., 1998). However, the recalcitrant structure of the lignocellulosic part should first be converted to fermentable sugars using a suitable pretreatment, which enhance the bio-digestibility and accessibility of the enzymes to the structure in the following hydrolysis process. This process should improve the enzymatic hydrolysis, consume minimal amounts of chemicals, the pretreating agent can be recycled and reused, produce minimal waste, facilitate the recovery of lignin, and consume low amounts of energy with reasonable operating and capital investment requirement (Taherzadeh & Karimi, 2008). In our previous study, ethanol organosolv delignification was employed for the pretreatment of OFMSW to improve ABE production (Farmanbordar et al., 2018). However, to our knowledge, catalyzed organosolv pretreatment has not been evaluated for improvement of ABE production from OFMSW and its effect on biohydrogen production has not been investigated. In this study, OFMSW was evaluated as a sustainable feedstock for the production of hydrogen, butanol, and other value-added chemicals by C. acetobutylicum. Ethanol pretreatment at different conditions was used prior to enzymatic hydrolysis and fermentation. Acetic acid and butyric acid, the fermentation byproducts, at different doses were used as the catalyst to increase the rate of delignification. The catalyzed organosolv pretreatment increased the fermentable sugars of OFMSW and also decreased the lignin and inhibitors contents for ABE production. Effects of the main influencing pretreatment parameters focusing on catalyst dose and temperature were investigated on the lignin and inhibitor removal, solid recovery, and the yield of enzymatic hydrolysis and subsequent fermentation. 5

2. Materials and methods 2.1. Raw material The organic fraction of municipal solid waste (OFMSW) used as the substrate in this study was collected in summer from a local MSW management site (Recycling Center, Isfahan, Iran) and a restaurant on the student campus of Isfahan University of Technology (Isfahan, Iran). The collected wastes mainly contained fruit skins, vegetables, stale bread, rice, beans, and garden wastes. The OFMSW was air dried, hammer milled, and sieved to achieve a particle size between 833 μm (20 mesh) and 177 μm (80 mesh). 2.2. Pretreatment The organosolv pretreatment enhances by soaking 20 g of OFMSW in 180 g of 85% (v/v) aqueous ethanol solution (solid-to-liquid ratio of 1:9). Different amounts of acetic acid and butyric acid in the range of 0-1% w/w (based on OFMSW dry weight) were added to the ethanolwater solution as a catalyst. The pretreatments were carried out in a 500 mL high-pressure stainless steel reactor, which was equipped with a pressure gauge and thermometer (Amiri et al., 2014). After loading the mixture of OFMSW and pretreatment solution, the reactor was heated to the desired temperatures (120 and 160 °C) at the rate of 4 °C/min in an oil bath. The reaction solution was then held for 30 min at the desired temperature. Afterward, the reactor was immediately cooled to room temperature in an ice bath. Then, the pretreated solids were removed by filtration, washed three times with 100 mL ethanol solution at 60 °C, and subsequently floated in distilled water until obtaining neutral pH. The pretreated solids were air dried overnight and stored in plastic bags at room temperature for future use. 2.3. Enzymatic hydrolysis 6

The untreated and organosolv-pretreated OFMSW were hydrolyzed at the initial substrate concentration of 50 g/L (based on dry weight) in 50 mM sodium citrate buffer (pH 4.8) in 118 mL sealable glass bottles. Bottles containing the mixture were autoclaved at 121 °C for 20 min. The hydrolysis of both pretreated and untreated OFMSW was conducted by Cellic® CTec2 and HTec2 (kindly provided by Novozymes A/S, Denmark) in a 9:1 ratio (Jafari et al., 2016). The cellulase activity of the enzyme mixture was 95 filter paper units (FPU) per mL, measured based on the standard method presented by Adney and Baker (1996). Afterward, 20 FPU enzyme mixture per gram of dry substrate was added to each sterilized bottle (Obama et al., 2012). Finally, the bottles were sealed and incubated in a shaking incubator at 45 °C and 120 rpm for 72 h. The suspensions obtained after 72 h enzymatic hydrolysis were subsequently fermented by C. acetobutylicum. 2.4. Microorganism and inoculum preparation Clostridium acetobutylicum NRRL B-591 spores were purchased from the Persian Type Culture Collection (Tehran, Iran) and used in all fermentations. The bacterial spores were cultivated in a 50 mL sterilized cooked meat medium (60 g/L cooked meat (Sigma-Aldrich) and 10 g/L glucose) at 37 °C and 160 rpm for 24 h. Afterward, the spore suspension was transferred to 1.5 mL vials and mixed with 15% glycerol. After quick-freezing the mixtures by using liquid nitrogen, the vials were maintained at -80 °C for future use. In order to prepare the inoculum, two cultivation steps were carried out. First, the spore suspension was cultivated in a 25 mL cooked meat medium. In this regard, the mixture of 1.5 g cooked meat and 0.25 g glucose in 25 mL distilled water was sterilized by autoclaving at 121 °C for 20 min. After cooling to 75 °C, the medium was inoculated with 0.5 mL spore suspension, subjected to heat shocking at 75 °C for 2 min, and subsequently cooled in cold water for 1 min. 7

Then, the spore cultivation was conducted at 37 °C for 18 h. For the second cultivation step, a 50 mL peptone-glucose-yeast extract (PGY) medium (containing (g/L); 3, peptone; 30, glucose; and 1, yeast extract) was autoclaved and supplemented with 1% (v/v) filter-sterilized P2 stock solution. The P2 stock solution contained (g/L): minerals (20, MgSO4.7H2O; 1, MnSO4.7H2O; 1, FeSO4.7H2O; and 1, NaCl), buffer (220, C2H3O2NH4; 50, KH2PO4; and 50, K2HPO4), vitamins (0.1, para-aminobenzoic acid; 0.1, thiamin; and 0.001, biotin), and 0.15 cysteine (Jafari et al., 2016). Then, the bottle containing the PGY-P2 solution was sealed and purged with pure nitrogen to obtain anaerobic conditions. Afterward, 5 mL of precultured solution (prepared in the first cultivation step) was added to the medium and incubated at 37 °C and 160 rpm for 18 h to prepare actively-growing inoculum for the fermentation. 2.5. Fermentation The hydrolysate (both solid and liquid phases) obtained after enzymatic hydrolysis was subjected to the fermentation by Clostridium acetobutylicum NRRL B-591. After the adjustment of pH to 6.8 using 5 M NaOH, 20 mL of hydrolysates was supplemented with 1 g/L yeast extract and 3 g/L peptone in 118 mL serum bottles. The bottles were sealed with a butyl rubber stopper fastened with an aluminum crimp and autoclaved at 115 °C for 15 min. The bottles were then purged with pure nitrogen gas passed through a hot copper column (190-200 °C) to remove any trace oxygen. The autoclaved media were supplemented with 1% (v/v) filter-sterilized P2 stock solution and inoculated with 6% (v/v) actively-growing inoculum at sterile conditions under a sterile laminar flow hood. The fermentation was then conducted at 37 °C for 72 h in a shaking incubator at 160 rpm agitation (Jafari et al., 2016). 2.6. Analytical methods

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The untreated and organosolv-pretreated OFMSW were analyzed for total solids (TS), carbohydrate, lignin, and starch contents according to the methods provided by the NREL (Sluiter et al., 2008a; Sluiter et al., 2008b; Sluiter & Sluiter, 2008). Moreover, the concentration of starch in the hydrolysates was determined by applying two commercial enzymes of α-amylase (Liquezyme, Novozymes A/S, Denmark) and glucoamylase (Dextrozyme GA, Novozymes A/S, Denmark). The difference between glucose concentration before and after hydrolysis of starch indicated the starch concentration in the hydrolysates (Mahmoodi et al., 2018). Methods presented by Rakić et al. (2007) and Velioglu et al. (1998) were applied for measuring total phenolic compounds and tannins contents of solid materials, respectively. The concentrations of monomeric sugars as well as fermentation products were measured by a high performance liquid chromatograph (HPLC), equipped with a refractive index (RI) detector (Jasco International Co., Tokyo, Japan). To determine the concentration of sugars, an Aminex HPX-87P column (Bio-Rad, Richmond, CA, USA) was used at 80 °C during the mobile phase of deionized water with 0.6 mL/min flow rate. The concentration of produced solvents and acids was detected using an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) at 60 °C by 0.005 M H2SO4 as a mobile phase with the flow rate of 0.6 mL/min. The compositions of H2 and CO2 in gas samples were analyzed by a gas chromatograph (GC2552, Teif Gostar Faraz Co., Iran) equipped with a thermal conductivity detector (TCD) and a 3 m (length) × 3 mm (internal diameter) packed column (Porapak Q, Chrompack, Germany). The gas samples were taken using a 250 μL pressure lock syringe (VICI Precision, Louisiana, USA). The column, injector, and detector temperature were maintained at 40, 100, and 150 °C, respectively. Nitrogen was used as a carrier gas with the flow rate of 50 mL/min. All

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experiments were carried out in duplicate, and the presented results are the average of two replicates. 2.7. Statistical analysis Statistical analysis of the results was performed by one-way analysis of variance (ANOVA) and Tukey’s test to compare the significance between the means of results at the 95% level of confidence (p ˂ 0.05).

3. Result and discussion 3.1. Pretreatment The biodegradable fraction of municipal solid waste was subjected to ethanol organosolv pretreatment at 120 and 160 °C for 30 min. In the pretreatment, acetic acid and butyric acid at different concentrations of 0, 0.5, and 1% (w/w) were used as a catalyst. The pretreatment conditions were selected by considering the results of a previous study (Farmanbordar et al., 2018) and preliminary experiments. First, the effect of acetic acid and butyric acid addition on ethanolic pretreatment was investigated at 120 and 160 °C. The pretreatment conditions, solid recovery, and composition of untreated and pretreated OFMSW, i.e., starch content, total phenolic compound, and lignocellulose fractions, are summarized in Table 2. As shown in Table 2, ethanolic pretreatment significantly affected the lignocellulosic fraction of OFMSW, through which 41.5-68% of hemicellulose and 36-65% of lignin were removed from OFMSW, leading to the pretreated solids with 48-61% glucan, depending on the pretreatment conditions. The pretreatment without acid at 120 °C removed 104 g of total lignin and 60 g of xylan from each kg of OFMSW. Through the pretreatment with addition of 1% (w/w) butyric 10

acid at 120 °C, 111 g of total lignin and 66 g of xylan from each kg of OFMSW were removed. When 1% (w/w) acetic acid was added to the pretreating solution at 120 °C, 117 g total lignin and 75 xylan were removed. The pretreatment with the mixture of 1% (w/w) butyric acid and 1% (w/w) acetic acid at 120 °C increased the lignin and xylan removal to 123 g and 79 g, respectively, from each kg of OFMSW. In other words, the higher dose of the acid catalyst decreased the lignin and hemicellulose content of the OFMSW, leading to a pretreated solid residue with higher glucan content. This agreed with the previous study that showed that the lignin and xylan removal in the ethanolic pretreatment of wheat straw were significantly enhanced by higher acid catalyst doses (Wildschut et al., 2013). Note that acetic acid was more effective in lignin and xylan removal compared to butyric acid. Increasing the pretreatment temperature improved the delignification and hemicellulose hydrolysis of OFMSW, similar to the results obtained by Salapa et al. (2017) for ethanolic pretreatment of wheat straw. As can be seen in Table 2, the pretreatment of each kg of OFMSW with 1% (w/w) acetic acid at 160 °C resulted in 176 g and 99 g of lignin and xylan removal, respectively, which was higher than those from the pretreatment with 1% (w/w) acetic acid at 120 °C. The highest amount of released lignin and hemicellulose was observed after the pretreatment with the mixture of 1% (w/w) butyric acid and 1% (w/w) acetic acid at 160 °C. The glucan content of the OFMSW was also affected by the pretreatment. The glucan fraction of pretreated OFMSW was increased, as a result of partial removal of other constituents, mainly lignin and hemicellulose. As can be concluded from the data in Table 2, the pretreated solids with higher glucan fractions were obtained by increasing both the acid catalyst dose and the temperature. The pretreatment with the mixture of 1% (w/w) butyric acid and 1% (w/w) acetic acid at 120 and 160 °C resulted in 8 and 16% higher glucan fraction, respectively, compared to

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that of untreated OFMSW. This result was in accord with the results obtained in previous studies (Amiri et al., 2014; Jafari et al., 2016). The untreated OFMSW consisted of 45.1% glucan, 14.5% xylan, 28.8% total lignin, and 41.5% starch. Solvent-producing Clostridium sp. can directly utilize unhydrolyzed starchy raw materials as a result of its sufficient extracellular amylolytic activity (Jang et al., 2012). As shown in Table 2, the highest amount of starch was detected after ethanol pretreatment with no acidic catalyst at 120 °C. The solid recovery and starch content were decreased by increasing the pretreatment temperature; however, they were similar for pretreated solids with different catalysts at the same pretreatment temperature. The pretreatment of OFMSW at 120 and 160 °C released 34-36% and 46-50% of solid into liquid, respectively. As shown in Table 2, the untreated OFMSW contained a relatively high amount of phenolic compounds (0.5 mM GAE), which could have originated from tea waste, fruits skins, and starchy materials in the OFMSW. Degradation of hemicellulose and lignin might also generate phenolic compounds in pretreated OFMSW (Sharma & Melkania, 2017; Ximenes et al., 2010). As can be seen in Table 2, total phenolic compounds were increased by boosting the pretreatment temperature as a result of hemicellulose or lignin degradation. Phenolic compounds induce significant inhibitory effects on microorganisms by affecting their metabolism to convert these inhibitors into less inhibiting chemicals until the reduction of their concentration is lower than a threshold amount (0.2 mM GAE) (Mirfakhar et al., 2017). The alteration in membrane permeability of the Clostridia is another effect of phenolic compounds, leading to the leakage of intracellular components and resulting in the inhibition of cell growth, DNA damage, and the inactivation of the several enzymes involved in the glycolytic pathway (Sharma & Melkania, 2017). Tannins are also known as polyphenolic compounds that exhibit severe inhibitory effects 12

on enzyme activities (Ximenes et al., 2011). Farmanbordar et al. (2018) reported that the biodegradable fraction of municipal solid waste contains considerable amounts of tannins, which exhibit crucial inhibitory effects on both amylolytic activity and ABE fermentation by C. acetobutylicum. As shown in Table 2, tannins formed the major fraction of the phenolic compounds content of both pretreated and untreated OFMSW. The organosolv-pretreated OFMSW resulted in solids with less than 0.2 mM GAE tannins. It has been previously shown that tannins at concentrations higher than 0.25 mM GAE have significant inhibitory effects on C. acetobutylicum, while it can tolerate a threshold tannin concentration of 0.2 mM GAE (Mirfakhar et al., 2017). Through the ethanolic pretreatment without acid at 120 °C, 91.8% of tannins were released into the liquid phase, which was increased to 92.3 and 92.9% by adding 0.5% (w/w) butyric acid and 0.5% (w/w) acetic acid to the pretreatment liquor, respectively. The ethanolic pretreatment with 1% (w/w) butyric acid at 120 °C resulted in the highest total phenolic compound removal of 95%, leading to the release of more than 0.4 mM GAE total phenolic compounds per each gram of OFMSW. 3.2. Enzymatic hydrolysis The untreated and pretreated OFMSW were converted to fermentable sugars through 72 h enzymatic hydrolysis by Cellic CTec2 and HTec2 enzymes using 5% (w/v) solid loading. The solid loading was selected based on a number of preliminary experiments and previous results (Mahmoodi et al., 2018; Yang et al., 2015). Fig. 1 shows the concentrations of sugars (glucose and xylose) as well as glucose yields. As a result of the pretreatment prior to enzymatic hydrolysis, the yield of glucose production and concentration of total sugars were increased by 15-71% and 30-82%, respectively. The removal of lignin and hemicellulose (Zhao et al., 2009) as well as polyphenolic compounds, particularly tannins (Ximenes et al., 2011), through the 13

pretreatment, might be the reasons for the improvement of enzymatic hydrolysis of ethanolpretreated OFMSW. The enzymatic hydrolysis of untreated OFMSW resulted in the production of hydrolysate with 20.5 g/L total sugar, containing 20.5 g/L glucose and 0.5 g/L xylose, where the glucose yield was 410 g/kg. Using pretreatment without acid at 120 °C, the glucose concentration and glucose yield in the hydrolysate were increased to 23 g/L and 471 g/kg, respectively, which were only 15% higher than those from untreated OFMSW. After increasing the acid catalyst to 1% (w/w), a gradual increase was observed in glucose production yield by enzymatic hydrolysis. The glucose yield of 701 g/kg was obtained from the ethanol-pretreated OFMSW with 1% (w/w) acetic acid at 160 °C, while it was 615 g/kg from the OFMSW pretreated with the mixture of 1% (w/w) acetic acid and 1% (w/w) butyric acid. Even though further increase in the acid catalyst dose to 2% (w/w) resulted in the higher lignin removal, it did not enhance the yield of enzymatic hydrolysis. It has been reported that besides the residual lignin content, its structure as well as its distribution on the surface of pretreated sweet sorghum bagasse (Yan et al., 2015), rice straw (Amiri et al., 2014), and poplar cellulose (Sun et al., 2014) has significant effects on the enzymatic hydrolysis.

(Taherzadeh & Karimi, 2008) These results were consistent with the result obtained in this study. As shown in Fig. 1, the highest glucose concentration of 35 g/L was obtained in the hydrolysate of ethanol-pretreated OFMSW with 1% (w/w) acetic acid at 160 °C, which was a 71% improvement compared to that of the untreated OFMSW. 14

Along with soluble sugars, starch was also presented in the hydrolysate of pretreated and untreated OFMSW (Fig. 2). As can be observed from Fig. 2, increasing the pretreatment temperature and acid catalyst dose decreased the concentration of starch in the hydrolysates, which is similar to the results obtained by Farmanbordar et al. (2018) for organosolv pretreatment of OFMSW. Starch concentration was reduced from 19 g/L in the hydrolysate obtained from ethanol-pretreated OFMSW without acid at 120 °C to 12 g/L in that obtained with the mixture of 1% (w/w) acetic acid and 1% (w/w) butyric acid at 160 °C. 3.3. ABE fermentation The hydrolysates obtained from the enzymatic hydrolysis of pretreated and untreated OFMSW were subjected to fermentation by C. acetobutylicum at 37 °C for 96 h, leading to acetone, butanol, ethanol, acetic acid, butyric acid, and hydrogen production. Fig. 3 shows the concentrations of produced solvents and acids. The pretreatment of the OFMSW improved the production of ABE. As depicted in Fig. 3, the pretreatment of OFMSW at 120 °C and 160 °C resulted in 4.6-5.3 and 5.1-6 fold higher total concentration of acetone, butanol, and ethanol in comparison to that obtained from untreated OFMSW, i.e., 2.5 g/L ABE. Even though the untreated OFMSW resulted in a hydrolysate with relatively high total sugar and starch concentration of 21 g/L and 14 g/L, respectively, the fermentation of the hydrolysate resulted in the production of only 0.9 g/L acetone, 1.2 g/L butanol, and 0.4 g/L ethanol. The inhibition of the amylolytic activity of C. acetobutylicum and ABE fermentation by a high concentration of tannins (0.3 mM GAE) could be responsible for the low yield of ABE production in spite of the high concentration of total sugar and starch in the untreated OFMSW hydrolysate. Previous studies have reported that the solvent-producing Clostridia are severely inhibited by phenolic compounds, and in particular tannins, at concentrations higher than the threshold amount of 0.2 15

mM GAE (Mirfakhar et al., 2017; Heidari et al., 2016). To assess the effect of phenolic compounds presented in sorghum grain on the amylolytic activity of C. acetobutylicum, Mirfakhar et al. (2017) utilized commercial α- amylase and glucoamylase for liquefaction and saccharification of the untreated and pretreated sorghum grain. They found that the inhibitors presented in untreated sorghum grain negatively affect the amylolytic activity, leading to 25% lower hydrolysis yield, resulting in 25% lower glucose production in comparison with the treated grain. Fermentation of the hydrolysates obtained from ethanol-pretreated OFMSW at 120 °C resulted in the production of 11.5-13.3 g/L ABE. The hydrolysate obtained from OFMSW pretreated without acid at 120 °C was fermented to 7.5 g/L butanol, 3 g/L acetone, and 1 g/L ethanol. Increasing the acid catalyst dose to 1% (w/w) resulted in the gradual improvement of both total sugar formations through the enzymatic hydrolysis (section 3.2) and ABE production through the fermentation of the hydrolysate. Hydrolysis of pretreated OFMSW using 1% (w/w) acetic acid at 120 °C resulted in a hydrolysate with 32 g/L sugar and 18.2 g/L starch, which was fermented to 13.3 g/L ABE. Therefore, the pretreatment of OFMSW at 120 °C resulted in more than 400% improvement in ABE production. However, the hydrolysate obtained from the pretreated OFMSW supplemented with the mixture of 1% (w/w) acetic acid and 1% (w/w) butyric acid at 120 °C not only failed to improve the enzymatic hydrolysis but also resulted in 13% higher starch removal, leading to 4% lower ABE production in comparison with that obtained by the pretreatment without acid. Increasing the pretreatment temperature from 120 °C to 160 °C enhanced ABE production gradually, which was highly dependent on the type and dose of acid catalyst. The hydrolysate obtained from the pretreated OFMSW with the mixture of 1% (w/w) acetic acid and 1% (w/w) butyric acid at 160 °C with 46 g/L total fermentable sugar was 5% higher than 16

that obtained from pretreated OFMSW at 120 °C, and was fermented to an almost similar ABE concentration of about 11.4 g/L. This observation could be related to both the 35% higher starch removal and the 67% higher total phenolic compounds formation through the pretreatment at 160 °C, leading to a hydrolysate with lower starch content and higher phenolic compounds. As Farmanbordar et al. (2018) reported, OFMSW hydrolysate obtained from pretreatment with 85% ethanol at 120 °C for 30 min with similar sugar content was fermented to 64% higher ABE, due to the 30% higher starch compared to that obtained by pretreatment with 75% ethanol. In another study, acetone pretreated sweet sorghum bagasse (SSB) at 180 °C for 90 min resulted in a hydrolysate containing a similar amount of sugar but lower ABE concentration after the fermentation in comparison with that obtained by the pretreatment for 30 min. This could be due to the higher inhibitory effect of phenolic compounds accompanied by higher lignin degradation of SSB pretreated for longer reaction time (Jafari et al., 2016). As can be seen in Fig. 3, the hydrolysate obtained from the pretreated OFMSW using 1% (w/w) acetic acid at 160 °C, containing 35 g/L glucose, 3 g/L xylose, and 15 g/L starch, was fermented to the highest ABE concentration, through which 3.9 g/L acetone, 9.7 g/L butanol, and 1.5 g/L ethanol along with 3.8 g/L acetic acid and 4 g/L butyric acid were produced. Therefore, the organosolv pretreatment with 85% ethanol containing 1% (w/w) acetic acid at 160 °C prior to enzymatic hydrolysis was accompanied with 500 % enhancement in the total concentration of ABE after 72 h fermentation. This process resulted in the ABE production yield of 0.28 g/g carbon source. Jin et al. (2019) utilized soluble sugars as well as hydrolysate of acid and alkali-pretreated residues of apple pomace for ABE fermentation by Clostridium beijerinckii P260. The fermentation resulted in the production of 202.8, 42.1, and 41.4 g ABE per each kg of dry apple 17

pomace from soluble sugars, acid-pretreated hydrolysate, and alkali-pretreated hydrolysate, respectively. Mixing the acid- and alkali-pretreated hydrolysates with water-soluble sugars alleviated the inhibitory effect of degradation products, leading to 260.1 and 262.2 g ABE per each kg of dry apple pomace, respectively. 3.4. Hydrogen production The time course of cumulative hydrogen production through the fermentation of hydrolysates obtained from untreated and pretreated OFMSW is illustrated in Fig 4. Hydrogen production from OFMSW was generally improved by the pretreatment. Using the pretreatment prior to enzymatic hydrolysis resulted in a 142-254% increase in the final accumulated hydrogen. Hydrogen was evolved after a short lag time of 6 h. It should be noted that the pretreatments had no significant effect on the lag time of hydrogen production. During the first 12 h of fermentation, less than 56 mL of hydrogen was evolved from each g of the substrate. More than 70% of the final accumulated hydrogen was produced within 20 h of fermentation. C. acetobutylicum produces hydrogen as well as acetic acid and butyric acid during the acidogenic phase (the exponential growth phase). When entering the solventogenic phase (the stationary growth phase), the metabolism of the cell shifts from the acid-hydrogen–producing pathway to the solvent-producing pathway (Chong et al., 2009; Marchal et al., 1992). Fermentation of the hydrolysate obtained from untreated OFMSW resulted in the production of only 49.1 mL hydrogen per each gram of the substrate with the productivity of 0.7 mL/h. From the hydrolysates obtained from the OFMSW pretreated at 120 °C, hydrogen production was enhanced from 129.3 to 150.4 mL per each gram of the substrate by increasing acid catalyst dose from 0 to 1% (w/w). The pretreatment with addition of 1% (w/w) acetic acid to 85% ethanol at 120 °C improved the yield of enzymatic hydrolysis to 584.7 g/kg, leading to 48.8 g/L total 18

carbon source (glucose, xylose, and starch), which was subsequently fermented to 150.4 mL hydrogen per each gram of substrate. This process increased the hydrogen production volume by 206.2%. A rise in the pretreatment temperature from 120 to 160 °C increased the concentration of fermentable sugars in the pretreated OFMSW, resulting in an increase in hydrogen production from the hydrolysates obtained after 72 h enzymatic hydrolysis. The fermentation of the hydrolysate obtained from OFMSW without addition of catalyst at 160 °C produced 101 mL hydrogen per each gram of the substrate within 20 h of fermentation, while prolonging the fermentation time from 20 to 72 h resulted in 135.7 mL hydrogen. The hydrolysate obtained from OFMSW pretreated with addition of 1% (w/w) acetic acid at 160 °C resulted in the highest hydrogen production volume and productivity of 174 and 2.4, respectively, which was 3.5 fold higher than those obtained from untreated OFMSW. The results of this study are comparable with the results obtained from the fermentation of 7 g/L pure glucose for fermentation by C. acetobutylicum ATCC 824, which obtained the yield of 222 mL H2/g substrate (Oh et al., 2009). Moreover, through the fermentation of fermentable sugars, i.e., glucose and xylose, obtained from acid hydrolysis of de-oiled rice bran by C. acetobutylicum YM1, accumulated hydrogen production volume of 572.5 mL H2/g substrate were achieved (Azman et al., 2016). 3.5. Mass balance The concentrations of produced solvents, i.e., acetone, butanol, and ethanol, as well as the cumulative hydrogen volume were generally increased by the pretreatments. Achieving higher ABE concentration and cumulative hydrogen volume was not necessarily accompanied by higher yields of solvents and hydrogen generation. Hence, the overall yield of products, i.e., the amount of products generated from each kg of raw OFMSW, should be considered as a key factor for 19

evaluating the efficiency of each pretreatment (Table 3). As can be observed from Table 3, compared with untreated OFMSW, the pretreated solids in all conditions resulted in the higher overall yields of ABE and hydrogen production, varying between 113.7 and 173 g ABE as well as 64.4 and 97.5 L H2 per each kg of OFMSW, respectively. The enzymatic hydrolysis and fermentation of each kg of untreated OFMSW resulted in the production of only 50 g ABE, 49.1 L hydrogen, as well as 29.8 g acetic acid and 21.3 g butyric acid. The pretreatment catalyzed by 1% w/w acetic acid at 120 °C and subsequent enzymatic hydrolysis and fermentation resulted in the highest yields of 173 g ABE and 97.5 L H2 from each kg of dry OFMSW. Even though the enzymatic hydrolysis and fermentation of pretreated OFMSW at the higher temperature (160 °C) were accompanied by the higher concentration of products, compared to that obtained at lower temperature (120 °C), an inverse relationship between the pretreatment temperature and the overall fermentation yield was observed. This was due to the lower solid recovery achieved after the pretreatment at 160 °C. Fig. 5 depicts the mass balance of an integrated process for ABE and hydrogen production as primary products, as well as acetic acid and butyric acid as byproducts, using the hydrolysate obtained from each kg of dry OFMSW pretreated with 1% w/w acetic acid–catalyzed ethanol at 120 °C. Additionally, the results of gasoline equivalent from one kg of dry OFMSW are shown in Fig. 5. The concentrations of glucose, xylose, and starch in enzymatic hydrolysate, and the concentration of fermentation products were obtained experimentally, while other ingredients were calculated based on mass balance over each unit. According to Fig. 5, 210.5 mL enzyme was consumed for hydrolysis and subsequent fermentation of each kg dry OFMSW, resulting in the production of 97.5 L (8.7 g) hydrogen (28 g gasoline equivalent), 114.1 g butanol (95 g gasoline equivalent), 15.1 g ethanol (9.5 g gasoline equivalent), 43.8 g acetone, 46.2 g acetic acid, and 48.3 g butyric acid. The organic fraction of municipal solid waste hydrolysate has been 20

previously evaluated for ethanol production using Saccharomyces cerevisiae and improved by thermal pretreatment, whereas 126 g ethanol (73 g gasoline equivalent) was produced from each kg OFMSW (Ballesteros et al., 2010). Using autoclave as a pretreatment process followed by enzymatic hydrolysis (38% glucose) and fermentation resulted in the production of 19.4 kg ABE and 0.9 kg hydrogen from one ton of MSW (Meng et al., 2019). The enzymatic hydrolysis of solid residue after autohydrolysis pretreatment of pine and elm resulted in the production of 162 and 295 g sugar from each kg pine and elm, respectively, which was fermented to 79.3 and 117.6 g ABE, respectively (Amiri & Karimi, 2015). Zhang et al. (2016) reported the production of 51.9 L hydrogen per each kg of corn stover by the combination of Clostridium cellulolyticum and Citrobacter amalonaticus after steam explosion pretreatment. Among different microorganisms producing H2, C. acetobutylicum offers the advantages of direct utilization of starch, the highest yield of H2 production, and the co-production of H2 and ABE. In the current study, the ethanolic pretreatment of each kg OFMSW catalyzed by addition of 1% w/w acetic acid at 120 °C for 30 min converted considerable amounts of carbohydrates to ABE and hydrogen. The total amount of gasoline equivalent from ethanol, butanol, and hydrogen production obtained after the pretreatment and subsequent hydrolysis and fermentation of each kg of dry OFMSW was 132.5 g. Even though the process of utilizing OFMSW for co-production of hydrogen and advanced biofuel has several interesting advantages from the environmental and economic point of view, a techno-economic analysis is required to evaluate the potential of the process for commercialization.

4. Conclusions 21

To have a more sustainable process, all the chemicals used for the pretreatment were the byproducts of the fermentation. It was found that the catalyzed organosolv pretreatment was a crucial process for the fermentation, not only to increase the fermentable sugars (up to 51%) but also to remove the inhibitors of OFMSW (95% total phenolic compound removal). The pretreatment at the best conditions (1% acetic acid at 120 °C) resulted in the highest energy yield of 6000 kJ (total gasoline equivalent of 132.5 g), mainly in the form of butanol and biohydrogen from each kg of dry OFMSW. Acknowledgments The authors are grateful to Novozymes, Denmark, for gifting the cellulases used in this study, and to the Institute for Biotechnology and Bioengineering, Isfahan University of Technology, for financial support.

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[38] Sluiter, A., Sluiter, J. 2008. Determination of Starch in Solid Biomass Samples by HPLC: Laboratory Analytical Procedure National Renewable Energy Laboratory. [39] Sun, Q., Foston, M., Meng, X., Sawada, D., Pingali, S.V., O’Neill, H.M., Li, H., Wyman, C.E., Langan, P., Ragauskas, A.J. 2014. Effect of lignin content on changes occurring in poplar cellulose ultrastructure during dilute acid pretreatment. Biotechnol. Biofuels., 7(1), 150. [40] Taherzadeh, M., Karimi, K. 2008. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int. J. molecular Sci., 9(9), 1621-1651. [41] Tonini, D., Hamelin, L., Alvarado-Morales, M., Astrup, T.F. 2016. GHG emission factors for bioelectricity, biomethane, and bioethanol quantified for 24 biomass substrates with consequential life-cycle assessment. Bioresour. Technol., 208, 123-133. [42] Velioglu, Y., Mazza, G., Gao, L., Oomah, B. 1998. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. Journal of agricultural and food chemistry, 46(10), 4113-4117. [43] Wildschut, J., Smit, A.T., Reith, J.H., Huijgen, W.J. 2013. Ethanol-based organosolv fractionation of wheat straw for the production of lignin and enzymatically digestible cellulose. Bioresour. Technol., 135, 58-66. [44] Ximenes, E., Kim, Y., Mosier, N., Dien, B., Ladisch, M. 2011. Deactivation of cellulases by phenols. Enzyme. Microb. Technol., 48(1), 54-60. [45] Ximenes, E., Kim, Y., Mosier, N., Dien, B., Ladisch, M. 2010. Inhibition of cellulases by phenols. Enzyme. Microb. Technol., 46(3-4), 170-176. [46] Yan, Z., Li, J., Li, S., Chang, S., Cui, T., Jiang, Y., Cong, G., Yu, M., Zhang, L. 2015. Impact of lignin removal on the enzymatic hydrolysis of fermented sweet sorghum bagasse. Appl. Energy, 160, 641-647. [47] Yang, M., Zhang, J., Kuittinen, S., Vepsalainen, J., Soininen, P., Keinanen, M., Pappinen, A. 2015. Enhanced sugar production from pretreated barley straw by additive xylanase and surfactants in enzymatic hydrolysis for acetone-butanol-ethanol fermentation. Bioresour. Technol., 189, 131-137. [48] Zhang, S.C., Lai, Q.H., Lu, Y., Liu, Z.D., Wang, T.M., Zhang, C., Xing, X.H. 2016. Enhanced biohydrogen production from corn stover by the combination of Clostridium cellulolyticum and hydrogen fermentation bacteria. J. Biosci. Bioeng., 122(4), 482-7. [49] Zhao, X., Cheng, K., Liu, D. 2009. Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis. Appl. Microbiol. Biotechnol., 82(5), 815-27.

25

Figure Captions: Fig. 1. ( ) glucose and ( ) xylose concentration as well as (○) glucose yield after 72 h enzymatic hydrolysis of OFMSW pretreated with 85% v/v ethanol at different temperatures and catalyst dose and untreated OFMSW Fig. 2. Concentration of starch after 72 h enzymatic hydrolysis of OFMSW pretreated with 85% v/v ethanol at different temperatures and catalyst dose and untreated OFMSW Fig. 3. Concentration of () acetone, ( ) butanol, ( ) ethanol, (×) butyric acid, and (○) acetic acid after 72 h fermentation of the untreated OFMSW and enzymatic hydrolysates of the pretreated solid obtained from organosolv pretreatment by ethanol 85% (v/v) ethanol with different catalysts at 120 and 160 °C for 30 min Fig. 4. Accumulated hydrogen production during 72h of fermentation for ( ) untreated, ( ) pretreated with 1% acetic acid at 120 °C, ( ) pretreated with 1% acetic acid at 160 °C, ( ) pretreated without catalyst at 120 °C, and (×) pretreated without catalyst at 160 °C Fig. 5. Overall mass balances for organosolv pretreatment, enzymatic hydrolysis, and fermentation processes

26

Table 1. Advantages and disadvantages of different hydrogen production processes H2 production process

Examples

Advantages

Disadvantages

Reference(s)

Based on fossil primary energy sources

Coal gasification, steam methane reforming, and gasification of heavy hydrocarbons

Highly efficient

The requirement of a huge amount of energy, environmental problems

(Ren et al., 2016)

Based on nonfossil primary energy source

Radiolysis, electrolysis, pyrolysis

Environmentally friendly, energyand costintensive

Low efficiency

(Dong et al., 2009; Ren et al., 2016).

Biological processes

Biophotolysis, photo fermentation, and dark fermentation

Environmentally friendly and sustainable

Low efficiency, high production price

(Ren et al., 2016)

27

Table 2 Composition of untreated and organosolv-pretreated OFMSW

Pretreatment Catalyst (% w/w) Acetic Butyric acid acid

Total Solid Recovery (%)

Starch (%)

0 0.5 1 0 0 0.5 1

66 66 65 65 65 66 64

45.8±0.9 45.5±1.0 45.3±1.1 45.4±1.2 44.8±1.0 44.9±1.2 43.7±1.1

0.07±0.00 0.06±0.00 0.04±0.00 0.05±0.00 0.07±0.00 0.08±0.00 0.10±0.00

0 0 0 0.5 0 1 0.5 0 1 0 0.5 0.5 1 1 Untreated

53 51 54 53 50 50 50 -

41.9±10 41.6±1.1 41.2±1.2 41.5±10 40.6±10 40.2±1.5 39.4±1.2 41.5±10

0.12±0.00 0.16±0.00 0.16±0.00 0.17±0.00 0.20±0.00 0.16±0.00 0.21±0.00 0.51±0.00

0 0 0 0.5 1 0.5 1

Total Phenolic (mM)

Tannin (mM)

Composition of lignocellulose fraction (%)

Glucan

Xylan

Other carbohydrates 85% v/v Ethanol-water solution at 120 °C 0.04±0.00 48.4±0.5 12.9±0.6 0.04±0.00 50.2±0.3 12.1±0.1 0.03±0.00 50.9±0.3 12.2±0.2 0.04±0.00 50.5±0.4 12.1±0.1 0.04±0.00 52.3±0.4 10.9±0.2 0.05±0.00 51.6±0.5 11.4±0.1 0.07±0.03 53.5±0.3 10.3±0.1 85% v/v Ethanol-water solution at 160 °C 0.08±0.00 52.4±1.2 11.9±0.7 0.10±0.00 54.4±0.6 11.2±0.1 0.11±0.01 55.5±0.3 10.4±0.4 0.11±0.01 55.4±0.2 10.6±0.3 0.12±0.00 58.5±0.4 9.3±0.3 0.10±0.01 57.3±0.5 9.3±0.3 0.13±0.00 61.3±0.5 8.2±0.2 0.33±0.01 45.1±0.5 14.5±0.2

28

ASL

AIL

10.6±1.1 10.1±0.5 9.8±0.3 10.1±0.2 10.5±0.2 10.3±0.1 10.7±1.2

1.9±0.1 1.8±0.4 1.7±0. 1 1.8±0. 1 1.7±0.1 1.7±0.1 1.6±0.1

26.1±0.1 25.8±0.1 25.4±0.1 25.5±0.3 24.6±0.1 25.0±0.5 24.0±0.6

10.2±1.7 9.2±0.7 9.7±0.7 10.0±0.7 9.9±0.6 10.2±0.3 10.1±0.9 11.6±0.8

1.7±0.1 1.5±0.2 1.3±0.1 1.3±0.2 1.1±0.1 1.2±0.1 1.0±0.1 2.1±0.1

23.9±0.1 23.7±0.4 23.1±0.2 22.7±0.6 21.2±0.1 21.9±0.1 19.0±0.2 26.7±0.1

Table 3 Overall yield of acetone, butanol, ethanol, acetic acid, butyric acid, and hydrogen Pretreatment Catalyst (%w/w)

Overall Yield (g/ kg MSW)

Acetone

Acetic acid 0 0 0 0.5 1 0.5 1

Butyric acid 0 0.5 1 0 0 0.5 1

Butanol

Ethanol

ABE

Acetic acid

Butyric acid

ABE yield (g/g carbon source)

H2 yield mL/g carbon source

H2 overall yield (L/kg MSW)

0.25 0.25 0.25 0.25 0.27 0.26 0.26

140.2 140.3 141.4 143.3 151.0 146.0 134.9

84.9±3.4 86.1±2.9 84.7±3.0 87.2±0.9 97.5±3.2 90.8±1.3 76.5±2.7

0.26 0.27 0.27 0.27 0.28 0.27 0.25 0.07

140.0 140.7 141.0 143.2 163.5 145.0 140.4 69.9

71.9±3.1 70.4±3.0 74.2±3.1 75.5±3.1 87.1±2.9 73.0±3.2 64.4±2.4 49.1±7.1

85% v/v Ethanol-water solution at 120 °C 38.8±1.2 39.5±1.2 38.8±1.0 39.5±0.8 43.8±1.4 40.6±1.5 36.1±0.9

98.3±3.3 99.7±4.2 97.9±2.4 100.9±1.6 114.1±1.4 106.4±0.5 96.3±2.1

14.5±2.3 14.7±2.2 14.5±2.2 14.4±2.1 15.1±2.3 15.0±2.1 13.4±1.2

151.6 153.9 151.2 155.0 173.0 162.1 145.8

30.4±2.1 41.8±0.8 41.5±1.1 42.3±0.9 46.2±1.2 41.3±1.1 24.9±0.7

31.9±1.2 41.0±1.0 39.6±1.3 41.2±1.4 48.4±0.9 47.7±1.6 37.1±1.3

85% v/v Ethanol-water solution at 160 °C 0 0 0 0.5 0 1 0.5 0 1 0 0.5 0.5 1 1 Untreated

34.4±2.1 33.5±2.0 35.1±2.1 35.4±2.4 38.7±2.4 35.3±2.2 28.0±2.0 18.4±5.5

86.9±5.4 85.3±5.2 89.5±4.4 89.6±3.4 96.8±3.7 87.9±3.5 74.8±4.0 23.6±5.0

14.4±3.5 14.0±3.4 14.8±3.4 14.8±3.3 14.8±3.3 14.4±2.7 10.8±2.4 8.1±2.1

135.7 132.8 139.4 139.8 150.3 137.6 113.7 50.0

36.2±1.3 39.4±1.5 38.3±0.9 33.0±1.5 38.6±1.9 37.3±0.8 22.8±1.4 29.8±0.9

29

34.5±0.9 33.9±1.1 33.9±1.2 32.9±0.9 39.9±0.9 36.4±1.4 28.9±1.1 21.3±1.3

200

No acid

0.5% Butyric 1% Butyric 0.5% Acetic acid acid acid Glucose

Xylose

Untreated

120 °C 160 °C

120 °C 160 °C

120 °C 160 °C

0

120 °C 160 °C

0

120 °C 160 °C

100

120 °C 160 °C

5

Glucose yield (g/kg dry OFMSW)

G

F

CD

F B

300

10

A

15

E

400 BC

20 F

500

CD

25

F

600

F

700

30

CDE

35

F DE

800

120 °C 160 °C

Sugare (g/L)

40

1% Acetic 0.5% Butyric 1% Butyric acid+ acid acid+ 0.5% 1% Acetic acid Acetic acid glucose yield

Fig. 1. ( ) glucose and ( ) xylose concentration as well as (○) glucose yield after 72 h enzymatic hydrolysis of OFMSW pretreated with 85% v/v ethanol at different temperatures and catalyst dose and untreated OFMSW. Means shown by the same letter are not significantly different for total sugar.

30

25

C

C 160 °C

Untreated

A

120 °C

160 °C

BC

160 °C

B

A

120 °C

D

BC

160 °C

120 °C

A

BC

160 °C

120 °C

A

BC

160 °C

120 °C

A

120 °C

A

BC

10

160 °C

15

120 °C

Starch (g/L)

20

5 0

No acid

0.5% Butyric acid

1% Butyric acid

0.5% Acetic 1% Acetic acid 0.5% Butyric 1% Butyric acid+ 1% acid acid+ 0.5% Acetic acid Acetic acid

Fig. 2. Concentration of starch after 72 h enzymatic hydrolysis of OFMSW pretreated with 85% v/v ethanol at different temperatures and catalyst dose and untreated OFMSW. Means shown by the same letter are not significantly different.

31

16

4

14

3.5

12

3

10

2.5

B

B

B

AB

A

AB

AB

B

B

AB

1.5

B

6

AB

2 B

8

1

2

0.5

No acid

0.5% Butyric acid

1% Butyric acid

Butanol

160 °C

120 °C

160 °C

120 °C

160 °C

120 °C

160 °C

120 °C

160 °C

120 °C

160 °C

120 °C

160 °C

120 °C

0

Untreated C

4

0

0.5% Acetic 1% Acetic acid 0.5% Butyric 1% Butyric acid+ 1% acid acid+ 0.5% Acetic acid Acetic acid

Acetone

Ethanol

Butyric acid

Acetic acid

Fig. 3. Concentration of ( ) acetone, ( ) butanol, ( ) ethanol, (×) butyric acid, and (○) acetic acid after 72 h fermentation of the untreated OFMSW and enzymatic hydrolysates of the pretreated solid obtained from organosolv pretreatment by ethanol 85% (v/v) ethanol with different catalysts at 120 and 160 °C for 30 min. Means shown by the same letter are not significantly different for total ABE.

32

Acetic acid (g/L), Butyric acid (g/L)

4.5

AB

ABE (g/L)

18

Cumulative Hydrogen (mL/g substrate)

200 180 160 140 120 100 80 60 40 20 0 0

10

20

30

40

50

60

70

Fermentation time (h) Untreated

Pretreated with 1% acetic acid at 120 °C

Pretreated with 1% acetic acid at 160 °C

Pretreated without catalyst at 120 °C

Pretreated without catalyst at 160 °C

Fig. 4. Accumulated hydrogen production during 72h of fermentation for ( ) untreated, ( ) pretreated with 1% acetic acid at 120 °C, ( ) pretreated with 1% acetic acid at 160 °C, ( ) pretreated without catalyst at 120 °C, and (×) pretreated without catalyst at 160 °C

33

80

OFMSW (1 kg dry) Ethanol (7743 g)

Liquid (10535 g)

Pretreatment

Water (2432 g) Acetic acid (10 g)

Ethanol recovery

dry solid (650 g)

Buffer (14214 g) Enzymatic Hydrolysis Enzyme (211 mL) Hydrolysate (15075 g)

Fermentable sugars Starch (237 g)

Xylose (31.07 g)

Glucose (380 g)

Nutrient (158 g)

Fermentation

Water (15737 g)

Inoculum (780 g)

Acetone (44 g)

Acetic acid (46 g)

Ethanol (15 g)

Butanol (114 g)

Butyric acid (48 g)

Hydrogen (8.7 g)

Total gasoline equivalent (132.5g)

Fig. 5. Overall mass balances for organosolv pretreatment, enzymatic hydrolysis, and fermentation processes 34

35

Highlights

 Ethanolic treatment was suitable for OFMSW pretreatment to produce ABE and H2.  The addition of acetic and butyric acids as catalysts improved the biofuel yields.  Phenolic compounds exhibited an inhibitory effect on the coproduction of ABE and H2.  Hydrogen was produced after a short lag time of about 6 h.  More than 132 g gasoline equivalent was obtained from each kg OFMSW.

36