Assis, September 14th, 2021 Dear Editor-in-Chief: We are submitting the manuscript entitled: “Chemical characterization, hydrolysis and bioethanol production from municipal solid waste”, by Fabíola Ribeiro de Oliveira, Bruna Escaramboni, Pedro de Oliva Neto for consideration in Energy Conversion and Management: X. The aim of this work was the production of bioethanol from the organic portion of the municipal solid waste (MSW), using a technology in conforms to the concept of biorefinery through enzymatic hydrolysis by R. oligosporus production of glucoamylases in solid-state cultivation and alcoholic fermentation of the hydrolysate by S. cerevisiae. Chemical characterization was initially applied to achieve this goal. The bioprocess proposed was: a) efficient in convert starch into reducing sugar (relative high levels of yields were obtained). b) useful for bioethanol production. c) innovative by use of a new technology patented by UNESP (BR 102014031591-8 A2) for the enzymatic hydrolysis reaction. d) sustainable for reuse of the MSW’s organic portion. Finally, the organic fraction of MSW was able to produce a high quantity of reducing sugars through hydrolysis and of bioethanol by fermentation. This manuscript is an original work of authors and has not been published before in any form. Besides, it is not under consideration by another journal at the same time. All the authors agree with this submission as well as the instructions and recommendations of the Journal were met. Best regards, *Corresponding author: Fabíola Ribeiro de Oliveira Bioenergy Research Institute (IPBEN), Bioprocess Unit, Department of Biotechnology, Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP), Campus Assis, Avenida Dom Antônio, 2100, 19806-900, Assis, SP. Brazil Ph: +55-11-989158530 Email: fabiola.oliveira@unesp.br Highlights • Characterization evidenced 67.21% of carbohydrates with potential for reuse. • Technology patented by UNESP effectively converted starch into reducing sugars. • Concentrated hydrolyzed liquid resulted in 57.75% of bioethanol yield. Chemical characterization, hydrolysis and bioethanol 1 production from municipal solid waste 2 3 Fabíola Ribeiro de Oliveiraa,*, Bruna Escarambonia, Pedro de Oliva Netoa 4 5 6 a Bioenergy Research Institute (IPBEN), Bioprocess Unit, Department of 7 Biotechnology, Universidade Estadual Paulista "Júlio de Mesquita Filho" 8 (UNESP), Campus Assis, Avenida Dom Antônio, 2100, 19806-900, Assis, SP, 9 Brazil. 10 11 12 13 14 * Corresponding author: Fabíola Ribeiro de Oliveira 15 ORCID: https://orcid.org/0000-0001-5152-3467 16 Tel.: +55 11 989158530 17 E-mail address: fabiola.oliveira@unesp.br 18 19 20 2 Abstract 21 Food waste is increasing in the world and this residue when inappropriately disposed, cause 22 serious problems in the environment, due to its toxicity for soil and water. Then, new 23 technologies for the use of municipal solid waste (MSW) can decrease its damage to nature 24 and expenses for society, besides open up the opportunity to obtain value-added products. 25 There is also a strong interest in the development of fuels from renewable sources, aiming at 26 lower environmental impacts than the widely used fossil fuels. In view of this, biomass from 27 MSW is known for its great potential as a source for biofuels and biomolecules production. 28 Therefore, the present work aimed to produce ethanol from the organic portion of the MSW. 29 The proposed technology conforms to the concept of biorefinery, and it consisted in the 30 enzymatic hydrolysis using glucoamylases produced by Rhizopus oligosporus in solid-state 31 cultivation, through technology patented by UNESP (BR 102014031591-8 A2), which 32 showed a conversion capacity of starch to reducing sugars (RS) of 67.38%, followed by 33 alcoholic fermentation by Saccharomyces cerevisiae, producing 0.25 g of ethanol per gram of 34 reducing sugar offered. Therefore, it was found that use of this lower cost source of enzyme 35 results in good hydrolysis yielding, and consequently, considerable bioethanol production 36 from MSW. 37 38 Keywords: Biomass, biorefinery, fermentation, reducing sugars, enzymatic hydrolysis 39 40 41 42 43 44 45 3 1. Introduction 46 The significant increase in the production of municipal solid waste (MSW) is an issue 47 that has been studied and analyzed worldwide due to difficulties involved in its disposal, 48 which requires appropriate locations, and searches for alternative ways for its reuse. It is 49 estimated each person discards approximately 170 kg of organic matter per year [1]. 50 Annually, 2.01 billion tons of MSW are generated in the world [2]. Only in Brazil 79.6 51 million tons of MSW were discarded in 2019, of which 29 million were destined for places 52 considered inadequate such as dumps or landfills [1]. 53 Inappropriate disposal of huge amounts of waste promotes accentuated environmental 54 impacts, as shown by studies carried out in China and the European Union, in which waste of 55 food consumption was estimated between 62.8 and 88 million tons per year. Noting that 56 environmental impacts, in terms of ecological footprint, are in the order of 186 million ton 57 CO2 equivalent, 1.7 million ton SO2 equivalent, 0.7 million ton PO4 equivalent, in addition to 58 16292 Mm³ of grey water footprint [3,4]. Other studies also reveal that places close to 59 landfills have high levels of organic pollutants in soils and water even after 20 years of 60 inactivity [5]. 61 In addition, the high need for energy, the use of non-renewable sources and growing 62 environmental pollution have generated an urgency in the search for energy alternatives, 63 resulting in what is known as the biofuels policy. This strategy aims to reduce the emission of 64 carbon monoxide and hydrocarbons and dependence on fossil fuels. Since it uses biomass as a 65 raw material, reduces the net production of acid rain by emitting nitrogen and a lower sulfur 66 content in its burning, thus releasing a lesser amount of greenhouse gases. [6,7]. 67 Based on this idea, use of solid waste as a raw material in the production of alcohol 68 has better environmental benefits compared to conventional methods. Once there is no 69 negative impact that can come from land use, growth, harvesting, and methods of rigorous 70 pre-treatment of the traditional process [8]. The organic part contained in MSW such as 71 4 bagasse, husks, oleochemicals and food waste has the potential to generate products of 72 renewable origin such as biofuels, biosurfactants and recyclable materials. This is due to its 73 heterogeneous composition, considering that, as a rule, contain important sources of sugars, 74 lipids, carbohydrates, mineral acids, inorganic compounds, dietary fibers, phenolic 75 compounds, carotenoids, and tocopherols, which can be used through bioconversion processes 76 [9]. 77 Despite having sufficient amounts of nutrients with potential for fermentation must, it 78 needs prior processing, as occurs with carbohydrates, once ethanol-producing yeasts, such as 79 Saccharomyces cerevisiae, are not able to use all types of carbohydrates present in wastes. 80 Therefore, polysaccharides as the starch must be enzymatically hydrolyzed into glucose 81 monomers [8–10]. 82 This saccharification process takes place through some enzymes, generally in two 83 stages, with alpha and beta amylases and glucoamylases being responsible for the complete 84 degradation of starch into glucose, by starch's α-1-4 and α-1-6 glycosidic bonds hydrolysis 85 [11]. An alternative to the conventional starch hydrolysis system is a single enzymatic extract 86 and temperature, with a shorter reaction time, making starch hydrolysis more advantageous. 87 This can be done through the enzymatic complex produced from the Rhizopus oligosporus 88 fungus by bringing together the enzymes in a single amylolytic extract, with high 89 performance, and also obtaining this input from agro-industrial residues or food waste as 90 substrate, highlighting the sustainability of the process [12,13]. 91 Ethanol biofuel can be obtained by the carbohydrate fraction of residues or sugars 92 directly. However, other biofuels, such as biodiesel, can be obtained from the lipid fraction of 93 biomass. Thus, from the enzymatic hydrolysis of residues, it is possible to separate them, 94 through centrifugation, into distinct fractions, such as soluble carbohydrates, lipids, and 95 fibers. 96 5 Knowing that the hydrolysate of the organic part of the MSW tends to be a rich must 97 for fermentation, the present work consisted of the production of bioethanol from MSW. 98 Biochemical characterization, hydrolysis of the organic portion under different conditions to 99 obtain fermentable sugars, and alcoholic fermentation was used to achieve this goal. 100 2. Materials and Methods 101 2.1. Collect, separation and quantification of Municipal Solid Waste - MSW 102 Municipal solid waste was collected from different residences in the municipality of 103 Assis, SP, Brazil. This material was autoclaved at a temperature of 121 ºC and pressure of 1 104 atm, during 20 min. Then the recyclable portion was separated from the organic one and both 105 were quantified by their respective weights in relation to the total. 106 2.2. Organic fraction chemical characterization 107 After separation, the organic material was submitted to drying in an oven at 45 ºC, 108 mixed and grinded, aiming at its homogenization for greater precision in chemical 109 characterization regarding the contents of moisture, crude protein, lipids, ashes, starch and 110 fibers [14]. For ashes’ characterization, a change in the protocol was necessary, since the 111 sample was kept in a muffle furnace at 900 ºC for 48 h, until weight became constant. 112 2.3. Enzymatic hydrolysis of MSW by R. oligosporus glucoamylases 113 For the enzymatic hydrolysis of the organic fraction, was used an amylolytic extract 114 from R. oligosporus produced through technology patented by UNESP (BR 102014031591-8 115 A2), which occurs by solid-state fermentation processes and enzymatic extraction, providing a 116 competitive lower-cost bioprocess [12,15]. 117 The organic residue was mixed with 0.05 M sodium acetate buffer (pH 4.5) to obtain 118 20% (w/v) of reactional medium and gelatinized at 90 ºC for 30 min in a thermostated water 119 bath (Te 183 – Tecnal – Piracicaba, SP, Brazil). Then, temperature was reduced to 50 ºC and 120 enzymatic extrat, from the R. oligosporus cultivation, was added at 15 U/g of the substrate on 121 6 a dry basis. One unit (1 U) was defined as the amount of enzyme capable of releasing 1 µmol 122 of reducing sugar per minute. The enzymatic hydrolysis occurred by 24 h incubation and, at 123 the ending time, enzyme was inactivated in a boiling bath for 5 min, followed by cooling and 124 centrifugation at 4000 rpm for 15 min, obtaining three fractions: lipidic, hydrolysate and 125 fibers. 126 This reaction was performed in two different scales, first in falcon-type flasks (50 mL) 127 then in a 2 L benchtop bioreactor (Tecnal – Tec-Bio Plus model) with a 500 mL working 128 volume. Mechanical agitation and temperature control by water bath. Both were tested 129 without or with manual stirring at intervals of 1 h during the first 10 h and in the last 4 h of 130 reaction, with a pause between the tenth and the twentieth hour of reaction. 131 The hydrolysis yield was calculated from the concentration of reducing sugars (RS, 132 g/L), the volume of hydrolysate (H, L), and the starch in the pre-hydrolysis sample (SPH, g), 133 according to the equation: 134 Yield (%) = [(𝑅𝑆 ∗ 𝐻)/𝑆𝑃𝐻] ∗ 100 135 A kinetic study was also performed, evaluating yield of starch to reducing sugars 136 conversion, through different hydrolysis conditions: with and without stirring, with 24 h and 137 30 h of reaction and work volumes of 30 mL and 500 mL. 138 The hydrolysate fraction was characterized reducing sugars’ quantification by the 139 method of Miller (1959) [16], ashes, moisture, proteins by Kjeldahl’s method [17], and 140 soluble fibers by difference [14]. 141 2.4. Alcoholic fermentation 142 Saccharomyces cerevisiae M26, isolated and stored by the team of the Industrial 143 Microbiology Laboratory of the Faculty of Sciences and Letters - UNESP Campus Assis, SP, 144 Brazil was spread in supplemented medium with the following composition (%, w/v): 2% 145 sucrose, 1% yeast extract, 0.1% ammonium sulfate, 0.0028% ZnSO4.7H2O, 0.114% 146 7 K2HPO4.3H2O, 0.024% de MgSO4.7H2O, 0.00104% MnSO4.H2O and pH 5.0 [18]. The 147 spread was carried out in 3 steps: in test tubes containing 5 mL of medium, in 250 mL 148 Erlenmeyers containing 100 mL of medium and, finally, in 1 L Erlenmeyers containing 500 149 mL of medium. Then, a centrifugation was performed at 4 ºC, 3500 x g for 25 minutes. 150 Fed-batch process was performed according to the parameters of successful 151 methodologies by Dorta et al. 2006. To prepare the must, the hydrolyzed liquid obtained was 152 concentrated by raising the temperature, to evaporate the water, and stir until reaching the 153 amount of soluble solids accounted for at 20º Brix. At the fermentation, 9 g of S. cerevisiae 154 dry biomass was suspended in 75 mL of distilled water and 15 mL of must was added during 155 the feedings at 0, 1, 2, 3, 4, 5, 6, 7, 8 and 9 hours of the process, which lasted 24 hours with 156 stirring at 80 rpm in a shaker at 31°C [18]. 157 At the end of fermentation, there was centrifugation at 4 ºC, 3500 x g for 25 minutes, 158 aiming to separate yeast and wine, followed by both must and wine distillation and evaluation 159 with a densimeter (Anton Paar DMA 35 Basic, São Paulo, SP, Brazil). Yeast samples were 160 taken at the beginning and at the end of fermentation to carry out moisture and cell viability 161 analysis in a Neubauer Chamber and Optical Microscope using methylene blue as dye. 162 Calculations were made according to the formulas: 163 Theoretical ethanol (g) = 𝐶𝑆 ∗ 0.511 164 Theoretical ethanol (mL) = 𝐶𝑆 ∗ 0.6475 165 Yield (%) = (𝑂𝐸/𝑇𝐸) ∗ 100 166 Ethanol productivity ( 𝑔 𝐿∗ℎ ) = (𝑂𝐸 – 𝐼𝐸) / (𝑅𝑇 ∗ 𝑀𝑉) 167 Where: 168 CS = consumed sugar (g); OE = obtained ethanol (g); IE = initial ethanol (g); RT = reaction 169 time (h); MV = must volume (L). 170 8 2.5. Statistical analyses 171 For data analyses, the analysis of variance statistical test (ANOVA) and comparison of 172 means by Tukey test at a significance level of 0.05 were used. For this, the software used was 173 BioEstat 5.0 (Institute for Sustainable Development Mamirauá, Tefé, AM, Brazil). 174 3. Results and Discussion 175 3.1. Collect, separation and quantification of MSW 176 In this study, 19.41 Kg of MSW were collected, in which 16.65 Kg were organic 177 material with moisture of 67.03±7.22% or 5.48 Kg of total solids and 2.76 Kg of recyclable 178 material. Therefore, MSW was composed of 85.8% organic compounds and 14.3% recyclable 179 ones, in which there was 1.79 Kg of plastic, 0.73 Kg of degradable material and 0.24 Kg of 180 metal (Table 1). After partial drying and grinding, 3.91 Kg of the organic fraction with 181 18.59% moisture was obtained, which is equivalent to 76.53% of the initial organic portion 182 due to water loss (Figure 2). 183 Table 1. Composition of the recyclable fraction in percentage (wet basis) 184 Recyclable Materials % (w/w) Metal 8.91 Glass - Plastic 64.66 Degradable 26.43 185 According to Tyagi et al. 2018, crude MSW, on a wet basis, after collection typically 186 contains 46% organic waste, including food waste, garden waste, wood and process waste, 187 followed by 17% paper, 10% plastic, 5% glass, 4% metal and 18% other materials [19]. 188 However, several factors contribute to changes in its composition, such as the organic 189 9 fraction, which varies according to the place of origin, with a percentage of 50-70% in low-190 income communities and 20-40% in high-income communities. 191 Factors such as local culture, climate and geographic conditions are also added as 192 influencers on the amount and composition of urban solid waste (USW) [20]. Another 193 example is found in the study carried out in the city of Addis Ababa, Ethiopia by Tassie et al. 194 2019, in which the total wet-based MSW generated, recyclable materials such as metal, glass, 195 plastic, paper, wood, and rubber were estimated in 15% of the total composition and 70% of 196 the mass represented the portion of organic waste [21], a value 18.4% lower than that 197 obtained in the present work. Probably in places where there is greater industrialization and/or 198 greater consumption habits of more industrialized products, the percentage of organic matter 199 is lower as the recyclable materials increase. 200 3.2. Organic fraction characterization 201 Chemical characterization of the pre-treated and homogenized organic MSW fraction 202 was performed aiming to determine the possible applications of this material, such as biofuels 203 production, the final objective of this study. 204 Carbohydrates, including fibers and starch, accounted for 67.2% of the organic 205 fraction on a dry basis, followed by proteins (21.3%) (Table 2). Acid detergent fiber (ADF) 206 and neutral detergent fiber (NDF) totaled 40.74%, of which 29.02% were hemicellulose and 207 11.72% cellulose and lignin. 208 10 Table 2. Comparison of the chemical composition of organic waste *nd: not determined References Moisture (%, w/w) Ash (%, w/w) Proteins (%, w/w) Lipids (%, w/w) Starch (%, w/w) Raw Fiber (%, w/w) Carbohydrates (%, w/w) [22] nd* 1.46 ± 0.10 7.89 ± 0.50 5.95 ± 0.09 56.51 ± 0.60 20.2 ± 0.7 68.2 [23] nd 5.7 ± 0.2 16.3 ± 6.2 nd 40.2 ± 4.4 nd nd [19] 72.8 ± 7.6 nd 17.7 ± 5.5 17.5 ± 6.6 nd 29.2 ± 15.0 55.5 ± 10.1 [24] nd nd 6.8 – 25.8 5.6 – 24.7 11.7 – 56.5 3.5 – 51.7 32.2 – 68.2 This study 18.59 ± 0.73 4.85 ± 0.15 21.34 ± 1.83 6.60 ± 1.25 26.55 ± 4.35 40.74 ± 1.67 67.21 11 Since it is well established that the composition of a raw material in terms of fat, 212 starch, hemicellulose and cellulose determines its biofuel potential, the characterization of the 213 organic fraction is essential. Aiming at it, is interesting to carry out a nutritional analysis as 214 the main component of urban waste is food. [24]. 215 Characterization of the MSW properties changes according to the regional, seasonal 216 and socioeconomic contexts. Campuzano; González-martínez 2016 compiled the physical, 217 chemical, elementary and chemical characteristics of 43 cities in 22 countries, showing the 218 variation in waste characteristics attributed to the different cultural lifestyles and waste 219 management systems found among these countries [25]. This variation does not allow, 220 therefore, a generalization of residues' characteristics [19]. 221 Knowing this, the results obtained in the present work are within the compositions 222 described in the literature (Table 2). The high carbohydrate content and the low amount of 223 lipids are also verified in those studies, while the ash content remained close to that described 224 by Nishimura et al. 2017 [23]. However, the percentage of starch was found below that of 225 some studies, although still within the range described by Barampouti et al. 2019 [19,20,24]. 226 To establish a form of universal application of the methodologies proposed in this work, 227 taking into account the heterogeneity of MSW, it would be interesting to standardize a 228 minimum amount of starch needed before proceeding with hydrolysis and fermentation. 229 3.3 Hydrolysis of MSW by R. oligosporus glucoamylases 230 As the organic fraction of MSW is a complex mixture of easily digestible compounds, 231 mainly starch materials, the use of carbohydrate hydrolysis processes highlights the potential 232 for the production of MSW biofuels [22]. 233 First, the amylolytic activity of the enzymatic extract of Rhizopus oligosporus, 234 previously produced according to Escaramboni et al (2018) had 24.9 U/mL [12]. Thus, for 235 12 there to be 15 U/g of glucoamylase enzyme in the hydrolysis of 30 mL of reaction volume, 236 with 20% (w/v) of residue, it was necessary to add 3.62 mL of the enzymatic extract. 237 After centrifuging the hydrolyzed material, the fractions were separated and quantified 238 in volume and mass (Table 3). For the quantification in volume, the 30 mL of reaction 239 medium was taken into account, while for the quantification in mass, the mass added in the 240 reaction on a dry basis (6 g) was taken into account. Figure 2 shows the result of the 241 gelatinization process, used for better effectiveness of the hydrolysis reaction. Proportion of 242 total solids present in each organic fraction after hydrolysis was 70.32% of fibers (non-243 hydrolyzed material), 24.79% of soluble solids present in the hydrolyzed liquid and 4.9% of 244 fat fraction. Figure 1 shows the phase separation after centrifugation. 245 Table 3. Quantification in volume and mass of Municipal Solid Waste (MSW) fractions after 246 hydrolysis by glucoamylases from R. oligosporus 247 248 Reducing sugars (RS) quantification in the hydrolysate resulted in 33.78±0.49 g/L 249 (3.38%). This result showed that only 40.42% of the starch was converted into RS. After 250 scaling up the processes 16.7 times, with a work volume of 500 mL in a 1 L reactor, an 251 increase on sugars yield was verified, once there was a 67.38% yield. The manual stirring 252 process of one in one hour, used only in the hydrolysis scaling, can be responsible for the 253 increase of the process efficiency. To evaluate this hypothesis, a kinetic study of the process 254 Components Reaction medium volume Dry mass (20% reaction medium) 30 mL % (v/v) 6.06 g % (w/w) Fat 3.25 10.83 0.3 4.89 Total hydrolysate 19.25 64.17 1.5 24.79 Insoluble fiber 7.5 25 4.26 70.32 Total 30 100 6.06 100 13 was carried out (Figure 3). This study found that stirring provides greater hydrolysis yield, as 255 it resulted in an increase in the concentration of reducing sugars reaching 5.4% after 24 h of 256 hydrolysis and 6.32% when extended to 30 h, being the maximum yield starch to RS 257 conversion rate of 75.62%. 258 Fig. 1. Phases’ separation of MSW after hydrolysis and centrifugation. 259 260 Finally, the process was again carried out in 1 L flasks with 500 mL of working 261 volume, increasing the frequency of agitation and the reaction time to see if there would be an 262 improvement in the efficiency of the process, but the glucose concentration was similar to 263 previous test, with 5.6% RS and 67% (w/w) yield. In addition, the amount of soluble solids 264 present in the hydrolysate was measured through a refractometer, which resulted in 12.5 265 °Brix. 266 Table 4 presents RS concentrations obtained in the hydrolysis, considering statistical 267 analysis of the observed variations. Thus, it was noted that there was no statistical difference 268 in the results obtained at 24 and 30 h in a 500 mL reactor with manual stirring. However, on a 269 smaller scale there was a difference related to the presence or absence of stirring and related 270 14 to the time of hydrolysis. Even so, it is possible to affirm that the enzymatic hydrolysis yield 271 is more related to the agitation than to the reaction time. 272 Fig. 2. (a) MSW’s processed organic fraction (b) Appearance of the product before (c) and 273 after 30 min of gelatinization of MSW at 90 ºC (d) Hydrolysate after 24 h fermentation. 274 275 276 277 278 279 280 (a) (b) (c) (d) 15 281 Fig. 3. Kinetic study of starch hydrolysis in MSW at 50 ºC in a 50 mL Falcon tube, with the 282 addition of 15 U/g of glucoamylase enzyme obtained from the cultivation of R. oligosporus. 283 Bars represent the standard deviation of tests performed in triplicate. 284 285 Table 4. Amount of reducing sugars (RS) and hydrolysis yield under different conditions of 286 time and agitation. 287 288 289 290 291 292 293 *Means followed by different letters indicate statistical differences (p < 0.05). 294 3.4. Chemical characterization of the fermentable fraction 295 0 20 40 60 80 100 0 5 10 15 20 25 30 35 Y ie ld ( % ) Hydrolysis time (h) Hydrolysis condition Hydrolysis time (h) Reducing sugars (g/L) Hydrolysis yield (%) Without stirring– Falcon 50 mL 24 33.79±0.49 a* 40.42 With stirring – Falcon 50 mL 24 53.97±4.25 b 64.60 With stirring – Falcon 50 mL 30 63.21±5.78 c 75.62 With stirring – Reactor 500 mL 24 56.32±9.95 b 67.38 With stirring – Reactor 500 mL 30 55.95±2.09 b 67.00 16 This step evidenced the proportion of nutrients existing in the hydrolyzed liquid 296 (with 78.69% moisture), which served as a must for fermentation. A large amount of soluble 297 fiber (12.2%) was found, probably rich in hemicellulose, followed by reducing sugars (5.6%), 298 1.04% fat, 12.16% soluble fiber, 1.54% protein and 0.97% mineral residue. 299 3.5. Alcoholic Fermentation 300 After the propagation stage, 232.48 g of wet yeast were obtained, with 82.50% 301 moisture. Thus, the amount of 9 g in dry basis required for fermentation was equivalent to 52 302 g in wet basis. Cell viability before (100%) and after (99.79%) fermentation was also verified, 303 proving that there was minimal interference of the hydrolysate on S. cerevisiae growth and 304 metabolism. 305 RS and total reducing sugars (TRS) were also determined after both concentration and 306 autoclaving, aiming to verify the occurrence of a Maillard reaction, with a consequent 307 reduction in the present RS (Table 5). Results show a concentration of 145.44 g/L in the must 308 used for fermentation. 309 No statistical difference between the RS and TRS concentrations of the concentrated 310 hydrolysate before and after autoclaving was found, proving that there was no significant loss 311 of sugars by Maillard reaction during heating at 121ºC in the autoclave. 312 During the fermentation, 150 mL of must were added considering all the feeds, this 313 amount resulted in the production of 177.3 mL of “wine” (fermented must), a volume 314 quantified after centrifugation. The wine produced contained 1.91% of TRS, thus, it is known 315 that the amount of total reducing sugar contained in the must was 14.54%. Therefore, it was 316 possible to conclude that 18,42 g of sugar was consumed during fermentation. 317 Distillation process proved that there was no alcohol in the must and about 3.93% (% 318 v/v) of alcohol content in the wine. Therefore, 177.3 mL of wine with 3.067% produced 5.44 319 17 g of ethanol or 57.75% yield. From these results, it was also possible to perform the 320 calculations of ethanol productivity, which resulted in 1.51 g/L.h. 321 Table 5. Concentration of reducing sugars and soluble solids after hydrolysis, concentration, 322 autoclaving and fermentation. 323 *Means followed by different letters indicate statistical differences (p < 0.05). 324 Table 6 shows the comparison of the results of this study with the literature, 325 highlighting the concentration of 30.68 g/L of ethanol produced in this study, since 5.44 g of 326 ethanol were obtained from 177.3 mL of wine. Thus, the results of this study are within the 327 standard obtained by the referenced articles, which also used urban solid waste as 328 fermentation must and Saccharomyces cerevisiae as inoculum [22,26–28]. 329 Although some studies use different chemical pre-treatments to improve ethanol 330 production and different enzymes for MSW hydrolysis, it can be seen that there is a certain 331 constancy in the amount of ethanol produced from this source, which varies only according to 332 the amount of must used in the fermentation process [22,28]. 333 If we consider 100% of the fermentable organic matter (ie, excluding recyclable 334 material) of the MSW we have 26.55% starch with hydrolysable potential. After enzymatic 335 Reducing sugars (g/L) Total reducing sugars (g/L) Soluble solids (°Brix) Hydrolysate liquid 56.32±9.95 a* 80.19±1.80 d 12.5 Concentrated hydrolysate (must) 136.52±1.93 b 155.54±8.13 e 20 Autoclaved must 132.63±2.36 b 145.44±1.11 e 20 Wine 14.36±0.75 c 19.12±0.42 f 10 18 hydrolysis of the starch present, we have 67.38% of the capacity to convert in reducing sugars 336 (RS) therefore 17.66%. If 21.81 g of sugar offered in alcoholic fermentation produced 5.44 g 337 of ethanol, we have a Yp/So (yield of ethanol produced by sugar offered) of 0.25 g/g. 338 Therefore, of the 17.66% of RS, 4.42% (w/w) of ethanol were produced, that is, for every 100 339 Kg of MSW (considering only the fermentable fraction) we will have 4.42 Kg of ethanol 340 produced. According to ABRELPE (2020), resources invested by Brazil in the collection of 341 municipal solid waste (MSW) in 2019 were R$ 25 billion [1]. This is equivalent to 342 approximately R$ 0.31/Kg (about 0.058 US $/Kg) of collected MSW, considering 79.6 billion 343 Kg of MSW collected. Besides, considering that this amount of MSW would be capable of 344 generating 3,52 billion Kg of bioethanol or 4,46 billion L and knowing that the average price 345 of ethanol in Brazil is R$5.31, it would be possible to acquire a revenue of 23.68 billion reais. 346 Table 6. Comparison of fermentation results with literature. 347 348 349 350 351 352 353 354 * Total reducing sugars 355 **nd: not determined 356 References Must Concentration* (g/L) Yield (%) Ethanol Concentration (g/L) Ethanol Productivity (g/L.h) This study 145.44 57.75 30.68 1.51 [26] 135.00 81.00 44.00 1.83 [22] 12.00 21.80 5.81 nd [27] 120.00 nd** 39.00 nd [28] 24.00 nd 9.50 nd 19 Thus, according to these values, if the carbohydrate and hydrolysis contents obtained 357 in this work are similar to those processed in Brazil, we could produce around 3.16 billion Kg 358 of ethanol per year. The Sugarcane Agroindustry Union (UNICA), based on data published by 359 the National Agency for Petroleum, Natural Gas and Biofuels (ANP) and by the Brazilian 360 Association of Pipeline Gas Distribution Companies (ABEGAS) compiles the quantity of 361 ethanol consumed by Brazilians per year. Through this source it is known that in 2017, 25.56 362 billion liters of ethanol were consumed, which when converted into Kg by multiplying this 363 volume in m3 by the density of 789 Kg/m3, is equivalent to 20.17 billion kg of ethanol. Thus, 364 the production of 3.16 billion Kg of ethanol from MSW would be enough to supply 15.67% 365 of the national demand. 366 According to FAO (2011) 1.3 billion tons of food waste are produced in the world 367 [29], if the numbers of this work are close to the global average, it is possible to produce 368 something close to 57.46 billion Kg of ethanol. 369 From the results obtained in this study, it was possible to verify several aspects related 370 to the composition of municipal solid waste and its possibility of application, showing its 371 viability in contributing to the reduction of the amount of incorrectly discarded MSW and its 372 potential application in ethanol production. It is also important to stand out that RS 373 concentration could be substantially increased if we consider the use of fibrolytic enzymes as 374 xylanases and cellulases, although this would increase the cost of the process. 375 4. Conclusion 376 With regard to the organic portion of municipal solid waste from Assis-SP, Brazil, 377 chemical characterization evidenced the presence of fibers and starches with potential for 378 reuse, mainly through hydrolysis and subsequent fermentation. Use of the enzymatic extract 379 produced from Rhizopus oligosporus fungus, through technology patented by UNESP, proved 380 20 to be effective in converting starch into reducing sugars. Finally, the concentrated hydrolyzed 381 liquid obtained in this process resulted in good bioethanol yield, proving its potential for the 382 production of this biofuel. Therefore, the work highlights the importance of separating 383 recyclable and organic waste in the collected MSW, in view of the savings made both in the 384 treatment of these wastes and in their use as raw material for obtaining liquid fuels 385 (bioethanol). 386 387 388 Funding 389 This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo 390 (FAPESP, SP, Brazil, grant number 2019/24970-5). 391 Acknowledgments 392 The authors are thankful to the Studentship and São Paulo Research Foundation (FAPESP, 393 SP, Brazil, grant number 2019/24970-5) for the funding. The first author acknowledges her 394 parents, Marlene and Jucelino for the encouragement to research. 395 Author Contributions. 396 PON and BE conceived and designed the research. FRO conducted experiments as well as 397 organized and analyzed the data with the help of PON and BE. FRO and BE wrote the 398 manuscript. BE contributed with data statistical processing and edited the paper. All authors 399 reviewed and approved the manuscript. 400 401 Declaration of competing interest 402 The authors declare that they have no known competing financial interests or personal 403 relationships that could have appeared to influence the work reported in this paper. 404 21 Ethical Approval 405 This article does not contain any studies with human participants or animals performed by any 406 of the authors. 407 22 References [1] ABRELPE - Associação Brasileira de Empresas de Limpeza Pública e Resíduos Especiais. Panorama dos resíduos sólidos no Brasil 2020. [2] Kaza S, Yao L, Bhata-Tata P, Woerden FW. What a Waste 2.0. A Global Snapshot of Solid Waste Management to 2050. 2018. [3] Scherhaufer S, Moates G, Hartikainen H, Waldron K, Obersteiner G. Environmental impacts of food waste in Europe. Waste Manag 2018;77:98–113. https://doi.org/10.1016/j.wasman.2018.04.038. [4] Sun SK, Lu YJ, Gao H, Jiang TT, Du XY, Shen TX, et al. 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