Waste Management 137 (2022) 304–311 Contents lists available at ScienceDirect Waste Management journal homepage: www.elsevier.com/locate/wasman Enzymatic hydrolysis of the organic fraction of municipal solid waste: Optimization and valorization of the solid fraction for Bacillus thuringiensis biopesticide production through solid-state fermentation Esther Molina-Peñate a, b, Antoni Sánchez a, Adriana Artola a, * a GICOM Research Group, Department of Chemical, Biological and Environmental Engineering, School of Engineering, Edifici Q, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain b Aeris Tecnologías Ambientales S.L, Carrer Santa Rosa, 38, local, 08290 Cerdanyola del Vallès, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: To reach a more sustainable society, the implementation of a circular economy perspective in municipal waste Enzymatic hydrolysis management becomes essential. In this work, the enzymatic hydrolysis of source-separated organic fraction of OFMSW municipal solid waste (OFMSW) has been optimized as a sugar-releasing step. A liquid sugar concentrate, with a Solid-State Fermentation maximum reducing sugar concentration of 50.56 g L-1, and a solid hydrolyzed fraction were obtained. The effect Bacillus thuringiensis Biopesticide of the harshness of the hydrolysis conditions was evaluated on the performance of the resulting solid fraction as a substrate for Bacillus thuringiensis biopesticide production through solid-state fermentation. A production of 3.9 × 108 viable cells g− 1 dry matter with a 33% sporulation ratio was achieved for milder hydrolysis conditions, highlighting the potential of the solid fraction of hydrolysis as a substrate of SSF processes. The proposed valorization pathway for the OFMSW results in a sugar concentrate with potential for fermentative processes and a fermented solid containing biopesticides from Bacillus thuringiensis. 1. Introduction successfully as a pretreatment for enhancing biogas yield (Mlaik et al., 2019). Recently, the focus has been shifted to the production of higher The organic fraction represents nearly half of the globally generated market value products and enzymatic hydrolysis has emerged as a tool municipal solid waste (MSW), which is produced at a rate of 2.01 billion for extracting functionalized compounds, such as sugars, fatty acids, or tonnes per year (Kaza et al., 2018). This organic fraction of municipal proteins (Pleissner and Peinemann, 2020). Sugars are of special interest solid waste (OFMSW) comprises green and food waste from parks, as they can be the platform for the production of bioproducts through households, commerce, and restaurants (Al Seadi et al., 2013). It is an fermentation processes (Cabas Candama et al., 2020; Zhang et al., abundant organic waste, with high moisture content and complex 2020). Cellulases are the most commonly used enzymes for the hydro­ composition in polysaccharides, lignocellulose, sugars proteins, lipids, lysis of OFMSW but, considering its complex composition, the use of and macro/micronutrients (Campuzano and González-Martínez, 2016). complementary enzymatic activities, such as xylanases or amylases, is From a circular economy perspective, in which organic waste as a source advisable to increase the rate and yield of fibers conversion (de la Torre for new bioproducts must be kept within the production cycles as long as et al., 2017; Hu et al., 2013). possible (Sánchez et al., 2015), the OFMSW becomes a potential feed­ Most studies dealing with enzymatic hydrolysis as a step to release stock for biotechnological processes. sugars from the OFMSW have been focused on the use of the liquid To date, the valorization of the OFMSW has been focused mostly on fraction of the hydrolysis. Different valorization scenarios have been composting and biogas production through anaerobic digestion. Pre­ considered with relative success, for example, the production of succinic treatments have appeared as a tool to reduce the chemical complexity of acid (Stylianou et al., 2020), acetic acid (López-Gómez et al., 2019), or the OFMSW and to increase its biodegradability by enhancing access to lipids (Ghanavati et al., 2015). Conversely, few studies have also taken individual components (Romero-Cedillo et al., 2017; Romero-Güiza into consideration the valorization of the solid fraction, which has been et al., 2016). In this sense, enzymatic hydrolysis has been applied either diluted and fermented together with the liquid fraction * Corresponding author. E-mail address: adriana.artola@uab.cat (A. Artola). https://doi.org/10.1016/j.wasman.2021.11.014 Received 29 July 2021; Received in revised form 8 November 2021; Accepted 10 November 2021 Available online 22 November 2021 0956-053X/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). E. Molina-Peñate et al. Waste Management 137 (2022) 304–311 (Ebrahimian and Karimi, 2020) or simply directed to anaerobic diges­ Units (FBGU) per gram with an operating pH range of 3.5–5.5 and a wide tion processes (Mahmoodi et al., 2018). To attain the implementation of operating temperature range of 25–55◦ C. enzymatic hydrolysis as an extractive step and provide a zero-waste Experiments were conducted in sterile conditions using 500 mL alternative to the current model, it is necessary to ensure the full Erlenmeyer flasks containing 100 g of sterile ssOFMSW diluted with exploitation of the OFMSW potential. In this regard, solid-state 0.05 M sodium citrate buffer to reach a 10% (w v-1) solid to liquid ratio. fermentation (SSF) appears as a potential technology for the utiliza­ Experiments were conducted for 24 h at 180 rpm and temperature, tion of unhydrolyzed solids. initial pH, and enzyme load were adjusted according to the experimental SSF, defined as a process that occurs in the absence or near absence design (Table 2). The adjustment of the pH was done by changing the pH of free water (Pandey, 2003), has been established as an efficient and of the sodium citrate buffer. Immediately after the enzymatic hydrolysis, environmentally friendly tool for the valorization of various solid samples were centrifuged at 6000 rpm for 15 min at 4◦ C, both the su­ organic waste (Yazid et al., 2017) to produce different marketable pernatant (liquid fraction) and the pellet (solid fraction) were collected. products, such as aroma compounds (Martínez-Avila et al., 2021), bio­ The liquid fraction was centrifuged at 8000 rpm for 10 min, and the surfactants (Jiménez-Peñalver et al., 2016), or biopesticides (Mejias supernatant was collected and stored at − 20◦ C for RS determination. et al., 2020; Sala et al., 2020). Among them, biopesticides derived from The solid fraction was collected for RS determination, characterization, Bacillus thuringiensis sp. (Bt), the most widely used microbial biopesti­ and further use in the SSF process. cide, have shown a robust production on SSF using OFMSW or similar wastes because of their content in easily biodegradable organic matter 2.2.1. Optimization of the enzymatic hydrolysis procedure and a great variety of macro and micronutrients (Ballardo et al., 2017; Enzymatic hydrolysis conditions were evaluated and optimized by Rodríguez et al., 2019; Zhang et al., 2013; Zou et al., 2016). During the RSM using a Box-Behnken design. Temperature, initial pH, and enzyme sporulation phase, Bt species produce crystal inclusions containing toxic load were chosen as the three independent variables of the design and proteins (Cry or Cyt protein) (Bravo et al., 2011). These proteins are were tested at three different levels (low, medium, and high). For tem­ selectively toxic for a wide spectrum of hosts including insects of Cole­ perature (25◦ C, 40◦ C, 55◦ C) and pH (3.5, 4.5, 5.5), the selected levels optera, Lepidoptera, Diptera, Hymenoptera, Hemiptera, and Orthoptera were based on the operating ranges specified by the enzyme cocktail orders, as well as phytopathogenic nematodes and terrestrial gastropods supplier (Novozymes, Denmark). The enzyme load levels (0.01, 0.05, (Malovichko et al., 2019). The toxicity is produced by ingestion causing 0.1 mL g− 1 DM) were equivalent to 1.2, 6.6, 12 FBGU g− 1 dry matter gut cell lysis. Bt-derived biopesticides are already available in the (DM) according to the supplier and were based on the range proposed by market commercialized under different names and forms, such as Arapoglou et al. (2010) and Cabas Candama et al. (2020) and assessed DiPel®or XenTari® (Kenogard), Agree®(Bioamvac), or Deliver®(Cer­ on preliminary experiments (data not shown). Time was fixed in 24 h, tis), and produced through submerged fermentation employing defined agitation in 180 rpm, and solid load in 10% (w v-1) because it was found synthetic media. as the maximum percentage ensuring a proper mixing under the This paper aims to evaluate the enzymatic hydrolysis of OFMSW as a experimental set-up. The final design consisted of 15 runs with a trip­ sugar-releasing step to obtain a sugar-rich solution, and the subsequent licate in the central point to allow for the estimation of the pure error use of the unhydrolyzed solid fraction to produce Bt-derived biopesti­ (Box and Behnken, 1960). Two responses were considered, RS concen­ cide. First, an optimization of the hydrolysis to increase the amount of tration in the liquid fraction (mg gi-1 DM) and RS concentration in the reducing sugars (RS) is presented, providing two optimum scenarios for solid fraction per initial gram of DM (mg gi-1 DM). Calculations were the Viscozyme L® enzymatic cocktail. Then, the suitability of the done considering the 18 g of initial dry OFMSW in all the experiments resulting solid fraction to be used as a substrate of a SSF process pro­ and the % of recovered wet solids after centrifugation (around 70%). A ducing Bt spores is analyzed. To the best of our knowledge, this is the second-order polynomial model, as presented in Equation (1), was fitted first work using SSF technology to valorize the solid fraction resulting for the results of each response. from enzymatic hydrolysis of the OFMSW. ∑3 ∑3 ∑2 ∑3 Y = β0 + β Xi + i=1 i β X2 + i=1 ii i i=1 β Xi Xj j=i+1 ij (1) 2. Materials and methods where Y is the predicted response; β0 is model constant and βi , βii , βij are 2.1. OFMSW collection and preparation regression coefficients of linear, quadratic, and cross-product terms; Xi and Xj are coded independent variables. The quality of fit of the poly­ Source separated OFMSW (ssOFMSW) samples, kindly provided by nomial model equations was expressed by the coefficient of determi­ Mancomunitat La Plana (Malla, Barcelona), were collected upon arrival nation (R2) and their prediction capability by the predicted R2. at the MSW treatment plant. First, samples were screened manually for To verify the obtained models, the enzymatic hydrolysis was per­ the presence of inert materials such as glass, plastics, metals, or textiles. formed in triplicate under the optimum conditions predicted. The Bones, hard shells, hair, and excess paper were also removed. Then, experimental and predicted response values were compared and the samples were homogenized mechanically using a home composting predictive capability of the model was assessed. The efficiency of the shredder (Tecoinsaen SL, Spain), packed into 1 kg bags, and stored at hydrolysis is reported through a performance index computing the in­ − 20◦ C for a maximum of two months. Initial characterization of the two crease of RS in both fractions after the hydrolysis with respect to the RS homogenized OFMSW batches collected for this study (September and initially present in the substrate. November 2020) was performed. Before use, samples were defrosted overnight at 5◦ C and sterilized by autoclaving at 121◦ C for 30 min. 2.2.2. Statistical analysis Results were analyzed using the Design-Expert® statistical software 2.2. Enzymatic hydrolysis (Stat-Ease, Inc, United States). With the aid of the program, multiple regression analysis for the construction of the model, and statistical For the enzymatic hydrolysis, the commercial cocktail Viscozyme L® analysis of variance (ANOVA) for evaluating its significance, were from Novozymes (Denmark) was selected. This cocktail has been gaining conducted. Linear and quadratic effects of the variables and their in­ interest in the pretreatment of food waste, a major component of teractions on the release of RS were calculated and plotted into three- OFMSW, (Cabas Candama et al., 2020; Chua et al., 2021; Gabiatti Junior dimensional and contour plots. Maximum RS concentration in each et al., 2020) because of its wide range of carbohydrases, including fraction was estimated using numerical optimization. cellulase, β-glucanase, hemicellulase, xylanase, arabinase, and pecti­ nase. The declared activity by the provider is 100 Fungal Beta Glucanase 305 E. Molina-Peñate et al. Waste Management 137 (2022) 304–311 2.3. Solid-state fermentation of the solid fraction of hydrolysis system (Arduino® based). With the recorded oxygen concentration, the specific oxygen uptake rate (sOUR) was calculated as an indicator of the 2.3.1. Microorganism, inoculum preparation, and growth assessment biological activity as stated by Ponsá et al. (2010). Experiments lasted Bacillus thuringiensis var israelensis (Bti) (CECT 5904) was obtained 72 h, which has been established as the maximum spore count time for from Colección Española de Cultivos Tipo (Valencia, Spain) and preserved Bt (Cerda et al., 2019). At this time, the final pH, viable cells, and spores at − 80◦ C using a seed lot system in cryo-pearls (DeltaLab, Barcelona, were determined. Spain). A summary of the experimental process described can be seen in Inoculum preparation was performed according to the methodology Fig. 1. presented by Mejias et al. (2020). Shortly, one cryo-pearl was inoculated in 100 mL of sterilized Nutrient Broth N◦ 2 (Oxoid CM0067B, England) 2.4. Analytical methods and incubated for 20 h, at 130 rpm and 30◦ C until it reached an optical density of 2.5–3. Afterward, the culture was centrifuged for 10 min at RS were measured using the DNS method (Miller, 1959) both in the 3500 rpm and 4◦ C. The obtained pellet was resuspended in 3 mL of liquid and solid fractions of the enzymatic hydrolysis. For the solid supernatant. To reach an inoculum concentration on the solid substrate fraction analysis, a solid–liquid extraction was performed using distilled of approximately 107 CFU g− 1 DM, the resuspended inoculum was water in a 1:10 (w v-1) ratio for 30 min at 50◦ C and 150 rpm. The liquid diluted at 1:10 (v v-1) with supernatant. The final inoculum contained phase from the extraction, together with the supernatant obtained from around 108 CFU mL− 1 and no spores were detected. the centrifugation of the liquid hydrolysate were filtered through a 0.45 Bti growth was assessed in terms of viable cells and spores. The µm membrane filter and diluted with water to obtain a concentration in procedure used is as described by Mejias et al., 2020. First, a solid–liquid the range of the calibration curve (glucose, 0.2–3.3 g L-1). RS in the solid extraction was performed using Ringer solution in a 1:10 (w v-1) ratio at fraction is always expressed per initial gram of DM before the hydrolysis 150 rpm for 20 min. For spore determination, 20 mL of the previous was performed. extract were submitted to a thermal shock by incubating them at 80◦ C Solid fractions were characterized physiochemically in terms of for 10 min and then placing them into ice. Serial dilution banks of both moisture content (MC), dry matter (DM), organic matter (OM), ashes, extracts were prepared using Ringer and plated in triplicates onto Petri and pH according to standard procedures (Thompson et al., 2001). dishes containing Nutrient agar medium (Oxoid CM0003B, England). Cellulose, hemicellulose, and lignin content were determined using an The plates were incubated for 20 h at 30◦ C and viable cells or spores Ankom200 Fiber Analyzer incubator (Ankom Technology, Macedon, were estimated in terms of colony-forming units (CFUs). The sporulation NY). C/N ratio was determined using a CHNS elemental analyzer Flash ratio at a certain time was calculated considering that the viable cell 2000 (Thermo Scientific). Biodegradability was assessed through the count includes vegetative cells and spores. dynamic respiration index (DRI), which represents the average oxygen uptake rate during the 24 h of maximum activity observed, as described 2.3.2. Substrate preparation elsewhere (Ponsá et al., 2010; Sala et al., 2020). DRI is expressed in mg The solid fractions resulting from the selected as optimal conditions of oxygen consumed per g of dry matter per hour. All measurements of the enzymatic hydrolysis were collected sterile and mixed with sterile were conducted in triplicates. wood chips of particle size between 0.5 and 5 cm (Acalora, Palets Pla d’Urgell). Wood chips act as a bulking agent and are necessary to pro­ 3. Results and discussion vide porosity to the solid matrix. The resulting SSF substrate for each reactor consisted of 95 g of solid fraction and 15 g of wood chips Table 1 summarizes the characteristics of the two batches of manually mixed and inoculated with 2.6 mL of diluted Bti inoculum. ssOFMSW used in this study. The majority of the physicochemical values Triplicates for each condition studied were performed. were in close agreement with the average values from 43 cities in 22 countries presented by Campuzano and González-Martínez (2016). 2.3.3. Experimental SSF set up Except for the reducing sugars content in batch 1 (p = 0.008), the lignin SSF experiments were conducted in 0.5 L packed bed reactors under (p = 0.049), and nitrogen (p = 0.041) content in batch 2, which showed aseptic conditions. Filled reactors were placed in a temperature- significant differences (Table S.1). These parameters were reported as controlled water bath at 30◦ C and connected to a mass flow meter highly variable by the same authors. Sugars are a very easily degradable (Bronkhorst, Netherlands), which continuously supplied a specific compound and their amount fluctuates depending on the time from airflow saturated through a humidifier to prevent drying in the solid waste generation to analysis (Hansen et al., 2007). This higher RS value matrix. Airflow was set to 37 mL h− 1 g− 1 DM in all the experiments for can also explain the higher biodegradability of batch 1, measured by the ensuring aerobic conditions (Mejias et al., 2017). Exhaust gases exited DRI, which is a measure of biological activity in terms of oxygen con­ from the top of each reactor, went through a water trap, and reached an sumption. It is important to highlight that the OFMSW is an inherently oxygen sensor (Alphasense, UK) connected to a custom-built acquisition variable material that does not only variates among countries or cities, Fig. 1. Scheme of the experimental process conducted for the valorization of source-separated OFMSW (ssOFMSW). 306 E. Molina-Peñate et al. Waste Management 137 (2022) 304–311 Table 1 liquid fraction and the solid fraction (Table 2). Characterization of the two batches of source-separated OFMSW used in this The highest value of RS concentration in the solid fraction was ob­ study together with average values from 43 cities in 22 countries reported by tained in run 13, 133.2 mg gi-1 DM, corresponding to temperature 40◦ C, Campuzano and González-Martínez, 2016. initial pH 4.5, and enzyme load 0.05 mL g− 1 DM. By contrast, the Parameter Batch 1 Batch 2 (Campuzano and González- minimum value was obtained in run 9, 60.8 mg gi-1 DM, corresponding Martínez, 2016) to temperature 40◦ C, initial pH 3.5, and enzyme load 0.01 mL g− 1 DM. Moisture content 81.0 ± 72.9 ± 72.8 ± 7.6 Comparison with literature values is difficult because, among the few (%) 0.1 1.0 publications dealing with the solid fraction of enzymatic hydrolysis, DM (%) 19.1 ± 27.1 ± 27.2 ± 7.6 none of them measured the RS concentration in the solid (Ghanavati 0.1 1.0 OM (%*) 90.1 ± 89.7 ± 84.6 ± 9.9 et al., 2015; Zhang et al., 2020). As detailed in Table 2, the highest value 1.0 1.7 of RS concentration in the liquid fraction was obtained in run 4, 365.1 RS (%*) 24.1 ± 15.7 ± 10.5 ± 6 mg gi-1 DM, corresponding to temperature 25◦ C, initial pH 4.5, and 0.5a 1.3 enzyme load 0.1 mL g− 1 DM. By contrast, the minimum value was ob­ C (%*) 45.4 ± 45.5 ± 46.6 ± 4.4 tained in run 3, 167.4 mgi g− 1 DM, corresponding to temperature 25◦ C, 0.3 1.5 H (%*) 6.5 ± 6.4 ± 0.2 6.6 ± 0.62 initial pH 4.5, and enzyme load 0.01 mL g− 1 DM. Comparing both of 0.03 them, the effect of enzyme concentration is evident as at equal condi­ N (%*) 2.1 ± 0.7 1.9 ± 0.3a 2.9 ± 0.6 tions the amount of liberated RS in the liquid fraction doubled. In S (%*) 0.1 ± <0.1 0.3 ± 0.26 contrast to the solid fraction, the maximum release of RS occurred at the 0.04 maximum enzyme concentration. The maximum obtained RS concen­ C/N ratio 17 24 16 Cellulose (%*) 15.9 ± 7.4 ± 0.1 18.6 ± 15 tration in the liquid fraction, 50.56 mg mL− 1 is comparable to other 0.2 studies performing enzymatic hydrolysis of OFMSW. For example, Hemicellulose (%*) 10.0 ± 6.8 ± 0.1 8.6 ± 4.6 López-Gómez et al. (2019) obtained 55.41 mg mL− 1 of glucose from a 0.1 source selected waste, and Ebrahimian and Karimi (2020) a maximum of Lignin (%*) 7.8 ± 0.3 2.1 ± 0.2a 9.7 ± 5.3 pH 5.25 ± 5.31 ± 5.02 ± 0.95 35 mg mL− 1 of glucose. Generally, the achieved RS concentration is 0.05 0.04 lower than those experiments that employed selected food waste, which DRI 7.5 ± 0.2 4.2 ± 0.3 NA reached between 130 and 170 mg mL− 1 (Yan et al., 2011; Zhang et al., 1 (mg O2 g− DM 2020), because this type of waste is lesser in complexity and amount of h− 1) impurities (Alibardi and Cossu, 2015). An exception was found in Cabas * Dry basis. NA, not available. Data presented as mean values ± standard Candama et al. (2020), who reached 25 mg mL− 1 of glucose using waste deviation (n = 3). a significantly different parameter (p < 0.05) based on the from fruit and vegetables and the same enzymatic cocktail as the Tukey test analysis. employed in this paper. In every run the concentration of RS obtained in the solid fraction was lower than that initially present in batch 1 (241 but also within the same region by other factors such as weather con­ mg g− 1 DM), indicating the solubilization of the sugars towards the ditions, seasonal changes, nutritional habits, or recollection system liquid fraction. The total amount of released RS increased almost two- (Campuzano and González-Martínez, 2016; Hansen et al., 2007). fold in runs 4 (467.2 mg gi-1 DM) and 8 (466.9 mg gi-1 DM) suggesting gradual hydrolysis of the fibers present in the OFMSW. Those runs (3, 7, 9, 12) without enzyme addition did not show an increase in RS after the 3.1. Enzymatic hydrolysis as sugar releasing step enzymatic hydrolysis as displayed by the performance index of 1, proving the hydrolysis effect of the cocktail to a greater or lesser extent The evaluation of the enzymatic hydrolysis as a sugar-releasing step depending on the conditions of the hydrolysis. was performed using batch 1 of ssOFMSW and based on three opera­ In general, low enzymatic load and high pH lead to lower RS con­ tional parameters for two response variables. The operational parame­ centrations in both fractions whereas medium and high enzyme loads ters studied were temperature, pH, and enzyme load, previously and medium and low pHs lead to larger amounts of RS released. identified as significant factors affecting the enzymatic hydrolysis of Reaching an agreement between the sugar release in both fractions, as organic wastes (Guan and Yao, 2008; Yan et al., 2011). These parame­ seen in runs 1, 5, and 8 in Table 2, would maximize the potential of ters were evaluated based on their effect on the release of RS in the Table 2 Design matrix of the Box-Behnken model and observed responses: reducing sugars in the solid and liquid fractions. The performance of the hydrolysis is expressed as fold increase of RS. Run Temp (◦ C) pH Enzymatic load RS solid fraction RS liquid fraction Hydrolysis performance* (ml g1 DM) (mg gi-1 DM) (mg mL− 1) (mg gi-1 DM) 1 25 3.5 0.05 121.8 43.5 314.3 1.8 2 25 5.5 0.05 121.6 38.0 274.8 1.6 3 25 4.5 0.01 80.2 23.2 167.4 1.0 4 25 4.5 0.1 102.1 50.6 365.1 1.9 5 55 3.5 0.05 130.8 45.2 326.1 1.8 6 55 5.5 0.05 94.4 31.5 227.3 1.3 7 55 4.5 0.01 66.8 25.2 182.0 1.0 8 55 4.5 0.1 128.5 46.9 338.5 1.9 9 40 3.5 0.01 60.8 23.7 171.5 1.0 10 40 3.5 0.1 124.5 40.8 295.0 1.7 11 40 5.5 0.01 69.2 25.1 181.2 1.0 12 40 5.5 0.1 82.2 41.6 300.8 1.6 13 40 4.5 0.05 133.2 33.2 239.5 1.3 14 40 4.5 0.05 119.7 35.4 256.0 1.6 15 40 4.5 0.05 122.3 30.7 221.4 1.4 * 1 Calculated as the total amount of RS (liquid and solid fraction) with respect to the initially measured in batch 1 (241 mg g− DM). 307 E. Molina-Peñate et al. Waste Management 137 (2022) 304–311 OFMSW hydrolysis as a pretreatment of fermentative steps. whole temperature range described by the provider. This observation has also been recently reported by Cabas Candama et al. (2020) for waste of fruits and vegetables. The effect of the pH, reported as a 3.2. Process optimization and verification parameter with significant effects in Table 3, can be seen in Fig. 2.A and Fig. 2.C. The RS concentration increases when pH decreases, reaching The RSM Box-Behnken design was implemented to optimize the the maximum at pH 3.5. However, as shown in Fig. 2.C, for low tem­ sugar release from OFMSW, both in the liquid and solid fractions of perature (25◦ C) the maximum RS concentration is reached at pH around hydrolysis. The variables were analyzed by a multiple regression anal­ 4.5. The adequate performance on milder conditions, i.e. lower tem­ ysis to obtain a regression equation that could predict the response perature and not so acidic pH, makes Viscozyme L a notable cocktail for within the specified range (Guan and Yao, 2008). Analysis of variance OFMSW hydrolysis as a reduction in energy and chemical requirements (ANOVA) was used to investigate the significance of fit for the model highly influences the process economic cost (Alvira et al., 2010). Lastly, equations. As the goal of this paper is the valorization of the solid enzyme load was found the most significant parameter (Table 3) as fraction of hydrolysis the following discussion is focused on it but the evident in the steeper surfaces observed in Fig. 2.A and Fig. 2.B model and ANOVA for the liquid fraction can be found in the supple­ compared with Fig. 2.C. It can be seen that RS concentration increases as mentary material (Table S.2). enzyme load does until a certain value after which decreases. This The experimental results were fitted with a quadratic equation. The observation suggests that the enzymes had limited access to the solid ANOVA analysis presented in Table 3 resulted in a significant regression fraction so all attachment sites in the solid were occupied reaching an model (p < 0.05) and a not-significant lack of adjustment (p > 0.05). enzyme saturation effect, which has also been previously reported by Consequently, the model presents a good adjustment with the experi­ Cabas Candama et al. (2020) for waste of fruits and vegetables. At the mental data reported. A logarithmic transformation of the response for same time, it is important to highlight that the hydrolysis of the complex analyzing the data was performed to achieve a better adjustment in the composition of OFMSW requires several enzymatic activities. For food quadratic model (Joglekar and May, 1987). The parameters enzyme waste, the major contributor to OFMSW, it has been shown that poly­ load and pH showed significant effects, specifically, the linear effect of saccharides such as xylan and pectin, can interfere in the hydrolysis of the enzyme load was the most significant as the smaller the p-value, the cellulose and hemicellulose by masking them (de la Torre et al., 2017; more significant the corresponding coefficient (Haber and Runyon, Van Dyk et al., 2013). Viscozyme L contains a wide range of carbohy­ 1973). According to the R2, which is a measure of the degree of fit drases addressing these fractions, however, the design of tailor-made (Haber and Runyon, 1973), the model could explain 98.76% of the cocktails with ratios of enzymatic activities specific for OFMSW or variability in the response. The model also has the capacity to explain with increased substrate specificity by protein engineering, might bring 88.40% of the variations in new observations according to the predicted further the release of sugars from OFMSW (Chapman et al., 2018). R2, which is in reasonable agreement (<0.2) with the adjusted R2 Finally, optimization of the RS concentration in the solid fraction (96.51%) (Haber and Runyon, 1973; Joglekar and May 1987). In addi­ was conducted by a numerical optimization method using the Design- tion, it shows a low coefficient of variation (1.11%), being indicative of Expert® software. The target was to maximize the RS concentration the reliability of the experimental design. The resulting regression while keeping temperature, initial pH, and enzyme load within the study equation for the response of RS concentration in the solid fraction for the range. The optimum operating conditions were close to temperature conditions studied is presented in Equation (2). 55◦ C, initial pH 3.5, and enzyme load 0.08 mL g− 1 DM, and correspond log10 RS(mgg− 1 DM) = + 2.10 − 0.007X1 − 0.033X2 + 0.10X3 − 0.035X1 X2 to 157.0 mg gi-1 DM. Considering that milder conditions would benefit the economy and energy balance of the process, another optimum that + 0.045X1 X3 − 0.059X2 X3 + 0.011X12 − 0.042X22 − 0.146X32 minimizes temperature and enzyme load was also selected. Mild con­ (2) ditions were temperature 25◦ C, enzyme load 0.06 mL g− 1 DM, and initial pH 4.5, which correspond to a prediction of 131.9 mg gi-1 DM. where X1 , X2 and X3 represent the temperature (◦ C), initial pH, and Both conditions, extreme and mild, were verified experimentally. enzyme load (mL g− 1 DM), respectively. Considering the inherent variability of the OFMSW, it was decided to The interaction effects of the three variables were corroborated in verify the optimum conditions using batch 2 of ssOFMSW to also assess three-dimensional response surface plots (Fig. 2). Based on the ANOVA the reproducibility of the process. The experimental results obtained analysis (Table 3), the temperature was not a significant parameter as were 122.6 ± 13.4 mg gi-1 DM for extreme conditions and 105.2 ± 9 mg illustrated in plots showing the interaction of temperature with enzyme gi-1 DM for mild conditions. The experimental RS concentrations load and pH (Fig. 2.B and Fig. 2.C, respectively). Therefore, the Visco­ observed using conditions predicted by the model were lower than ex­ zyme L cocktail was suitable for the hydrolysis of OFMSW through the pected but within the 95% confidence interval for the extreme and the 99% for the mild. It should be noted that batch 2 contained considerably Table 3 fewer sugars (157.7 mg g− 1 DM) than batch 1, which might explain the ANOVA for the response surface quadratic model when RS concentration in the lower RS concentrations achieved. These results are quite satisfactory solid fraction was used as a response. considering the complexity of working with such a variable substrate as Source DF Mean Square p-value OFMSW (Hansen et al., 2007). This lower initial amount of sugars Model 9 0.1154 0.0003* resulted in greater hydrolysis performance, 2.7 for mild conditions and X1 – Temperature (◦ C) 1 0.0023 0.3908 2.9 for the extreme. X2 – pH 1 0.0474 0.0081* 1 These conditions were selected for maximizing the RS concentration X3 – Enzyme load (mL mg− DM) 1 0.3978 0.0001* X1⋅X2 1 0.0264 0.0247* in the solid fraction, however, they also led to high values in the liquid X1⋅X3 1 0.0429 0.0099* fraction (304.2 ± 9.5 mg gi-1 DM for mild conditions and 339.4 ± 7.2 mg X2⋅X3 1 0.0739 0.0032* gi-1 DM for extreme conditions, equivalent to concentrations of 42 and X1 2 1 0.0024 0.3867 47 g L-1respectively). This can be explained because the hydrolysis of the X2 2 1 0.0347 0.0149* X3 2 1 0.4175 0.0001* solid fibers is a gradual process, and partially hydrolyzed fibers in the Residual 5 0.0026 solid fraction might be solubilized during the extraction required for RS Lack of Fit 3 0.0022 0.6315 analysis. The partial hydrolyzation of fibers results in easily accessible Pure Error 2 0.0033 sugars during the SSF. R2 = 98.76%, R2 (adj) = 96.51%, R2 (pred) = 88.40%, and C.V. = 1.11%. *Significant parameters (p < 0.05). 308 E. Molina-Peñate et al. Waste Management 137 (2022) 304–311 Fig. 2. Combined effect of (A) enzyme load and pH; (B) enzyme load and temperature; and (C) pH and temperature in RS concentration in the solid fraction. Other factors were at medium levels. 3.3. Assessment of the solid fraction of hydrolysis as a substrate for spore count and 3.9 × 108 CFU g− 1 DM for viable cell count. Viable cell biopesticide production through SSF value was in close agreement with those obtained by Mejias et al. (2020), but there still was potential for improvement in the sporulation A preliminary assessment has been performed to evaluate the capa­ ratio (33%). Conversely, the solid hydrolysate derived from the extreme bility of the obtained solid hydrolysate to support Bti growth and conditions did not support Bti growth but resulted in a 12-fold decrease. sporulation. Even though spore count and endotoxicity are not always This could be related to the low initial pH which was 5.2 compared with proportional, spore count can be considered an indirect estimation the 6.3 of the mild treatment. This pH value was lower than the optimum method because during sporulation each Bt cell produces a spore that reported for Bti sporulation (7.0) and close to inhibitory (5.5) (De might contain toxic proteins (De Lourdes Tirado Montiel et al., 2001). Lourdes Tirado Montiel et al., 2001; Özkan et al., 2003). Therefore, an The SSF operation parameters were based on the optimization per­ additional step of pH adjustment using 1 M NaOH to neutral values after formed by Mejias et al. (2020) using biowaste mixed with digestate as the enzymatic hydrolysis was evaluated in the SSF. Results from this SSF substrate. The performance of the solid hydrolysate fractions from experiment can also be seen in Fig. 3. It is illustrated how the growth of extreme and mild pretreatment conditions of batch 2 was evaluated on a Bti was favored by the change of pH, reaching values of viable cells of triplicate after 72 h. The solid fractions were characterized before the 2.5 × 107 CFU g− 1 DM and spores of 1.6 × 106 CFU g− 1 DM, with a 6% of SSF in terms of DM and pH, resulting in 23.2 ± 0.9 and pH 6.3 for the sporulation ratio. Therefore, after a pH adjustment, the solid hydroly­ mild hydrolysis and 23.9 ± 1.1 and pH 5.2 for the extreme hydrolysis sate derived from extreme conditions also appeared as a suitable sub­ conditions. strate for Bti growth but not as promising as the one derived from mild The fermentation started with no spore presence in the solid matrix pretreatment conditions. Regarding the RS concentration, values at 72 h and, as seen in Fig. 3, the solid hydrolysate derived from the mild con­ were below the detection levels of the method, which indicates con­ ditions reached an average concentration of 1.3 × 108 CFU g− 1 DM for sumption of over 90% of the RS. Compared to a previous work using 309 E. Molina-Peñate et al. Waste Management 137 (2022) 304–311 1e+9 Acknowledgments Viable cells t=0h Viable cells t=72h Esther Molina thanks Generalitat de Catalunya for her pre-doctoral Viable cells & Spores (CFUs g DM) Spores t=72h grant (DI 7, 2019). The authors thank Mancomunitat La Plana, for 1e+8 -1 providing the source-separated OFMSW. Financial support was provided by the Spanish Ministerio de Ciencia e Innovación (Ref. PID2020- 114087RB-I00). 1e+7 Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi. 1e+6 org/10.1016/j.wasman.2021.11.014. References 1e+5 Mild Extreme Extreme + pH Al Seadi, T. 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