Waste and Biomass Valorization (2023) 14:1433–1445 https://doi.org/10.1007/s12649-022-01978-5 ORIGINAL PAPER Bacillus thuringiensis Production Through Solid‑State Fermentation Using Organic Fraction of Municipal Solid Waste (OFMSW) Enzymatic Hydrolysate Esther Molina‑Peñate1,2 · Nil Arenòs1 · Antoni Sánchez1 · Adriana Artola1 Received: 8 July 2022 / Accepted: 6 November 2022 / Published online: 16 November 2022 © The Author(s) 2022 Abstract This paper aims to explore an alternative pathway to valorize the organic fraction of municipal solid waste. It is based on the use of enzymatic hydrolysis to obtain a sugar-rich fraction with the potential for liquid fermentative processes and a partially hydrolyzed solid that is evaluated as a substrate for solid-state fermentation. Different strategies to modify the pH of the solid substrate to reach a neutral pH, suitable for the growth of biopesticide producer Bacillus thuringiensis, have been explored. The use of alkaline cosubstrates was evaluated on two different scales and temperature was assessed as a preliminary indicator of the scale-up viability of the process strategy. By ensuring a proper pH throughout the process, the growth and sporulation of Bacillus thuringiensis were achieved. The best cosubstrates and mixing ratios were 50% of digested sewage sludge and 25% of digested organic fraction of municipal solid waste, which led to a spore concentration of 1.1 × ­109 spores ­g−1 of dry matter and 6.4 × ­108 spores ­g−1 of dry matter, respectively. Overall, a reproducible and flexible solid-state fermentation process has been achieved for hydrolyzed organic municipal waste based on the use of alkaline urban wastes as cosubstrates. This valorization pathway fits with the concept of urban biorefineries. Graphical Abstract Keywords Enzymatic hydrolysis · Solid-state fermentation · Biowaste · Biopesticide · Bacillus thuringiensis * Adriana Artola adriana.artola@uab.cat Extended author information available on the last page of the article 13 Vol.:(0123456789) 1434 Waste and Biomass Valorization (2023) 14:1433–1445 Statement of Novelty succinic acid [17], bacterial poly(3-hydroxybutyrate) [18], and biofuels [19]. The remaining partially hydrolyzed and The present research provides a novel, flexible and repro- non-hydrolyzed fibers could also be used in fermentation ducible solid-state fermentation process for the valorization processes, even though the literature on the topic is scarce. of solid hydrolysates from the organic fraction of munici- Solid-state fermentation (SSF) is described as the aero- pal solid waste into biopesticides in the context of an urban bic fermentation process that takes place in near absence waste biorefinery. The results obtained will aid in the search of free water [20]. It has been gaining research interest as and implementation of alternative valorization pathways for it can use different solid organic wastes as substrate and specific byproducts of urban waste with large environmental does not always demand strict sterile conditions [21–24]. impacts associated. For instance, it has been used to produce fungal and bacterial biopesticides [25, 26], aroma compounds [27], bioplastics [28], and biosurfactants [29, 30]. When working with urban Introduction organic wastes, complete sterilization is an arduous and energy-intensive task. Therefore, robust microorganisms that With the increasing municipal solid waste (MSW) genera- can thrive in non-sterile environments would facilitate pro- tion over the years, significant political and social efforts cess implementation in real scenarios. In this sense, Bacil- have been made to introduce selective collection systems lus thuringiensis (Bt) species have been grown successfully that allow for its recycling. In the case of the organic fraction under not completely sterile conditions in soy residues [31], (OFMSW), which represents nearly half of the total MSW OFMSW [24], digestate [22], and wastewater [32]. Biopes- [1], selective collection systems prevent its natural biodeg- ticides derived from Bt constitute almost 90% of the world’s radation associated with the release of greenhouse gases and biopesticide market [33], a market growing annually at a other major environmental impacts [2]. Besides, it improves rate of 15% [34]. These microorganisms are Gram-positive the quality of OFMSW and facilitates its recycling, which soil bacteria that produce endotoxin proteins accumulated is commonly performed through composting or anaerobic in parasporal crystals during the sporulation phase, which digestion (AD) [3, 4]. Considering the complex composition are selectively toxic for pest insects mainly belonging to the of the OFMSW, rich in polysaccharides, lignocellulose, pro- orders Lepidoptera, Diptera and Coleoptera [35]. Specifi- teins, lipids and macro/micronutrients [5], this waste frac- cally, Bacillus thuringiensis var. israelensis (Bti) has been tion could be exploited to a larger extent for the production described as the most environmentally friendly agent for of higher-value bioproducts in a biorefinery-like scenario the control of larval mosquitoes [35] Conventionally, Bt has [6]. Besides contributing to the urgent need for ensuring been produced in liquid media through submerged fermen- materials and energy supply [7], such a scenario would also tations but its sporulation process can benefit from solid justify the significant economic investment required to build cultivation [33]. Also, SSF opens up doors for more sim- a robust and efficient source-separated collection system [3, ple solid formulation possibilities. Like many bacteria, Bt 6]. In this urban biorefinery context, the widely implemented thrives at a pH near neutrality [36, 37]. However, pH control technology of AD for biogas production can still be used as and monitorization during the course of SSF processes are a complementary technology to handle intermediates [8]. difficult due to the heterogeneity of the solid matrix, a lim- Also, due to its robustness, it can be considered a tool to ited amount of free water and a lack of suitable online pH reduce the risk derived from the heterogeneity and variabil- measurement methods. Also, the solid substrate itself might ity of the OFMSW as a substrate. have a buffering effect due to its complex chemical composi- Enzymatic hydrolysis has emerged as a powerful tech- tion [12]. Another major challenge of this technology is the nology during the development of lignocellulosic biorefin- heat and mass transfer-related issues that arise during the eries to break down macromolecules into their functional scale-up. Therefore, monitoring process temperature from units [9]. So, its application to municipal waste streams has early implementation stages is important to prevent heavy also been gaining interest [10–13]. The main components efficiency losses at larger scales [38, 39]. Also, an adequate of the OFMSW are carbohydrates and fibers, representing temperature of around 30 °C must be ensured during the up to 85% of its composition [5], hence their fractioning growing phase of Bt [36]. can generate a variety of fermentable sugars [14]. From a In this paper, the use of the solid hydrolysate from enzy- biorefinery perspective, these sugars act as building blocks matic hydrolysis of OFMSW is evaluated as a substrate for for different fermentative processes, especially when col- Bt growth via SSF. Two operational strategies have been lected in a liquid fraction [15]. Several papers have explored tested to adjust pH near neutrality and maximize Bt growth the use of sugars obtained from the OFMSW in submerged and sporulation at 0.5 L: chemical pH modification and the fermentation, for instance, the production of acetic acid [16], use of cosubstrates with high buffering capacity. Specifi- cally, byproducts of the AD process of urban wastes have 13 Waste and Biomass Valorization (2023) 14:1433–1445 1435 been selected as cosubstrates. Process development to a Table 2  Characterization of the solid hydrolysates obtained from 1.5 L reactor without temperature control and using non- each batch of OFMSW sterile substrates has also been done to study its suitability Parameter Batch 1 (n = 2) Batch 2 (n = 3) for full-scale implementation in a biorefinery-like environ- MC (%) 75.3 ± 1.3 74.7 ± 4.5 ment for OFMSW. DM (%) 24.7 ± 1.3 25.4 ± 4.5 OM (%)a 88.7 ± 1.4 91.7 ± 1.7 pH 5.6 ± 0.2 5.3 ± 0.1 Materials and Methods RS (%)a 14.0 ± 2.9 13.3 ± 1.7 RS liquid fraction (g/L) 35.6 ± 4.2 46.4 ± 4.4 OFMSW Collection and Hydrolysate Production Data presented as mean values of the different hydrolysis rounds for The substrate of the enzymatic hydrolysis was the OFMSW each batch ± standard deviation collected from a door-to-door collection system selected MC moisture content, DM dry matter, OM organic matter, RS reduc- due to its high quality. It was collected upon arrival at the ing sugars a MSW treatment plant of Mancomunitat La Plana (Malla, Dry basis Barcelona). The few inert materials (< 1% w ­w−1), such as plastic, metals, glass or textiles, were removed manually as solid-to-liquid ratio was set to 10% (w ­v−1). Erlenmeyer well as hard shells, bones, hair and excess paper. Then, the flasks were incubated for 24 h at 180 rpm and rapidly cen- OFMSW was shredded mechanically using a home compost- trifuged at 6000 rpm for 15 min at 4 °C. Then, both frac- ing shredder (Tecoinsaen SL, Spain) and stored at − 20 °C tions were separated and the solid fraction was collected for a maximum period of three months. Before use, samples and stored at 5 °C until its use in the SSF for a maximum were defrosted overnight at 5 °C and sterilized by autoclav- time of 48 h. Characterization of the final hydrolysate can ing at 121 °C for 30 min. Two different batches of OFMSW be seen in Table 2. (October 2021 and January 2022) were used in this study and their initial characterization can be seen in Table 1. Solid‑State Fermentation Enzymatic hydrolysis of homogenized OFMSW was performed using a tailor-made enzymatic cocktail kindly Microorganism and Inoculum Preparation provided by ASA Spezialenzyme GmbH (Wolfenbüttel, Germany) and composed mainly by a blend of cellulases All tests were carried out using Bacillus thuringiensis var and pectinases but also hemicellulase, ß-glucosidase and israelensis (Bti) strain CECT 5904 obtained from Colección α-amylase. Experiments were conducted under sterile con- Española de Cultivos Tipo (CECT, Valencia, Spain) and pre- ditions in 2 L Erlenmeyer flasks following the provider’s served at − 80 °C using a seed lot system in cryo-pearls recommendations at 50 °C and initial pH of 4.5, modified (DeltaLab, Barcelona, Spain). For inoculum preparation, using 0.05 M sodium citrate buffer, and 0.05 mL of enzy- one cryo-pearl was inoculated in 100 mL of sterile Nutri- matic cocktail per g of initial dry matter (DM). The initial ent Broth nº2 (Oxoid CM0067B, England) and incubated at 30 °C and 130 rpm for 20 h when an optical density of 2.5–3.0 was reached. The culture was centrifuged for 10 min Table 1  Characterization of the batches of source-separated OFMSW at 3500 rpm and 4 °C. First, the obtained pellet was resus- collected in this study and average values from 43 cities in 22 coun- tries reported by Campuzano et al. [5] pended in 3 mL of the exhausted media and then, diluted 1:10 (v v.1) also with supernatant to reach approximately a Parameter Batch 1 (10/21) Batch 2 (01/22) Literature concentration of 1­ 08 CFU m­ L−1. No spores were detected MC (%) 77.2 ± 0.5 76.4 ± 1.0 72.8 ± 7.6 at this point. DM (%) 22.8 ± 0.5 23.6 ± 1.0 27.2 ± 7.6 OM (%)a 88.2 ± 1.1 89.7 ± 0.7 84.6 ± 9.9 Chemical pH Modification of the Solid Hydrolysate RS (%)a 17.0 ± 0.7 16.6 ± 0.9 10.5 ± 6 pH 5.6 ± 0.2 5.6 ± 0.1 5.02 ± 0.95 Chemical pH modification was done using calcium carbon- DRI24h 4.9 ± 0.7 3.5 ± 0.5 NA ate ­(CaCO3). First, the required amount to reach a pH of 7 (g ­O2 ­kg−1 DM ­h−1) was determined by adding increasing amounts (1–12 mL) Data presented as mean values ± standard deviation (n = 3). of a 1 M solution to the solid hydrolysate and thoroughly mixing it manually. A 10% of MC increase was set as the MC moisture content, DM dry matter, OM organic matter, RS reduc- ing sugars, DRI24h, dynamic respiration index average in the 24 h of maximum modification possible. The solid was left for 1 h maximum activity, NA not available at a cold temperature (5 °C) to settle and then pH was meas- a Dry basis ured according to standard procedures. 13 1436 Waste and Biomass Valorization (2023) 14:1433–1445 Cosubstrates and Mixtures Preparation The different amounts of cosubstrates used for preparing the mixture were calculated based on wet weight (25 and 50%). Three different organic materials related to urban wastes Materials were mixed manually and inoculated with 3 mL were evaluated as cosubstrates for the SSF process: (i) of diluted Bti to reach approximately ­107 CFUs ­g−1 DM. digested and dewatered sewage sludge coming from the Then, the best-performing scenarios were validated in AD process of a municipal wastewater treatment plant triplicate at 1.5 L and the effect of the mixtures on the tem- (Sabadell, Spain), (ii) digestate from the AD process of a perature was assessed as a preliminary step for scaling up. In source selected OFMSW treatment plant (Consorci per a la this case, 360 g of substrate mixture and 80 g of the bulking Gestió dels Residus del Vallès Oriental, Granollers, Spain), agent were mixed and inoculated with 14 mL of diluted Bti and (iii) compost from a composting plant of source selected inoculum. OFMSW (Planta de compostatge de Sant Cugat, Barcelona, Spain). Materials were characterized in terms of dry matter, organic matter (OM), pH, conductivity and biodegradability Monitoring Parameters upon arrival, as detailed later, and stored frozen until use for a maximum period of 3 months. Once defrosted overnight at All experiments lasted 72 h, which has been established 5 °C, both digestate and digested sewage sludge were sub- previously as the maximum spore counting time for Bt jected to a hygienization pretreatment (1 h at 70 °C) before [21] and were evaluated in terms of viable cells and spores their use in SSF processes to pasteurize them as specified in production. First, a solid-liquid extraction was performed the European Regulation Nº 142/2011. using Ringer solution in a 1:10 (w ­v−1) ratio at 150 rpm for These materials were mixed individually with the solid 20 min. Then, the extract was appropriately diluted and 50 hydrolysate at two different weight ratios, 25 and 50%, to µL plated in triplicate onto Petri dishes containing a Nutri- prepare the final mixtures used in the SSF processes. The ent agar medium (Oxoid CM0003B, England). To measure same amount of sterile wood chips of particle size between spores, 20 mL of the previous extract were submitted to a 0.5 and 5 cm (Acalora, Palets Pla d’Urgell, Spain) was added thermal shock by incubating them at 80 °C for 10 min and to the mixtures as a bulking agent to provide porosity to the then placing them into ice before plating [22]. All plates solid matrices. were incubated at 30 °C for 20 h and viable cells or spores were estimated in terms of colony-forming units (CFUs) and Experimental SSF Set‑Up related to the DM of the sample, following the equation: SSF experiments were performed at cylindrical polyvinyl Viable cells & spores concentration (CFUs per g DM) chloride packed-bed reactors of two different scales, 0.5 and n◦ CFUs ⋅ D ⋅ Ex = 1.5 L. Reactors were completely sealed and equipped with 0.05 ⋅ DM an air inlet and outlet port on the bottom and the top respec- where, nº CFUs is the average of counted CFUs in the tively. A humidified airflow was provided through a mass Petri dishes, D is the dilution factor of the extract, Ex is the flow meter (Mass-Stream D-6311, Bronkhorst, NL) set to extraction factor (9 mL per g of wet solid), 0.05 is the mL constant aeration of 37 mL g­ −1 DM h­ −1 for ensuring aerobic plated and DM is the sample dry matter per g of wet solid. conditions [40]. The oxygen concentration of the exhausted The sporulation ratio at a certain time is calculated con- gases was measured after a water trap by an ­O2–A2 oxygen sidering that the viable cell count includes both vegetative sensor (Alphasense, UK) connected to a custom-built data cells and spores according to the following equation: acquisition system (Arduino® based). The main difference between both scales was that, at 0.5 L, the temperature was spores g−1 DM Sporulation ratio(%) = controlled and kept constant at 30 °C by placing the reac- viable cells g−1 DM tor in a water bath, whereas at 1.5 L temperature was not controlled but only monitored using button temperature sen- The sporulation yield that expresses the concentration of sors (Maxim Integrated, U.S.) to obtain accurate temperature spores produced per initial viable cell inoculated is calcu- profiles at different reactor heights (10 and 20 cm). lated using the final spores concentration and the initial cell Initial experiments were performed at 0.5 L evaluating concentration. both the effect of the chemical modification of pH and the final spores use of cosubstrates on Bti growth and sporulation. Experi- Sporulation yield (spores per viable cell inoculated) = inital cells ments were performed in duplicate for each condition. The total amount of the final mixture was kept constant for all With the measured oxygen concentration at the outlet port the conditions and was 90 g of the substrate and 20 g of of the reactor, the specific oxygen uptake rate (sOUR) was the bulking agent, corresponding to a ratio of 1:2 v ­v−1. 13 Waste and Biomass Valorization (2023) 14:1433–1445 1437 calculated as an indicator of the biological activity according consumption of the four days after the lag phase expressed to the following equation [41]: ­ 2 ­kg−1 DM, as described elsewhere [40, 41]. All meas- in g O urements were conducted in triplicates. ( ) P ⋅ 32 ⋅ 60 ⋅ 1000a sOUR = F ⋅ 0.209 − yO2 ⋅ R ⋅ T ⋅ DM ⋅ 1000b where, sOUR is the specific oxygen uptake rate (g ­O2 ­kg−1 Results and Discussion DM ­h−1); F, airflow rate into the reactor (ml ­min−1); ­yO2, oxygen molar fraction in the exhaust air (mol O ­ 2 ­mol−1); P, The characterization of the OFMSW samples used in this pressure of the system assumed constant at 101,325 (Pa); study was in line with average literature values (Table 1). 32.6, oxygen molecular weight (g ­O2 ­mol−1 ­O2); 60, the During enzymatic hydrolysis, the complex carbohydrates conversion factor from minute to hour; ­1000a, conversion that comprise the OFMSW are converted to monomeric from ml to L; R, ideal gas constant (8310 Pa L ­K−1 ­mol−1); sugars released to the liquid fraction. The efficiency of this T, the temperature at which F is measured (K); DM, dry conversion process depends on many parameters, such as the matter of solids in the reactor (g); 1­ 000b, conversion factor enzymatic activities selected, the solids load and the time, from g to mg. among others. However, there is always a remaining solid fraction containing partially hydrolyzed and non-hydrolyzed Analytical Methods fibers with the potential to be used in further conversion processes. As can be seen in Table 2, the obtained solid Substrates and fermentation samples were characterized in hydrolysate is humid and rich in organic matter. Therefore, terms of dry matter, moisture content, organic matter and a potential substrate for SSF processes that require enough pH according to standard procedures [42]. Reducing sug- water to promote microbial growth [37]. From the RS meas- ars (RS) of the solid samples were measured after a solid- urement, it can be seen that a significant amount of eas- liquid extraction with distilled water using the DNS method ily accessible sugars remains solubilized in the free water and expressed per gram of DM [13, 43]. Biodegradability content [13]. The pH is rather acidic, which represents a was assessed through two respiration indexes and com- challenge for growing microorganisms that prefer pH near pared among the different substrates: the dynamic respira- neutrality such as bacteria, and specifically Bti [36, 37]. tion index (­ DRI24h), which represents the average oxygen Therefore, a pH adjustment step is required before grow- uptake rate during the 24 h of maximum activity observed ing Bti as was confirmed during an initial experiment in expressed in g ­O2 ­kg−1 DM ­h−1, and the cumulative oxygen which unmodified solid hydrolysate was used as a substrate consumption index ­(AT4), which is the cumulative oxygen (Fig. 1). In fact, not even the expected amount of around Fig. 1  Process parameters evolution (sOUR, outlet oxygen, viable cells, spores and pH) during the initial evaluation of Bti growth on unmodified solid hydrolysate. Initial spore count was 0 13 1438 Waste and Biomass Valorization (2023) 14:1433–1445 ­107 viable cells per g of DM in the sample of day 0 was and robust [44]. Increasing the amount of ­CaCO3, or other reached, indicating that cell death was almost immediately. substances involves an increase in the moisture content of Even though Bti was not observed after 24 h it can be seen the sample, which should not exceed 80% [37]. Besides, how other opportunist microorganisms started consuming it would also increase the production cost and difficult the oxygen. operation at larger process scales. Therefore, it was decided to test a different strategy for pH modification based on the Chemical Modification of the pH use of high-buffering capacity cosubstrates. The first attempt to modify the pH of the solid hydrolysate Selection and Screening of Cosubstrates was done using chemicals as it is the most straightfor- ward and commonly used methodology. A substance with The use of cosubstrates to improve process efficiency is a strong buffering capacity (­ CaCO3) was selected over a a widely researched and used practice in the anaerobic strong base, such as NaOH, due to the difficulties in moni- digestion of municipal wastes [45, 46]. For instance, the toring and modifying the pH during the course of SSF pro- rapid hydrolysis of food waste leads to an inhibitory pH cesses [37]. To reach a pH of around 7 it was necessary that can be overcome with the use of sewage sludge or ani- to add 8.5 mL of 1 M ­CaCO3 to the 90 g of hydrolysate, mal manure as a cosubstrate [45]. The use of cosubstrates which only increased the moisture content of the sample in SSF has been researched to a lesser extent [29, 47]. by around 2%. Results from this fermentation can be seen Nutrient supplementation is more common but it should in Fig. 2. As the bar graph shows, Bti was not able to thrive be highlighted that these studies are rarely conducted at and in only 24 h the viable cells were drastically reduced. larger scales [48]. For this study, the cosubstrate needed This can be explained by the drop in pH below 5.6, reported to provide supplemental alkalinity and, ideally, nutrients. as inhibitory for Bt species [33], as a consequence of the It was decided to evaluate only biomaterials in the frame- production of organic acids at the early stages of fermenta- work of municipal waste, as it would ease the implemen- tion [36]. Spores appeared at 24 h, reaching the maximum tation of such processes in industrial environments. The concentration at 72 h as expected [21, 26]. At his point, selected cosubstrates to be studied were: OFMSW diges- viable cells could not be measured because they could not tate, digested sewage sludge and OFMSW compost. Their be distinguished from opportunist microorganisms in the characterization alongside an average for the solid hydro- Petri dishes, which explains the sharp rise of sOUR after lysate (Table 2) is presented in Table 3. All three came 48 h. Even though spores are produced, it has been reported from large and well-established municipal waste treatment that spores developed in acidic environments are less viable plants, in fact, sewage characterization reported similar Fig. 2  Process parameters evolution (sOUR, outlet oxygen, viable cells, spores and pH) during the time course of solid hydrolysate with chemically modified pH. Initial spore count was 0 13 Waste and Biomass Valorization (2023) 14:1433–1445 1439 Table 3  Characterization of the Parameter Hydrolysate Compost Sewage sludge Digestate hydrolysate and the different cosubstrates used in this study MC (%) 75.4 ± 2.1 a 32.2 ± 0.2 83.5 ± 0.1 77.2 ± 0.4a DM (%) 24.6 ± 2.1a 67.8 ± 0.6 16.5 ± 0.1 22.8 ± 0.4a OM (%)* 86.9 ± 1.0 59.8 ± 5.4b 68.8 ± 1.1ab 72.3 ± 6.3a pH 5.5 ± 0.1 8.1 ± 0.1a 8.3 ± 0.2a 8.5 ± 0.2a Conductivity (mS/cm) 2.6 ± 0.1a 3.0 ± 0.1 1.1 ± 0.1 2.6 ± 0.1a DRI24h 1.2 ± 0.1a 0.7 ± 0.0 1.6 ± 0.2b 1.4 ± 0.1ab (g ­O2 ­kg−1 DM ­h−1) AT4 (g ­O2 ­kg−1 DM) 39.9 ± 2.4 50.1 ± 3.3 94.8 ± 4.0a 91.1 ± 2.4a Data presented as mean values ± standard deviation (n = 3). Same letters indicate not significantly different parameters (p > 0.05) based on the Tukey test analysis NA not available, MC moisture content, DM dry matter, OM organic matter, RS reducing sugars, DRI24h, dynamic respiration index average in the 24 h of maximum activity, AT4 cumulative oxygen consumption during the 4 days after the lag phase *Dry basis Fig. 3  a Dynamic respiration index (DRI) profiles and b cumulative oxygen consumption (COC) profiles, for the solid hydrolysate and the cosubstrates evaluated values to those obtained in a study performed 5 years ago oxygen consumed over a four-day period beyond the initial from the same treatment plant [40]. Montejo et al. [49] lag phase, which gives a deeper understanding of the sam- evaluated 30 compost samples from 10 MSW treatment ple’s biodegradability [40]. For instance, both digestate and plants and reported average values of 22 ± 9, 45 ± 8 and sewage sludge did not present significant differences with 7.6 ± 0.4 for MC, OM and pH respectively. Thus, com- the hydrolysate in terms of ­DRI24h but there was a strik- parable to the compost evaluated in this study (Table 2). ing difference in the ­AT4 values. This might indicate that All three cosubstrates presented a slightly alkaline pH, the hydrolysate contained easily accessible matter but was around three units above the solid hydrolysate. These alka- overall more exhausted. A better insight into the biodeg- line materials are well known for their buffering capacity radability potential can be seen in the respiration curves [45, 50]. Digestate was the cosubstrate presenting the high- (Fig. 3). The DRI profile of the hydrolysate drops after the est amount of OM, but there were no significant differences first 10 h, while the cosubstrates remain more active during with sewage sludge. The measure of biodegradability is that time leading to higher cumulative oxygen consumptions. expressed through the DRI index and is an indirect measure Therefore, compost, digestate and sewage sludge represent of the biodegradable organic matter content [41]. The ­AT4 an increase of 25.6, 128.3 and 137.6% in cumulative oxygen is another useful respiration index that indicates the total consumption, respectively. 13 1440 Waste and Biomass Valorization (2023) 14:1433–1445 Fig. 4  Process parameters evolution at 0.5 L scale: a sOUR profile, indicate no significantly different parameters for each group (p > 0.05) b oxygen profile, c initial and final viable cells and final spore count based on the Tukey test analysis (initial spore count was 0), and d initial and final pH. Same letters Table 4  SSF lag-phase and Mixtures Lag phase (h) Sporulation (%) Sporulation yield spores production at 0.5 and (spores/CFU inocu- 1.5 L for the two different ratios lated) of cosubstrates 0.5 L 1.5 L 0.5 L 1.5 L 0.5 L 1.5 L 25% Compost 5.0 ± 0.1 NA 28 ± 2 NA 0±0 NA 50% Compost 3.2 ± 0.1 NA 78 ± 2 NA 0.6 ± 0.3 NA 25% Digested sewage sludge 25.1 ± 4.8 33.4 ± 4.9 66 ± 2 78 ± 10 27 ± 15 5±1 50% Digested sewage sludge 10.0 ± 0.5 24.8 ± 0.4 100 ± 1 103 ± 9 196 ± 4 150 ± 13 25% Digestate 12.4 ± 0.9 17.5 ± 2.0 87 ± 19 73 ± 7 42 ± 7 44 ± 10 50% Digestate 8.5 ± 0.5 11.3 ± 0.9 82 ± 27 86 ± 11 27 ± 4 90 ± 7 The lag phase is calculated as the time it takes to reach 25% of the maximum sOUR. Data presented as mean values ± standard deviation (n = 2 at 0.5 L and n = 3 at 1.5 L) NA not applicable 13 Waste and Biomass Valorization (2023) 14:1433–1445 1441 An initial evaluation of the effect of the selected cosub- metabolic heat production [53]. Besides, one of the major strates on the SSF of the solid hydrolysate was performed challenges when scaling-up SSF processes is the intense by assessing them at two different wet weight ratios, 25 heat generation and its inefficient removal, alongside mass and 50%. The initial pH (Fig. 4d) for all the mixtures was transfer issues [20, 48]. Therefore, for the next experiment, above six and therefore, not inhibitory for Bti. The final pH, we decided to observe the evolution of temperature during after 72 h of fermentation remained above six except for the the fermentation course on a three-times increased scale mixture containing 25% of compost, which presented a pH (1.5 L) under uncontrolled temperature conditions. To do of 5.5. Therefore, this mixture was not alkaline enough to so, triplicate reactors for each condition were incubated at surpass the initial pH drop of the fermentation, which led to room temperature (23 °C ± 2) and sensors were distributed a drastic reduction of viable cells at the end of the fermenta- inside the solid matrix to monitor changes. For this experi- tion (Fig. 4c). In terms of oxygen consumption, the maxi- ment, compost was discarded as a cosubstrate due to its poor mum values were presented by both mixtures with diges- performance at 0.5 L. This experiment was also conducted to tate and were four times higher than the minimum values, study the reproducibility of the process on a different batch which were obtained for the 25% of compost. None of the of OFMSW and subsequent enzymatic hydrolysis runs. fermentations reached oxygen-limiting conditions (Fig. 4b), As can be seen in Fig. 5a, the maximum sOUR achieved the lowest oxygen concentration recorded was 12% achieved remained consistent with those obtained at 0.5 L (Fig. 4a), with a 50% of digestate mixture, as expected from the sOUR which makes sense considering that the airflow supplied (Fig. 4a). From Fig. 4, it can be seen how compost was not per gram of DM was maintained between scales. However, an adequate cosubstrate. Even though the mixture with 50% the profiles of the mixtures using sewage sludge changed presented a pH similar to that of other cosubstrates mixtures, at 1.5 L presenting narrower peaks that may be explained it did not lead to Bti growth but only to sporulation at a by different microbial profiles as a consequence of uncon- yield of almost 1 spore C ­ FU−1 inoculated with a sporula- trolled temperature. In terms of Bti growth and sporulation tion percentage of 78%. A similar effect has been previously (Fig. 5c), 50% sewage sludge still appeared as the best-per- observed when using biowaste digestate as a substrate for forming mixture but by a narrower margin and was not sig- growing Bt var kurstaki [26]. This may be explained by the nificantly different from the mixture with 50% of digestate low biodegradability of the sample, as this parameter has in terms of final concentration of viable cells. The sporula- been shown to positively influence the growth of Bt species tion achieved was also 100% and the yield dropped to 150 in SSF [22]. There were no significant differences in the compared with the 0.5 L scale (Table 4). The 25% of sewage growth of Bti in the other cosubstrates mixtures (Fig. 4c), sludge mixture also showed lower performance in compari- except for 50% of sewage sludge which outperformed. This son with the 0.5 L scale. It led to poor growth of Bti, which mixture also led to the highest production of spores (Table 4) was significantly lower than the other mixtures using diges- which also surpassed previous studies that presented a pro- tate. This can be explained by the relatively low pH achieved duction similar to the other mixtures (around 30 spores per at the end of the fermentation (Fig. 5d). Contrarily, the mix- CFU inoculated) [22]. tures using digestate increased their performance concerning At this point, it should be highlighted that the cosubstrates the 0.5 L scale. Therefore, sewage sludge as a cosubstrate were not added sterile, as this would have not made sense appeared to be more affected by the scale-up than digestate. in terms of process efficiency due to their nature. Instead, This may be explained because digestate has higher alka- they were added after a thermal hygienization step, which is linity [54] than sewage sludge [55] and thus prevents more known to reduce the microbial population of digestate and effectively the pH drop at the early stages of the fermentation sewage sludge but not completely, as they contain a great promoting the development of Bti. variety of microorganisms that arose from their respective Regarding the average temperature profiles within the anaerobic digestion processes [51, 52]. This implies that, packed bed reactors (Fig. 4b), the maximum temperatures with the use of this kind of cosubstrates, an autochthonous reached ranged from 34 °C for the mixture with 50% of population of microorganisms is also being added to the digestate and 27 °C for 25% of digestate. Minimum tem- solid matrix [22]. A direct effect can be seen in the consider- peratures (21 °C) were achieved by the 25% of sewage able reduction of the lag phase (Table 4) observed for those sludge, which corresponded to the longer lag phase observed mixtures with a higher amount of cosubstrates. (Table 4). Both mixtures with the higher ratio of cosub- strates increased faster their temperature reaching higher val- Process Verification and Temperature Evaluation ues, especially when using digestate. This can be explained because an increase in metabolic activity leads to an increase The increment in microbial activity caused by the use in temperature, which in turn stimulates microbial activ- of anaerobically digested cosubstrates can in turn cause ity. This phenomenon can be observed by comparing how an increase in temperature as a consequence of higher 13 1442 Waste and Biomass Valorization (2023) 14:1433–1445 Fig. 5  Process parameters evolution at 1.5 L scale: a sOUR profile, Same letters indicate no significantly different parameters for each b average temperature profile, c initial and final viable cells and final group (p > 0.05) based on the Tukey test analysis spore count (initial spore count was 0), and d initial and final pH. the maximum values for sOUR (Fig. 5a) and temperature yield (Table 4). Also, lower ratios of non-sterile cosubstrates (Fig. 5b) are obtained at the same time. imply lesser microbial load into the reactor and thus lesser Except for 25% of sewage sludge, process robustness competitive pressure microbial benefiting the growth of Bti. using a different batch of OFMSW has been demonstrated The final choice might also be influenced by a deeper char- at a larger scale, which is of major importance considering acterization of the biopesticide activity because even though the high variability of this type of waste [5]. Though 50% spore count is considered an indirect estimation method, it of sewage sludge reported the best results, both growth and does not completely predict the endotoxic potential [56]. sporulation yield were not reproducible and were lower than Overall, both digestate and sewage sludge, at a 25% and 0.5 L. It would be essential to evaluate if this downward 50% ratio respectively, appeared as suitable cosubstrates for trend continues as process scale-up does. It should also be the SSF of an acidic hydrolysate. This gives flexibility to the given special attention to the use of a 50% digestate mixture process and the final choice would depend on the availability for further scale-up because it led to the highest tempera- of each cosubstrate. From an urban solid waste biorefinery tures. It is well known that in SSF processes temperature perspective, the use of digestate from the AD process of increases as the scale does due to mass and heat transfer OFMSW makes more sense as it would lead to better process issues, especially for non-sterile substrates [20, 48]. This integration [6]. For instance, the higher quality OFMSW becomes even more relevant when the room or ambient tem- received into the treatment plant can be used for the enzy- perature increase, for instance with seasonality. Therefore, matic hydrolysis coupled with SSF and submerged fermen- for digestate, the 25% ratio appears as a lower-risk choice as tation for the liquid fraction while the OFMSW with less it presented milder changes in temperature and still retained quality could be treated through AD. This leads to a multi- the pH above 7 (Fig. 5d) with a comparable sporulation platform configuration that not only enhances the recovery 13 Waste and Biomass Valorization (2023) 14:1433–1445 1443 of resources but also provides the biorefinery with more flex- were made. The images or other third party material in this article are ibility and resources to adapt to energy requirements and included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in price fluctuation [57, 58]. However, digested sewage sludge the article's Creative Commons licence and your intended use is not is a far more abundant material that in many cases requires permitted by statutory regulation or exceeds the permitted use, you will treatment before its safe disposal, and therefore novel val- need to obtain permission directly from the copyright holder. To view a orization pathways. copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. Conclusion References 1. Kaza, S., Yao, L., Bhada-Tata, P., Van Woerden, F.: What a waste The use of alkaline cosubstrates has been implemented as 2.0: a global snapshot of solid waste management to 2050. The a successful and reproducible strategy to overcome the pH World Bank, Washington, DC (2018) drop during the SSF of Bacillus thuringiensis var. israelen- 2. Wilson, D.C.: Development drivers for waste management. Waste sis using solid hydrolysate of OFMSW as substrate. Two Manag. Res. 25, 198–207 (2007). https://​doi.​org/​10.​1177/​07342​ 42X07​079149 byproducts of urban waste treatments, digestate and digested 3. Mayer, F., Bhandari, R., Gäth, S.A., Himanshu, H., Stobernack, sewage sludge, appeared as adequate cosubstrates providing N.: Economic and environmental life cycle assessment of organic the process with certain implementation flexibility. Spores waste treatment by means of incineration and biogasification. Is concentration of 1.1 × ­109 spores ­g−1 DM and 6.4 × ­108 source segregation of biowaste justified in Germany? Sci. Total Environ. (2020). https://​doi.​org/​10.​1016/j.​scito​tenv.​2020.​137731 spores ­g−1 DM were obtained for sewage sludge and diges- 4. Cerda, A., Artola, A., Font, X., Barrena, R., Gea, T., Sánchez, tate respectively, which corresponds to yields of 112 and 48 A.: Composting of food wastes: status and challenges. Bioresour. spores per inoculated Bti cell. This work represents a further Technol. 248, 57–67 (2018). https://​doi.​org/​10.​1016/j.​biort​ech.​ step in novel valorization options for the organic fraction of 2017.​06.​133 5. Campuzano, R., González-Martínez, S.: Characteristics of the municipal solid waste, in line with a biorefinery scenario organic fraction of municipal solid waste and methane produc- for waste management as the future requires. Future studies tion: a review. Waste Manag. 54, 3–12 (2016). https://​doi.​org/​10.​ have to evaluate the biopesticide activity and safety of the 1016/j.​wasman.​2016.​05.​016 final product, as well as its formulation. 6. Molina-Peñate, E., Artola, A., Sánchez, A.: Organic municipal waste as feedstock for biorefineries: bioconversion technologies Acknowledgements Esther Molina thanks Generalitat de Catalunya for integration and challenges. Rev. Environ. Sci. Biotechnol. 21, her pre-doctoral Grant (DI 7, 2019). The authors thank ASA Spezial- 247–267 (2022). https://​doi.​org/​10.​1007/​s11157-​021-​09605-w enzyme GmbH, for providing the enzymatic cocktail; Mancomunitat 7. Jowitt, S.M., Mudd, G.M., Thompson, J.F.H.: Future availabil- de la Plana, for providing the OFMSW; Consorci per a la Gestió dels ity of non-renewable metal resources and the influence of envi- Residus del Vallès Oriental, for providing the digestate; Àrea metro- ronmental, social, and governance conflicts on metal produc- politana de Barcelona for the OFMSW compost; and Aigues Sabadell; tion. Commun. Earth Environ. (2020). https://​doi.​org/​10.​1038/​ for providing the sewage sludge. s43247-​020-​0011-0 8. Mahmoodi, P., Karimi, K., Taherzadeh, M.J.: Hydrothermal pro- Author Contributions EMP, AS and AA contributed to the study con- cessing as pretreatment for efficient production of ethanol and ception and design. Material preparation, data collection and analysis biogas from municipal solid waste. Bioresour. Technol. 261, were performed by EMP and NA. The first draft of the manuscript was 166–175 (2018). https://​doi.​org/​10.​1016/j.​biort​ech.​2018.​03.​115 written by EMP and AS and AA commented on previous versions of 9. Alvira, P., Tomás-Pejó, E., Ballesteros, M., Negro, M.J.: Pretreat- the manuscript. All authors read and approved the final manuscript. ment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101, Funding Open Access Funding provided by Universitat Autònoma de 4851–4861 (2010). https://d​ oi.o​ rg/1​ 0.1​ 016/j.b​ iorte​ ch.2​ 009.1​ 1.0​ 93 Barcelona. This research was funded by the Horizon 2020 research and 10. López-Gómez, J.P., Latorre-Sánchez, M., Unger, P., Schneider, innovation Program within the SCALIBUR project (Grant Agreement R., Coll Lozano, C., Venus, J.: Assessing the organic fraction of No 817788). municipal solid wastes for the production of lactic acid. Biochem. Eng. J. (2019). https://​doi.​org/​10.​1016/j.​bej.​2019.​107251 Data Availability Enquiries about data availability should be directed 11. Izaguirre, J.K., da Fonseca, M.M.R., Fernandes, P., Villarán, to the authors. M.C., Castañón, S., Cesário, M.T.: Upgrading the organic fraction of municipal solid waste to poly(3-hydroxybutyrate). Bioresour. Technol. 290, 121785 (2019). https://​doi.​org/​10.​1016/j.​biort​ech.​ Declarations 2019.​121785 12. Ebrahimian, F., Karimi, K., Kumar, R.: Sustainable biofuels and Conflict of interest The authors declare that they have no conflict of bioplastic production from the organic fraction of municipal solid interest. waste. Waste Manag. 116, 40–48 (2020). https://d​ oi.o​ rg/1​ 0.1​ 016/j.​ wasman.​2020.​07.​049 Open Access This article is licensed under a Creative Commons Attri- 13. Molina-Peñate, E., Sánchez, A., Artola, A.: Enzymatic hydrolysis bution 4.0 International License, which permits use, sharing, adapta- of the organic fraction of municipal solid waste: optimization and tion, distribution and reproduction in any medium or format, as long valorization of the solid fraction for Bacillus thuringiensis biopes- as you give appropriate credit to the original author(s) and the source, ticide production through solid-state fermentation. Waste Manag. provide a link to the Creative Commons licence, and indicate if changes 13 1444 Waste and Biomass Valorization (2023) 14:1433–1445 137, 304–311 (2022). https://​doi.​org/​10.​1016/j.​wasman.​2021.​11.​ substrate selection on the process. Waste Manag. 121, 403–411 014 (2021). https://​doi.​org/​10.​1016/j.​wasman.​2020.​12.​036 14. Yang, X., Choi, H.S., Park, C., Kim, S.W.: Current states and 28. Rivero, C.P., Hu, Y., Kwan, T.H., Webb, C., Theodoropoulos, C., prospects of organic waste utilization for biorefineries. Renew. Daoud, W., Lin, C.S.K.: Bioplastics from solid waste. Curr. Dev. Sustain. Energy Rev. 49, 335–349 (2015). https://​doi.​org/​10.​ Biotechnol. Bioeng. Solid Waste Manag. (2017) 1016/j.​rser.​2015.​04.​114 29. Jiménez-Peñalver, P., Gea, T., Sánchez, A., Font, X.: Production 15. Venkata Mohan, S., Nikhil, G.N., Chiranjeevi, P., Nagendranatha of sophorolipids from winterization oil cake by solid-state fermen- Reddy, C., Rohit, M.V., Kumar, A.N., Sarkar, O.: Waste biore- tation: optimization, monitoring and effect of mixing. Biochem. finery models towards sustainable circular bioeconomy: critical Eng. J. 115, 93–100 (2016). https://d​ oi.o​ rg/1​ 0.1​ 016/j.b​ ej.2​ 016.0​ 8.​ review and future perspectives. Bioresour. Technol. 215, 2–12 006 (2016). https://​doi.​org/​10.​1016/j.​biort​ech.​2016.​03.​130 30. Eras-Muñoz, E., Farré, A., Sánchez, A., Font, X., Gea, T.: Micro- 16. López-Gómez, J.P., Unger, P., Schneider, R., Venus, J.: From bial biosurfactants: a review of recent environmental applications. upstream to purification: production of lactic acid from the Bioengineered 13, 12365–12391 (2022). https://​doi.​org/​10.​1080/​ organic fraction of municipal solid waste. Waste Biomass Valoriz. 21655​979.​2022.​20746​21 (2020). https://​doi.​org/​10.​1007/​s12649-​020-​00992-9 31. Ballardo, C., Abraham, J., Barrena, R., Artola, A., Gea, T., 17. Stylianou, E., Pateraki, C., Ladakis, D., Cruz-Fernández, M., Sánchez, A.: Valorization of soy waste through SSF for the pro- Latorre-Sánchez, M., Coll, C., Koutinas, A.: Evaluation of organic duction of compost enriched with Bacillus thuringiensis with fractions of municipal solid waste as renewable feedstock for biopesticide properties. J. Environ. Manag. 169, 126–131 (2016). succinic acid production. Biotechnol. Biofuels 13, 1–16 (2020). https://​doi.​org/​10.​1016/J.​JENVM​AN.​2015.​12.​029 https://​doi.​org/​10.​1186/​s13068-​020-​01708-w 32. Prakash, J., Sharma, R., Patel, S.K.S., Kim, I.W., Kalia, V.C.: 18. Izaguirre, J.K., Barañano, L., Castañón, S., Santos, J.A.L., Bio-hydrogen production by co-digestion of domestic wastewater Cesário, M.T., da Fonseca, M.M.R., Alkorta, I., Garbisu, C.: Eco- and biodiesel industry effluent. PLoS ONE. 13, e0199059 (2018). nomic and environmental assessment of bacterial poly(3-hydroxy- https://​doi.​org/​10.​1371/​JOURN​AL.​PONE.​01990​59 butyrate) production from the organic fraction of municipal solid 33. Jallouli, W., Driss, F., Fillaudeau, L., Rouis, S.: Review on biope- waste. Bioresour. Bioprocess. 8, 1–12 (2021). https://​doi.​org/​10.​ sticide production by Bacillus thuringiensis subsp. kurstaki since: 1186/​S40643-​021-​00392-4/​FIGUR​ES/2 focus on bioprocess parameters. Process Biochem. 98, 224–232 19. Ebrahimian, F., Denayer, J.F.M., Karimi, K.: Efficient coproduc- (2020). https://​doi.​org/​10.​1016/j.​procb​io.​2020.​07.​023 tion of butanol, ethanol, and biohydrogen from municipal solid 34. Biopesticides Market - Growth, Trends, C.O.V.I.D.-., Impact: and waste through a cocultivated biorefinery. Energy Convers. Manag. Forecasts (2021). –2026 https://​www.​mordo​r inte​llige​nce.​com/​ 255, 115303 (2022). https://​doi.​org/​10.​1016/J.​ENCON​MAN.​ indus​try-​repor​ts/​global-​biope​stici​des-​market-​indus​try 2022.​115303 35. Bravo, A., Likitvivatanavong, S., Gill, S.S., Soberón, M.: Bacillus 20. Thomas, L., Larroche, C., Pandey, A.: Current developments in thuringiensis: a story of a successful bioinsecticide. Insect Bio- solid-state fermentation. Biochem. Eng. J. 81, 146–161 (2013). chem. Mol. Biol. 41, 423–431 (2011). https://​doi.​org/​10.​1016/j.​ https://​doi.​org/h​ ttps://​doi.​org/ https://​doi.​org/​10.​1016/j.​bej.​2013.​ ibmb.​2011.​02.​006 10.​013 36. Foda, M.S., Salama, H.S., Selim, M.: Factors affecting growth 21. Cerda, A., Mejias, L., Rodríguez, P., Rodríguez, A., Artola, A., physiology of Bacillus thuringiensis. Appl. Microbiol. Biotechnol. Font, X., Gea, T., Sánchez, A.: Valorisation of digestate from 22, 50–52 (1985) biowaste through solid-state fermentation to obtain value added 37. Kumar, V., Ahluwalia, V., Saran, S., Kumar, J., Patel, A.K., Sing- bioproducts: a first approach. Bioresour Technol. 271, 409–416 hania, R.R.: Recent developments on solid-state fermentation for (2019). https://​doi.​org/​10.​1016/j.​biort​ech.​2018.​09.​131 production of microbial secondary metabolites: challenges and 22. Mejias, L., Estrada, M., Barrena, R., Gea, T.: A novel two-stage solutions. Bioresour. Technol. 323, 124566 (2021). https://​doi.​ aeration strategy for Bacillus thuringiensis biopesticide produc- org/​10.​1016/j.​biort​ech.​2020.​124566 tion from biowaste digestate through solid-state fermentation. 38. Sala, A., Barrena, R., Sánchez, A., Artola, A.: Fungal biopesticide Biochem. Eng. J. 161, 107644 (2020). https://​doi.​org/​10.​1016/j.​ production: process scale-up and sequential batch mode operation bej.​2020.​107644 with Trichoderma harzianum using agro-industrial solid wastes 23. Yazid, N.A., Barrena, R., Komilis, D., Sánchez, A.: Solid-state of different biodegradability. Chem. Eng. J. 425, 131620 (2021). fermentation as a novel paradigm for organic waste valorization: https://​doi.​org/​10.​1016/J.​CEJ.​2021.​131620 a review. Sustainability 9, 224 (2017). https://​doi.​org/​10.​3390/​ 39. Zhang, W., Qiu, L., Gong, A., Cao, Y., Wang, B.: Solid-state Fer- su902​0224 mentation of Kitchen Waste for Production of Bacillus thuring- 24. Ballardo, C., Barrena, R., Artola, A., Sánchez, A.: A novel strat- iensis-based Bio-pesticide. BioResources 8, 1124–1135 (2013). egy for producing compost with enhanced biopesticide properties https://​doi.​org/​10.​15376/​biores.​8.1.​1124-​1135 through solid-state fermentation of biowaste and inoculation with 40. Mejias, L., Komilis, D., Gea, T., Sánchez, A.: The effect of airflow Bacillus thuringiensis. Waste Manag. 70, 53–58 (2017). https://​ rates and aeration mode on the respiration activity of four organic doi.​org/​10.​1016/j.​wasman.​2017.​09.​041 wastes: implications on the composting process. Waste Manag. 65, 25. Sala, A., Artola, A., Sánchez, A., Barrena, R.: Rice husk as a 22–28 (2017). https://​doi.​org/​10.​1016/j.​wasman.​2017.​04.​008 source for fungal biopesticide production by solid-state fermenta- 41. Ponsá, S., Gea, T., Sánchez, A.: Different indices to express tion using B. bassiana and T. harzianum. Bioresour. Technol. 296, biodegradability in organic solid wastes. J. Environ. Qual. 39, 122322 (2020). https://​doi.​org/​10.​1016/j.​biort​ech.​2019.​122322 706–712 (2010). https://​doi.​org/​10.​2134/​jeq20​09.​0294 26. Rodríguez, P., Cerda, A., Font, X., Sánchez, A., Artola, A.: Val- 42. Thompson, W.H., Leege, P.B., Millner, P.D., Watson, M.E.: Test orisation of biowaste digestate through solid state fermentation to methods for the examination of composting and compost.United produce biopesticides from Bacillus thuringiensis. Waste Manag. States Compost. Counc. Res. Educ. Found. United States Dep. 93, 63–71 (2019). https://​doi.​org/​10.​1016/j.​wasman.​2019.​05.​026 Agric. (2001) 27. Martínez-Avila, O., Muñoz-Torrero, P., Sánchez, A., Font, X., 43. Miller, G.L.: Use of dinitrosalicylic acid reagent for determination Barrena, R.: Valorization of agro-industrial wastes by produc- of reducing sugar. Anal. Chem. 31, 426–428 (1959). https://​doi.​ ing 2-phenylethanol via solid-state fermentation: influence of org/​10.​1021/​ac601​47a030 13 Waste and Biomass Valorization (2023) 14:1433–1445 1445 44. Saleh, S.M., Harris, R.F., Allen, O.N.: Fate of Bacillus thuring- digestate from codigestion of brewer’s spent grain and palm oil iensis in soil: effect of soil pH and organic amendment. Can. J. mill effluent by manure supplementation. Sustain. Environ. Res. Microbiol. 16, 677–680 (1970). https://​doi.​org/​10.​1139/​m70-​116 30, 1–11 (2020). https://​doi.​org/​10.​1186/​S42834-​020-​00056-6/​ 45. Karki, R., Chuenchart, W., Surendra, K.C., Shrestha, S., Raskin, TABLES/5 L., Sung, S., Hashimoto, A., Kumar, S.: Bioresource technology 53. Arora, S., Singh, P., Rani, R., Ghosh, S.: Oxygen uptake rate as a anaerobic co-digestion: current status and perspectives. Bioresour. tool for on-line estimation of cell biomass and bed temperature in Technol. 330, 125001 (2021) a novel solid-state fermentation bioreactor. Bioprocess. Biosyst. 46. Kasinath, A., Fudala-Ksiazek, S., Szopinska, M., Bylinski, H., Eng. 41, 917–929 (2018). https://​doi.​org/​10.​1007/​S00449-​018-​ Artichowicz, W., Remiszewska-Skwarek, A., Luczkiewicz, A.: 1923-2/​FIGUR​ES/9 Biomass in biogas production: pretreatment and codigestion. 54. Michele, P., Giuliana, D., Carlo, M., Sergio, S., Fabrizio, A.: Renew. Sustain. Energy Rev. 150, 111509 (2021). https://​doi.​org/​ Optimization of solid state anaerobic digestion of the OFMSW 10.​1016/j.​rser.​2021.​111509 by digestate recirculation: a new approach. Waste Manag. 35, 47. Fleuri, L.F., de Oliveira, M.C., de Lara Campos Arcuri, M., 111–118 (2015). https://​doi.​org/​10.​1016/J.​WASMAN.​2014.​09.​ Capoville, B.L., Pereira, M.S., Delgado, C.H.O., Novelli, P.K.: 009 Production of fungal lipases using wheat bran and soybean bran 55. Fonoll, X., Astals, S., Dosta, J., Mata-Alvarez, J.: Anaerobic co- and incorporation of sugarcane bagasse as a co-substrate in solid- digestion of sewage sludge and fruit wastes: evaluation of the state fermentation. Food Sci. Biotechnol. 23, 1199–1205 (2014). transitory states when the co-substrate is changed. Chem. Eng. J. https://​doi.​org/​10.​1007/​s10068-​014-​0164-7 262, 1268–1274 (2015). https://​doi.​org/​10.​1016/J.​CEJ.​2014.​10.​ 48. Soccol, C.R., Costa, E.S.F., da, Letti, L.A.J., Karp, S.G., 045 Woiciechowski, A.L., de Vandenberghe, L.P.: Recent develop- 56. De Montiel, L.T., Tyagi, M., Valero, R.D.: Wastewater treatment ments and innovations in solid state fermentation. Biotechnol. sludge as a raw material for the production of Bacillus thuringien- Res. Innov. 1, 52–71 (2017). https://​doi.​org/​10.​1016/j.​biori.​2017.​ sis based biopesticides. Water Res. 35, 3807–3816 (2001). https://​ 01.​002 doi.​org/​10.​1016/​S0043-​1354(01)​00103-8 49. Montejo, C., Costa, C., Márquez, M.C.: Influence of input mate- 57. Moncada, B., Aristizábal, J., Cardona, M.V.: Design strategies for rial and operational performance on the physical and chemical sustainable biorefineries. Biochem. Eng. J. 116, 122–134 (2016). properties of MSW compost. J. Environ. Manag. 162, 240–249 https://​doi.​org/​10.​1016/j.​bej.​2016.​06.​009 (2015). https://​doi.​org/​10.​1016/j.​jenvm​an.​2015.​07.​059 58. Alibardi, L., Astrup, T.F., Asunis, F., Clarke, W.P., De Gioan- 50. García-Gil, J.C., Ceppi, S.B., Velasco, M.I., Polo, A., Senesi, N.: nis, G., Dessì, P., Lens, P.N.L., Lavagnolo, M.C., Lombardi, L., Long-term effects of amendment with municipal solid waste com- Muntoni, A., Pivato, A., Polettini, A., Pomi, R., Rossi, A., Spagni, post on the elemental and acidic functional group composition and A., Spiga, D.: Organic waste biorefineries: looking towards imple- pH-buffer capacity of soil humic acids. Geoderma. 121, 135–142 mentation. Waste Manag. 114, 274–286 (2020). https://​doi.​org/​ (2004). https://​doi.​org/​10.​1016/J.​GEODE​RMA.​2003.​11.​004 10.​1016/j.​wasman.​2020.​07.​010 51. Astals, S., Venegas, C., Peces, M., Jofre, J., Lucena, F., Mata- Alvarez, J.: Balancing hygienization and anaerobic digestion of Publisher’s Note Springer Nature remains neutral with regard to raw sewage sludge. Water Res. 46, 6218–6227 (2012). https://d​ oi.​ jurisdictional claims in published maps and institutional affiliations. org/​10.​1016/J.​WATRES.​2012.​07.​035 52. Ndubuisi-Nnaji, U.U., Ofon, U.A., Ekponne, N.I., Offiong, N.A.O., Offiong, N.A.O.: Improved biofertilizer properties of Authors and Affiliations Esther Molina‑Peñate1,2 · Nil Arenòs1 · Antoni Sánchez1 · Adriana Artola1 1 2 GICOM Research Group, Department of Chemical, Aeris Tecnologías Ambientales S.L, Carrer Santa Rosa, 38, Biological and Environmental Engineering, School local, 08290 Cerdanyola del Vallès, Barcelona, Spain of Engineering, Universitat Autònoma de Barcelona, Edifici Q, 08193 Bellaterra, Barcelona, Spain 13