Assessment of different mechanical treatments for improving the anaerobic biodegradability of residual raspberry extrudate

Waste Management 139 (2022) 190–198 Available online 30 December 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/). Assessment of different mechanical treatments for improving the anaerobic biodegradability of residual raspberry extrudate ´ Angeles Trujillo-Reyes, ´ Erika Sinisgalli, Juan Cubero-Cardoso, Ana G. P´ erez, Antonio Serrano * , Rafael Borja, Fernando G. Fermoso Instituto de la Grasa, Spanish National Research Council (CSIC), Campus Universitario Pablo de Olavide– Ed. 46, Ctra. de Utrera, km. 1, Seville 41013, Spain ARTICLE INFO Keywords: Anaerobic digestion Ellagic acid Lignin Particle size distribution Pretreatment Raspberry achenes ABSTRACT Mechanical treatments can be simple and feasible methods for enhancing the anaerobic digestion of lignocellulosic substrates. This work aims to relate the direct effect of five different mechanical treatments, i.e., variation in the size and number of particles, with the variations in the chemical composition and, subsequently, the effect over the anaerobic digestion of residual raspberry extrudate, which was used as a model substrate. A high variation in the number of particles and the particle size distribution was achieved depending on the mechanical treatment applied, reaching the highest number of particles for the treatments with knife mills and mortar (around 8000 particles per gram). The higher number of particles was related to higher solubilisation, including phenolic compounds and sugars. The combination of knife mills and mortar pretreatment, which presented the highest number of particles, resulted in a 66% more of polyphenols in comparison to the raw substrate. However, the presence of anthocyanins was higher in mechanical treatments with less effect. The enhancement of the anaerobic digestion was clearly related to the increment in the number of particles of small size after the mechanical treatments. The highest methane yield coefficient (236 ±11 mL CH 4 /g volatile solids) was achieved for the raspberry extrudate treated with knife mills. 1. Introduction According to FAOSTAT, since 2000 worldwide berries production has been growing significantly, reaching a total production of up to 13 million tons in 2019 (FAOSTAT, 2021). Among the different berries, raspberries are one of the most important fruits due to their production volume and market value, i.e., a worldwide market value higher than 1500 million dollars in 2018 (FAOSTAT, 2021). Most of the production of raspberries are sold on the fresh market, but, in recent years, a significant part of this production is being used to elaborate processed products such as jams, yogurts, ice-cream, juices, etc. These products are mainly made from a fruit concentrate, which is obtained by extruding raspberries with various sieves with different mesh sizes (1.5 and 0.5 mm) (Cubero-Cardoso et al., 2020a). The sieves retain a residual fraction formed by the fibrous part of the raspberry and the achenes, called residual raspberry extrudate (RRE) (Cubero-Cardoso et al., 2020a). Achenes are the main component in the RRE, representing almost 80% by weight of the RRE (S´ ojka et al., 2016), although other authors indicated a higher percentage, i.e., approximately 90% in dry weight (Cubero-Cardoso et al., 2020a). The high concentration of organic matter in the RRE makes necessary to propose an adequate management method for this substrate. The free disposal in the soil or landfilling could result in the pollution of aquatic system due to lixiviates or release of unpleasant odours (Siles et al., 2013). The anaerobic digestion is a well-stablished valorisation technology that allows the conversion of organic substrates into biogas, which can be used as a renewable energy source (Ritigala et al., 2021). This technology has been proposed for different agricultural wastes, including strawberry extrudate (Cubero-Cardoso et al., 2020b; Siles et al., 2013) and raspberry extrudate (Trujillo-Reyes et al., 2019). Although the anaerobic digestion of these substrates was feasible and a high biogas production was achieved, some challenges have been also reported. For example, Siles et al. (2013) described that the presence of achenes in the strawberry waste favoured the destabilization of the anaerobic digestion performance, resulting also in around a 25% lower methane production than the strawberry extrudate after removing the achenes. This effect was due to the high content of lignin in the achenes, which limits the biological degradation due to the presence of inhibitory * Corresponding author. E-mail address: [email protected] (A. Serrano). Contents lists available at ScienceDirect Waste Management journal homepage: www.elsevier.com/locate/wasman https://doi.org/10.1016/j.wasman.2021.12.034 Received 28 June 2021; Received in revised form 9 November 2021; Accepted 20 December 2021 Waste Management 139 (2022) 190–198 191 phenolic compounds and a complex structure that difficult the enzymatic attack (Hendriks & Zeeman, 2009; Siles et al., 2013). Highintensive treatments have also been assessed for breaking down the lignocellulosic structures of the berries. In that sense, thermal treatments were the most commonly proposed, although other high-intensive treatments such as microwaves or pulsed electric fields have also been reported (B´ elafi-Bak´ o et al., 2012; Cubero-Cardoso et al., 2020a; Cubero-Cardoso et al., 2020b). These treatments affect different lignocellulosic components depending on the applied energy, being necessary a treatment with less effect to solubilize the hemicellulose than for cellulose and lignin. According to Hendriks and Zeeman (2009), thermal treatments at temperatures below 150 ◦C only causes the solubilization of the hemicellulose, being necessary higher temperatures and/or the application of steam-explosion treatments for the solubilization of the lignin. However, the application of high temperatures during the thermal treatments entails the formation of inhibitors for the anaerobic digestion process such as phenols and furans (Hendriks & Zeeman, 2009; Trujillo-Reyes et al., 2019). On another hand, the anaerobic digestion of berries showed a very limited improvement in many cases despite of the application of thermal treatments (Serrano et al., 2015; Trujillo-Reyes et al., 2019). Serrano et al. (2015) reported a slight increment in the methane yield coefficient of up to 16% after carrying out a thermal treatment at 120 ◦C during 15 min. Similarly, Trujillo-Reyes et al. (2019) reported an increment in the methane yield coefficient from 334 ±15 mL CH 4 /g VS (VS, total volatile solids) to 371 ±0 mL CH 4 /g VS in Table 1 Analytical characterization of RRE and raspberry extrudate after application of the different mechanical treatments carried out. Treatment pH %Moist. TS (g/kg RRE) MS (g/kg RRE) VS (g/kg RRE) COD (g/kg RRE) S COD (g/kg RRE) S COD/COD ratio RRE 3.4 ±0.1 43.1 ±2.0 569.1 ±19.8 9.4 ±0.2 559.7 ±19.6 944.1 ±12.0 43.3 ±0.3 0.05 MTBM 3.5 ±0.1 39.6 ±0.2 604.1 ±2.2 10.0 ±0.5 594.1 ±2.2 946.3 ±32.1 44.0 ±0.6 0.05 MTHM 3.5 ±0.1 32.1 ±0.1 678.9 ±0.56 11.0 ±0.3 667.9 ±0.5 1,073.7 ±9.7 63.1 ±0.9 0.06 MTKM 3.8 ±0.1 37.1 ±1.0 628.8 ±10.1 10.6 ±0.4 618.2 ±9.8 1,093.2 ±28.4 71.3 ±1.0 0.07 MTMO 3.5 ±0.1 39.1 ±0.3 609.3 ±2.8 9.9 ±0.4 599.4 ±3.1 1,056.2 ±18.1 50.4 ±1.7 0.05 CKMO 3.9 ±0.1 34.3 ±0.0 656.8 ±0.3 10.9 ±1.3 645.9 ±1.4 1,121.9 ±15.5 74.9 ±0.7 0.07 RRE: Residual raspberry extrudate; MTBM: treatment with ball mills; MTHM: treatment with hammer mills; MTKM: treatment with knife mills; MTMO: treatment with mortar; CKMO: combination of MTKM and MTMO; TS: total solids; MS: mineral solids; VS: volatile solids; COD: chemical oxygen demand; sCOD: soluble chemical oxygen demand. Fig. 1. The total number of particles quantified per gram of substrate after application of different mechanical treatments (RRE: Residual raspberry extrudate; MTBM: treatment with ball mills; MTHM: treatment with hammer mills; MTKM: treatment with knife mills; MTMO: treatment with mortar; CKMO: a combination of MTKM and MTMO). Size of particle (mm) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Number of particles/g 0 1000 2000 3000 4000 5000 RRE MTBM MTHM MTKM MTMO CKMO Fig. 2. Particles distribution obtained after the application of different mechanical treatments (RRE: Residual raspberry extrudate; MTBM: treatment with ball mills; MTHM: treatment with hammer mills; MTKM: treatment with knife mills; MTMO: treatment with mortar; CKMO: a combination of MTKM and MTMO). ´ A. Trujillo-Reyes et al. Waste Management 139 (2022) 190–198 192 the anaerobic digestion of RRE after applying a thermal treatment of 150 ◦C for 60 min followed by a phenol extraction process. The slight effect of the mild thermal treatments might indicate that the limitation for the anaerobic digestion of the berries extrudates is the access of the microorganisms to the more recalcitrant fractions, such as the lignin in the achenes. A possible alternative to treat the RRE could be the mechanical treatments in order to break the structure of the achenes, and thus release the compounds contained in achenes. There is a great variety of mechanical treatment techniques that can be differentiated by the type of strength used, which would be interesting to use (Alessi et al., 2020). In previous studies, where different types of treatments were applied to waste enriched in cellulosic material, as is the case of this waste, mechanical treatment was the most effective (Bong et al., 2018). Another study has shown that particle size reduction processes, such as ball and ultrafine grinding, increase the solvent extractable phenolic content of cereal grains, due to the increase of the specific surface area (Wang et al., 2014). As an extra advantage, mechanical treatments do not produce microbial inhibitors such as furfural or hydroxymethylfurfural (Hendriks & Zeeman, 2009). Consequently, an intriguing and promising option could be the combination of mechanical treatment and subsequent anaerobic digestion process. In that sense, particle size reduction during the mechanical treatment step would increase the surface area available to the anaerobic microorganisms, resulting in greater microbial access and, thus, an increase in the anaerobic biodegradability (Izumi et al., 2010; Jain et al., 2015). Therefore, the main objective of this study is to evaluate the suitability of different mechanical methods for the fractionation and disaggregation of the achenes presented in the RRE for enhancing its anaerobic degradability. For that, the effect of different mechanical treatments over the number of particles and particle size distribution obtained after these were related with the chemical characterization of the treated substrates. Subsequently, the performance, stability, and yield of anaerobic digestion of the treated substrates were also evaluated to compare the effect of the different proposed mechanical treatments. To the best of our knowledge, there are not reports in the literature comparing the effects of the above-mentioned pre-treatments on the chemical characteristics of RRE assessing simultaneously their influence on their anaerobic biodegradability. 2. Materials and methods 2.1. Residual raspberry extrudate and anaerobic inoculum RRE was collected in the factory HUDISA S.A, located in Lepe (Huelva, Spain). RRE was obtained in 2018–2019 season. During industrial processing, RRE was sieved with a 0.5 mm sieve, and it underwent to thermal treatment for enzymatic inactivation at 65 ◦C. RRE was kept under freezing conditions (-20 ◦C) before their use in order to prevent their self-fermentation and deterioration. Once the mechanical treatments had been carried out, the resulting fractions were stored in a cold room at 4 ◦C, in order to preserve them and avoid undesirable fermentations that would significantly alter their composition. As an inoculum source, a fresh sludge from the full-scale anaerobic treatment of wastewater with hydrolytic, acidogenic, and methanogenic capacity from “HEINEKEN SPAIN, S.A.” (Seville, Spain) beer industry was used. The main anaerobic inoculum characteristics were: pH =7.3 ±0.1; alkalinity =2730 ±40 mg CaCO 3 /L; total solids (TS) =63,000 ± 820 mg/kg; total volatile solids (VS) =45,605 ±420 mg/kg. 2.2. Mechanical treatments A total of five different mechanical treatments were carried out: treatment with ball mills (MTBM), treatment with hammer mills (MTHM), treatments with knife mills (MTKM), treatment with mortar (MTMO) and combination of MTKM and MTMO (CKMO). For each mechanical treatment, 60 g of substrate were used and processed. B) C) Total number of particles/g 020004000 6000 800010000 Soluble phenols (mg gallic acid eq./kg RRE) 2000 4000 6000 8000 10000 12000 CKMO MTKM MTHM MTMO RRE MTBM Total number of particles/g 02000400060008000 10000 Water-soluble total sugars (mg glucose eq./kg RRE) 10000 15000 20000 25000 30000 35000 CKMO MTKM MTHM MTMO RRE MTBM D) Total number of particles/g 02000400060008000 10000 Acid sugars (g galacturonic acid eq./kg RRE) 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 CKMO MTKM MTHM MTMO RRE MTBM A) Total number of particles/g 020004000 6000 8000 10000 sCOD (mg O2/kg) 30000 40000 50000 60000 70000 80000 MTHM MTKM CKMO MTMO RRE MTBM Fig. 3. (A) Soluble chemical oxygen demand (sCOD), (B) soluble phenols, (C) water-soluble total sugars, and (D) acid sugars quantified after the application of the different mechanical treatments (RRE: Residual raspberry extrudate; MTBM: treatment with ball mills; MTHM: treatment with hammer mills; MTKM: treatment with knife mills; MTMO: treatment with mortar; CKMO: a combination of MTKM and MTMO). ´ A. Trujillo-Reyes et al. Waste Management 139 (2022) 190–198 193 For MTBM, a planetary ball mill model S1 (Retsch, Haan, Germany) was used. The sample was dumped into a stone grinding vessel (50 mL) together with a total of four 1 cm diameter balls of the same material. The grinding vessel was placed inside the equipment eccentrically on the main wheel. The main wheel rotates in the opposite direction to the grinding bowl with a speed ratio of 1: −2. The movement of the balls inside the container is affected by a Coriolis effect due to the different rotational motions of the balls to the main wheel. The difference in speed between the balls and the bowl results in a combined action of shock and frictional forces that release a large amount of dynamic energy. The high interaction between these forces is responsible for the high grinding performance of planetary ball mills. The equipment was operated for 20 min at maximum speed (main wheel speed: 100 – 650 min −1 ). A micro impact hammer mills model DFH 48 (Culatti A, Steinen, Switzerland), was used for MTHM. The sample was placed in a storage vessel at the top of the equipment. The storage vessel is separated from the internal chamber of the mill (grinding chamber) by a metal separation. When the separating is removed, the sample passes into the grinding chamber, which makes up a toothed rim and blades with toothed ends. The equipment was operated for 30 s at maximum speed (controllable rotational speed between 50 and 6000 min −1 ). After 30 s, over 10–20 s, the speed was gradually slowed down until the equipment stopped operating. The equipment was put into operation following these indications three times. A stainless-steel coffee grinder model Aromatic (Taurus, Oliana, Barcelona, Spain) was used as a knife mill for MTKM. Two sharp, stainless-steel blades rotate in the centre of the grinding container (50 g of capacity). The equipment was operated for five minutes in three different periods, i.e., a total of 15 min. An electronically maintained preselected speed guarantees reproducible results. For MTMO, a 550 mL porcelain mortar for domestic use was employed (HDN ceramics). The manual work was carried out for 10 min. Finally, for the combination of mechanical treatments (CKMO), the sample was firstly grounded with the above-mentioned coffee grinder, and then, the pounded sample was crushed with the porcelain mortar was applied. For both treatments, the times described in the corresponding individual treatments were applied. 2.3. Quantification of the effect of mechanical treatment The size of untreated and treated particles of the RRE was measured through the automatic colony counter Ultra-HD Scan 4000 (Interscience, Saint Nom (France)) to evaluate the breaking effect of the different treatments. This method is frequently used for the quantification of microorganism colonies (Jacobsson et al., 2021; Siddiky et al., 2021). For this, about 1 g of sample from each treatment and from untreated RRE were dried in an oven at 105 ◦C for 24 h. Afterward, about 0.05 g of dried particles were individually distributed in a Petri plate in triplicate, which contained 5 mL of 7% Agar solution, to avoid movement of the particles. A detection range from 0 to 3 mm was used and the total number of particles was counted every 0.25 mm. The higher the number of particles counted with the minimum detection range, the more the treatment affected the residual raspberry extrudate. 2.4. Anaerobic digestion experimental procedure The anaerobic digestion of the untreated substrate (RRE) and of the phases obtained after the application of mechanical treatments with ball mills (MTBM), hammer mills (MTHM) and knife mills (MTKM) were evaluated in batch mode through biochemical methane potential (BMP) tests. BMP tests were carried out with a volatile solid ratio of inoculum/ substrate of 2 (3 g of inoculum/ 1.5 g of the substrate). The inoculum and substrate mixture were supplemented with 20% (v/v) of micro and macronutrients solutions as described in Raposo et al. (2006). In addition, 20 mL of 0.6 M NaHCO 3 was added to the inoculum to regulate the pH of the medium (Raposo et al., 2011). Finally, the working volume (240 mL) of the reactors (Erlenmeyer flask), was completed with distilled water. Reactors were submerged in a water bath at mesophilic conditions (35–37 ◦C) regulated with a thermostat, and continuously stirring (300 rpm). Methane production was measured using 0.5 and 1.0 L gasometers immersed in a solution of NaOH 2 N. Owing to the NaOH property of chemically absorbing the CO 2 present in the biogas, a correct measurement of the methane volume can get by liquid displacement. More details are described in Raposo et al. (2011) and Trujillo-Reyes et al. (2019). The BMP tests were run during the time interval needed to deplete the production of methane, i.e., 33 days. After completion of the test, the biodegradability of the substrates was calculated from the total chemical oxygen demand (COD) of the substrates (Table 1), considering that at temperature and pressure conditions of 25 ◦C and 1 atm, respectively, 382 mL CH 4 /g O 2 are generated. 2.5. Extraction of water-soluble compounds In order to analyse the soluble fraction of the untreated RRE and the different fractions obtained after the application of mechanical treatments, a method widely used for the analysis of soluble compounds in composted materials based on water extraction was applied (Leege et al., 2002). At 20 g of sample 160 g of distilled water was added, remaining in agitation at 300 rpm for 24 h. After this time, the sample obtained was centrifuged (4500 rpm during 10 min) and filtered with 0.45 µm nylon filters. 2.6. Extraction of polyphenolic fractions The extraction and subsequent fractionation of the polyphenolic compounds was carried out according to the method described by P´ erez et al. (2017) slightly modified. 20 mL of an 80% methanol/water mixture were added to 10 g sample (two replicates per sample were extracted). This mixture was kept in constant agitation at 300 rpm for 24 h at room temperature, and in the absence of light in order to avoid degradation of those compounds that are photosensitive (Aguilera-Carbo et al., 2008). After this time, the sample obtained was filtered using 47 mm microfiber filters vacuum. Aliquots of 5 mL of the filtered extract were evaporated in the dark to dryness at 40 ◦C. The sample obtained after the evaporation was redissolved in 1 mL of 0.2 N H 2 SO 4 containing 0.05% EDTA and loaded onto a previously conditioned C18 Sep-Pak cartridge (Supelco, Table 2 Phenolic composition (mg/kg of sample) after application of different mechanical treatments. Treatment Cyanidin 3sophoroside Cyanidin 3-(2glucosylrutinoside) Cyanidin 3-sophoroside-5rhamnoside Cyanidin 3glucoside Ellagic acid derivative Free ellagic acid RRE 22.69 ±1.70 2.54 ±0.50 6.26 ±0.45 1.30 ±0.06 10.64 ±1.04 9.66 ±0.53 MTBM 19.95 ±0.26 2.03 ±0.20 5.14 ±0.21 0.81 ±0.12 9.73 ±0.34 8.91 ±0.06 MTHM 16.95 ±1.25 2.27 ±0.11 4.35 ±0.31 0.60 ±0.03 10.25 ±2.29 9.74 ±0.90 MTKM 15.75 ±0.45 3.34 ±0.54 4.60 ±0.72 0.73 ±0.23 13.01 ±1.27 14.19 ±0.87 MTMO 19.23 ±0.25 3.89 ±0.17 5.42 ±0.30 0.96 ±0.10 11.27 ±0.82 9.90 ±0.77 CKMO 13.96 ±0.20 2.79 ±0.17 3.50 ±0.08 0.50 ±0.03 12.41 ±0.75 14.10 ±0.91 RRE: Residual raspberry extrudate; MTBM: treatment with ball mills; MTHM: treatment with hammer mills; MTKM: treatment with knife mills; MTMO: treatment with mortar; CKMO: combination of MTKM and MTMO. ´ A. Trujillo-Reyes et al. Waste Management 139 (2022) 190–198 194 Bellefonte, PA). Anthocyanins and other polyphenolic compounds were absorbed into the column while sugars and organic acids were eluted with up to 4 mL of the same solution. After complete drying of the column polyphenolics compounds were eluted with 2 or 3 mL of methanol. Extracts were filtered through 0.22 nylon filters before HPLC analysis. 2.7. Analytical methods The determination of pH, alkalinity, the concentration of total solids (TS), mineral solids (MS) and volatile solids (VS), total chemical oxygen demand (COD) and soluble chemical oxygen demand (sCOD) were carried out following the recommendations of the Standard Methods of the American Public Health Association (APHA) (APHA, 2017). These chemical analyses were used for the characterization of the untreated substrate (RRE), phases obtained after the application of mechanical treatments and inoculum, as well as for the final effluents from each BMP test. Anthrone colorimetric method was used for determining water-soluble total sugars using a spectrophotometer (Biorad iMark Microplate Reader, USA) (Ludwig & Goldberg, 1956). Results were A) ) B Total number of particles/g 0200040006000 8000 10000 mg total anthocyanins/kg of sample 15 20 25 30 35 40 CKMO MTKM MTHM MTBM MTMO RRE Total number of particles/g 0200040006000 8000 10000 mg free ellagic acid/kg of sample 8 9 10 11 12 13 14 15 16 MTHM MTMO MTBM RRE CKMO MTKM Fig. 4. (A) Total anthocyanins content and (B) free ellagic acid content quantified after the application of the different mechanical treatments (RRE: Residual raspberry extrudate; MTBM: treatment with ball mills; MTHM: treatment with hammer mills; MTKM: treatment with knife mills; MTMO: treatment with mortar; CKMO: a combination of MTKM and MTMO). ´ A. Trujillo-Reyes et al. Waste Management 139 (2022) 190–198 195 expressed as milligram of glucose equivalents per kilogram of RRE. M−hydroxybiphenil Chromogen method, as described by Blumenkrantz and Asboe-Hansen (Blumenkrantz & Asboe-Hansen, 1974) was used for quantifying water-soluble acid sugars. Results were expressed as grams of galacturonic equivalents per kilogram of RRE. Total polyphenols were determined through the Folin-Ciocalteau method (Singleton et al., 1999), after extraction with an 80% methanol/water solution, previously described. Results were expressed as milligrams of gallic acid equivalents per kilogram of RRE. Identification of the individual polyphenolic compounds was performed in a Beckman Coulter high efficiency liquid chromatography (HPLC) system, as described by CuberoCardoso et al., (2020a). 3. Results and discussion 3.1. Effect of mechanical treatment on particle distribution. Fig. 1 shows the total number of particles per gram of substrate quantified after each of the mechanical treatments. As it can be seen, MTBM and MTMO were the treatments with lowest effects over the number of particles, showing values very similar to the untreated RRE. MTHM allowed an increment in the number of total particles per gram of substrate of more than double in comparison with RRE, meaning a significant effect as for the breakage of the achene structure (Fig. 1). Finally, the highest breakage effects of the achene structure were observed for MTKM and CKMO, being these treatments the ones that caused the greatest effects as for the breakage of the achene structure, i. e., around 6 times a greater number of particles than in RRE (Fig. 1). Fig. 2 shows particle distribution obtained after quantification, i.e., number of particles per gram of substrate quantified by particle size in a range from 0 to 3 mm, every 0.25 mm. Particle distribution after MTBM and MTMO mechanical treatments was very similar to RRE (Fig. 2). MTHM also presented a particle size distribution similar to RRE, except for the range from 0 to 0.25 mm, where it had a greater number of particles per gram of substrate (Fig. 2). The high number of particles in this range explains the differences in the total number of particles between RRE and MTHM previously described (Fig. 1). MTKM and CKMO presented a parallel particle size distribution, showing the highest number of particles per gram of substrate with sizes lower than 1.75 mm and, specially, within the range from 0.25 to 0.50 mm, i.e., 32% and 42% of the total particles for MTKM and CKMO, respectively (Fig. 2). The slightly higher effect in CKMO respect MTKM would be a consequence of the consecutive subjection of the REE to two different treatments, where the particles obtained after the first treatment (MTKM) would be broken again during the subsequent MTMO treatment, resulting in a higher number of particles than where MTKM or MTMO treatment were individually applied. The effect of a treatment has been previously related with the rate of reduction in the particle size and the breakage of the lignin structure (García et al., 2012; Zhang et al., 2010). According to the results shown in Figs. 1 and 2, the impact and friction forces applied during MTBM and MTMO were not enough to achieve the breakage of the structure of the achenes, resulting in a poor reduction in size. In opposite, remarkable results were observed with MTHM, where 44% of the total quantified particles presented sizes lower than 0.25 mm, while the size of the remaining particles is like those quantified in RRE, MTBM and MTMO. This fact may be due to some of achenes could be retained between the toothed structure that conforms the edges of the milling chamber of the hammer mills due to their size and moisture, resulting unaffected during the process. The rest of the achenes were broken and reduced due to the impacts and friction between them caused by the rotation of the internal toothed structure blades. Finally, when comparing MTKM and CKMO, it can be observed that with CKMO improved the breakage of the achenes structure and Table 3 pH, alkalinity (mg CaCO 3 /L), and biodegradability (%) obtained at the end of the BMP test. Treatment pH Alkalinity (mg CaCO 3 /L) Biodegradability (%) RRE 7.4 ±0.1 5378 ±35 3 MTBM 7.6 ±0.1 5430 ±68 6 MTHM 7.4 ±0.1 5309 ±49 21 MTKM 7.4 ±0.1 5595 ±117 35 RRE: Residual raspberry extrudate; MTBM: treatment with ball mills; MTHM: treatment with hammer mills; MTKM: treatment with knife mills. Time (d) 0 5 10 15 20 25 30 35 mL CH4/g VS 0 50 100 150 200 250 300 RRE MTMB MTHM MTKM Fig. 5. Methane yield coefficient (mL CH 4 /g VS) for the different substrates studied (RRE: Residual raspberry extrudate; MTBM: treatment with ball mills; MTHM: treatment with hammer mills; MTKM: treatment with knife mills). ´ A. Trujillo-Reyes et al. Waste Management 139 (2022) 190–198 196 particle size reduction. In both MTKM and CKMO, the action of knives benefited the breakage of the achenes structure, as well as particle size reduction. In addition, the impact and friction forces applied with the mortar arm in the CKMO may also improve the pre-treatment effect. 3.2. Effect of mechanical treatment on the composition of the residual raspberry extrudate. The analytical characterizations of RRE and the substrates after application of the different mechanical treatments are shown in Table 1. pH values of the samples obtained after application of the mechanical treatments, which ranged from 3.4 and 3.9, varied very little compared to pH value of RRE. Regarding to moisture values, it can be observed that RRE was a relatively dry substrate, i.e., moisture =43.1 ±2.0%. The application of mechanical treatments resulted in a decrease of the moisture, which drop up to values of 32.1 ±0.1% after MTHM (Table 1). The moisture contained inside the achenes could have evaporated when destroying its structure or because of the energy applied during the treatment. In all cases studied, around 98% of TS were VS. Comparing the values of TS and VS between the different substrates, it is observed that whit respect to RRE, MTHM, MTKM and CKMO released a higher amount of organic matter, 16, 10 and 13%, respectively (Table 1). The sCOD/COD ratio indicated that approximately 90–95% of organic matter was not in soluble form for all the cases studied. In Fig. 3 (A to D), a positive relation between the number of particles and the concentration of sCOD, total polyphenolic compounds, watersoluble total and acid sugars is observed. The higher total number of particles per gram of substrate, i.e., smaller particle size, the greater solubilization of organic matter was obtained (Fig. 3A). A similar effect was previous reported, for example, in the study carried out by Palmowski and Müller (2003) with materials with a high content in fibre, i. e., hay stems, or in the study carried out by Izumi et al. (2010) with food wastes. Concretely, Palmowski and Müller (2003) reported an increment in the sCOD from 795 mg O 2 /L to 3929 mg O 2 /L after using a stirred ball mill with water addition during 1 h, whereas Izumi et al. (2010) reach a solubilisation in food waste of around 40% by applying a bead mill at different conditions (rpm varied from 300 to 40,000). Similarly, a positive relation was observed between the total number of particles per gram of substrate, which is related with a high presence of small size particles in the particle size distribution, and the polyphenolic compounds extracted from the RRE and after each mechanical treatment (Fig. 3B). In fact, CKMO, where the highest number of particles was quantified, a higher polyphenolic content extractable with solvent was obtained, i.e., 66% higher than in RRE (Figs. 1 and 3B). The effect derived from the mechanical treatments are consequence of the increase in the specific surface area of the particle materials, which facilitate the accessibility of the extraction solvents to the polyphenolic compounds (Wang et al., 2014). Fig. 3C and 3D also show a growing trend between the total number of particles and the water-soluble total and acid sugars, respectively. The increase of the water-soluble sugars can be a consequence of the breaking of covalent bonds during the mechanical pre-treatments, which decompose the complex lignocellulosic structures in simpler carbohydrates (Wang et al., 2014). Despite of this general trend, similar values of water-soluble total and acid sugars were obtained for MTHM, MTKM and CKMO, even with the latter two had a much higher number of total particles per gram of substrate (Fig. 3C and D). This may be since, from a certain particle size onwards, the release of these watersoluble compounds could not be significant. The presence of acid sugars, or uronic acids, is indicative of the existence of acid carbohydrates or pectins in the RRE (Cˆ ot´ e & Hahn, 1994), since galacturonic acid is the main component of pectins. Therefore, the extraction of these compounds could be interesting since pectins are compounds that are used in the food industry as gelling and thickening agents (Chasquibol Silva et al., 2008). 3.3. Effect of the mechanical treatments on the solubilization of polyphenolic compounds. The concentrations, expressed as mg/kg of sample, of identified individual polyphenolic compounds are shown in Table 2. After the breakage of the achenes structure with the application of the different mechanical treatments, the concentration of the identified anthocyanins decreased respect to RRE except for the cyanidin 3-(2-glucosylrutinoside), whose concentration varied in a wide range from 2.03 ±0.20 to 3.89 ±0.17 mg/kg of sample (Table 2). The general decrease on the anthocyanins could be a consequence of the thermal degradation of these compounds, which are thermosensitive (Oszmia´ nski et al., 2015). During the mechanical treatments applied, energy and force are supplied that cause an increase in temperature, which could give rise to the thermal degradation of these compounds. More of ellagic acid derivative and free ellagic acid respect to RRE were quantified when mechanical treatments with a knife mills were applied, i.e., MTKM and CKMO (Table 2). Total anthocyanins content according to the total number of particles per gram of substrate obtained after quantification is shown in Fig. 4A. In this case, particle size reduction did not favour the extraction of anthocyanins, since RRE presented a higher total anthocyanins content. Concretely, RRE presented a total anthocyanins content of 32.79 ±1.83 mg per kilogram of sample (3.28 mg per 100 g of sample), whereas the total anthocyanins content varied in a range from 20 to 30 mg per kilogram of sample after the mechanical treatments (Fig. 4A). Even at the RRE, the total anthocyanins values were in the lower range described for the raspberry fruit. According to Bobinait˙ e et al. (2016), total anthocyanins content in red raspberries usually varied between 20 and 100 mg per kg of fresh fruit. The low values determined in the present research may be a consequence of the application of temperature during the enzymatic inactivation heat treatment process that takes place in the industrial process where RRE is generated (Oszmia´ nski et al., 2015). Free ellagic acid content according to total number of particles per gram of substrate obtained after quantification is shown in Fig. 4B. Particle size reduction favoured the extraction of free ellagic acid. MTKM and CKMO, which generated a greater number or particles (Fig. 1), presented the higher content of ellagic acid (Fig. 4B). Concretely, for RRE, the free ellagic acid content was 9.66 ±0.53 mg per kilogram of sample for RRE, while for MTKM and CKMO the free ellagic acid content was around 14 mg per kilogram of sample, i.e., up to 32% higher than in RRE (Fig. 4B). The values obtained after MTKM and CKMO were higher than those usually contained in raspberry fruit. Concretely, although the values are highly variable depending on different factors such as fruit cultivar or ripening stage, the reported concentration of free ellagic acid in red raspberry fruit is usually lower than 11 mg per kilogram of fresh fruit (3.8 to 8.8 mg/kg (Anttonen & Karjalainen, 2005), 10.13 mg/kg (Zafrilla et al., 2001) and 1.1 mg/kg (Rao & Snyder, 2010)). The increase in the concentration of ellagic acid by MTKM and CKMO can be a consequence of the degradation of the cell structures during the mechanical treatments, since the ellagic acid is a polyphenolic compound strongly linked to cell walls (H¨ akkinen et al., 2000). The increment of the ellagic acid content as a consequence of a treatment application has been also reported by Mildner-Szkudlarz et al. (2019), which reported that roasting raspberry seeds resulted in an increase in free ellagic acid concentrations from 5.38 mg/kg to 10.07 mg/ kg. 3.4. Effect of mechanical treatment on the anaerobic digestion process. The effect of the different mechanical treatments over the anaerobic biodegradability was assessed by testing the biochemical methane potential of the raw RRE and the substrates obtained after the application of MTBM, MTHM and MTKM. These mechanical treatments were selected to represent the different effect on the particle size distribution described in Section 3.1 (Figs. 1 and 2). Despite of the mechanical ´ A. Trujillo-Reyes et al. Waste Management 139 (2022) 190–198 197 treatment, the analysis of the final effluent showed that the anaerobic digestion processes were stable, which pH values within the optimal ranges for the methanogenic activity, i.e., 7–8 (Wheatley, 1990), and an alkalinity concentration high enough (greater than5,300 mg CaCO 3 /L) to avoid possible acidification processes (Table 3). A clear relation between the number of particles in each substrate and the methane yield coefficient was found. The highest methane production corresponded to MTKM, i.e., 236 ±11 mL CH 4 /g VS (Fig. 5), in line with the highest number of particles quantified after this treatment respect the RRE and other treatments (Fig. 1). Likewise, RRE and MTBM, which presented a similar low total number of particles (Fig. 1), resulted in the lowest methane yield coefficient, i.e., 18 ±5 and 35 ±4 mL CH 4 /g VS, respectively (Fig. 5). The intermediate total number of particles in MTHM was also corresponded with an intermediate value of methane yield coefficient, i.e., 128 ±1 mL CH 4 /g VS (Fig. 5). These values were directly in line with the biodegradability of the substrate, which varied from very low values for RRE and MTBM, i.e., 3–6% (Table 3), to up to 35% for MTKM (Table 3). The increase in the methane yield coefficient and the biodegradability can be explained by the better accessibility of the microorganisms to the substrate due to an increase in the specific surface area at reducing the particle size (Izumi et al., 2010; Sun et al., 2019; Zheng et al., 2014). The higher biodegradability in MTKM can be also related with the higher increment in the concentration of soluble compounds, i.e., sCOD, sugars and phenolic compounds (Fig. 3), which would indicate that the substrate was partially hydrolysed during the mechanical treatment and, therefore, it is more susceptible of being biomethanised (Ortega et al., 2008). Similar results were also obtained by Palmowski and Müller (2003) with materials high in fibre, sunflower seeds, which reported an increment in the methane yield coefficientbiodegradability up to 19% by reducing the particle size from 5 to 2.04 mm. 4. Conclusions. A high variation in the number of particles and the particle size distribution was achieved depending on the mechanical treatment applied. The most effective treatments were MTKM and CKMO, reaching around 6 times a greater number of particles than in RRE. The higher number of particles was related to higher solubilisation of the RRE during the mechanical treatments, including an increase in sCOD, phenols, and sugars. However, the presence of anthocyanins was higher in the mechanical treatments with low effect over the RRE. The enhancement of the anaerobic digestion of the RRE was clearly related to the increment in the number of particles of small size after the mechanical treatments. Concretely, MTKM reached a methane yield coefficient of 236 ±11 mL CH 4 /g VS, more than 10 times higher than RRE. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was funded by the Spanish Ministry of Economy, Industry, and Competitiveness for funding this research through Project CTM2017-83870-R. Antonio Serrano is grateful to the Spanish Ministry of Science, Innovation, and Universities for his Juan de la CiervaIncorporacion fellowship (IJC2019-040933-I). Finally, Erika Sinisgalli wants to acknowledge the Erasmus Plus Programme of the EU for the fellowship received for her traineeship at the Instituto de la Grasa (CSIC), Seville, Spain. References Aguilera-Carbo, A., Augur, C., Prado-Barragan, L.A., Favela-Torres, E., Aguilar, C.N., 2008. Microbial production of ellagic acid and biodegradation of ellagitannins. Appl. Microbiol. Biotechnol. 78 (2), 189–199. 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