Biomass Conversion and Biorefinery https://doi.org/10.1007/s13399-020-00833-8 ORIGINAL ARTICLE Biogas production from co-digestion of different proportions of food waste and fresh bovine manure Monaliza Mirella de Morais Andrade 1 & Bárbara Ribeiro Alves Alencar 2 & Nathalia Pereira Leite 1 & Alessandra Lee Barbosa Firmo 3 & Emmanuel Damilano Dutra 1 & Everardo Valadares de Sá Barretto Sampaio 1 & Rômulo Simões Cezar Menezes 1 Received: 13 March 2020 / Revised: 17 June 2020 / Accepted: 19 June 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract The present study aimed to evaluate the process of anaerobic co-digestion of different proportions of food waste and fresh bovine manure. The experiments were carried out in the laboratory (using 250-mL reactors) and in a pilot-scale biodigester (8 m3). The laboratory experiment was conducted with batch feed systems testing three food waste (FW) and bovine manure (BM) ratios: 0:1, 1:2, and 3:1 (equivalent to 0, 33, and 75 % of food waste in the digester substrate, respectively). The pilot-scale biodigester had a continuous feed system and received a 1:2 FW:BM mixture ratio. The highest accumulated biogas production in the laboratory was 273 mL g-1 of volatile solids (VS) in the treatment with the 1:2 FW:BM ratio. Concentrations of volatile fatty acids (VFA) greater than 8 g L-1 inhibited methane production, except in the treatment without food waste. In the pilot-scale biodigester, concentrations of VFA were below the inhibitory threshold, and the biogas and methane yields were 271 L kgVS-1 and 220 L kgVS-1, respectively. Therefore, food waste can be successfully co-digested with fresh bovine manure in continuous feed digesters, with an initial organic load rate of at least 2.74 kgVS m-3 day-1. Future studies should aim to test progressive increases in food waste load to identify the threshold for inhibition. Keywords Organic residues . Anaerobic digestion . Biodigesters . Methane 1 Introduction The proper treatment and disposal of municipal solid waste are a challenge due to the high proportion of organic matter in its composition. The recycling and treatment of this organic fraction to recover energy are rapidly emerging as effective waste management strategies, diverting it from landfills, and generating income [1]. Currently, the most used treatment processes for the organic fraction of solid waste are * Monaliza Mirella de Morais Andrade [email protected] 1 Research Group on Biomass Energy, Department of Nuclear Energy, Federal University of Pernambuco, Av. Prof. Moraes Rego, 1235, Cidade Universitária, Recife, PE 50740-540, Brazil 2 Interdepartmental Research Group in Metabolic Engineering, Department of Genetics, Federal University of Pernambuco, Recife, PE 50670-901, Brazil 3 Federal Institut of Pernambuco, Recife, PE 50670-901, Brazil composting and anaerobic digestion. These processes not only represent an alternative to reduce the environmental impact caused by the inadequate disposal of these organic wastes but can also produce methane and biofertilizer [2–4]. Therefore, they represent an opportunity to recover energy and nutrients from organic residues. The process of anaerobic digestion can be influenced by several factors, which may inhibit the production of methane [5]. Due to the high content of readily biodegradable ingredients, food wastes are rapidly hydrolyzed, and this can lead to the accumulation of volatile fatty acids (VFAs), especially propionic acid, which can result in the failure of the process in a short period of time [6, 7]. An alternative to obtaining a better biogas yield is to co-digest food waste with other organic residues, seeking an adequate nutritional supply, the dilution of toxic compounds, and an increase of the biodegradable organic matter load [3, 8]. These organic residues can also be a ready source of inocula, providing microorganisms to accelerate the hydrolysis of organic compounds and biogas production and, consequently, reducing the residence time of the substrate in the biodigester and the time of operation [9]. Biomass Conv. Bioref. Several sources of inocula can be used in anaerobic biodigesters, such as sewage treatment plant effluent, stabilized effluents from operating anaerobic biodigesters, or manure from cattle or other ruminants [10–12]. Anaerobic sludge from sewage plants may be difficult to be obtained in many locations, requiring prior authorization from the management company of the sewage treatment plant and transportation to the place of biogas production, sometimes distant and difficult to access. Obtaining inoculum from substrate stabilization takes time and requires an appropriate area to store food waste before feeding to the biodigester [13], and some regions may not have operating biodigesters to supply stabilized effluents. On the other hand, fresh animal manure, in particular from ruminants, is a ubiquitous substrate in most areas of the world, and the co-digestion of food waste with fresh manure can be performed in a stable manner without the addition of anaerobic sludge [14]. Bovine manure, for example, in addition to being a good nitrogen source, has microbial communities that can assist in anaerobic digestion. Therefore, the use of fresh manure eliminates the cost and time to obtain inoculum from sewage plants or from substrate stabilization and facilitates the adoption of anaerobic biogas production in rural properties and small remote communities. However, the co-digestion of food waste with different sources of inoculum may affect the biodegradability results, as a consequence of different microbial communities and substrate chemical composition [13]. Little has been published about the behavior and the possibility of co-digesting without the addition of adapted inoculants, such as sewage sludge or stabilized biodigester effluents. Considering this lack of information, the objective of this study was to evaluate the process of anaerobic co-digestion of different proportions of food waste and fresh bovine manure both in batch feeding digesters in laboratory assays and in a pilot-scale (8 m3) continuous feeding system biodigester. the food preparation, such as rice, beans, and meats, were the same almost every day, but there was some variation in minor components, such as fruit type, so that the proportions of the components in the food waste had some daily variation. Once a week, all the waste generated during the day in the restaurant was collected and piled on a large plastic canvas in a screening yard. The waste was then mixed and removed from the pile by applying the blocking technique, according to NBR 10007 [15], in which the waste contained in two opposite quadrants is separated and mixed again, repeating the process until a uniform sample of approximately 2 kg was obtained. The samples were ground in a food grinder and dried in a forced ventilation oven at 40 ° C. The dry mass was milled, sieved in a 2-mesh screen, and stored in a screw-cap plastic container containing silica gel until the time to feed the small scale biodigesters (AD1). A subsample was analyzed in relation to its chemical composition (Table 1). Food waste collected daily in the restaurant was also used to feed the pilot-scale biodigester (AD2). After sorting and removing inorganic materials, the organic wastes were ground in knife mills and transferred to the feed box coupled to the biodigester for dilution with water and subsequent pumping into the biodigester. Two sources of bovine manure were used in the experiments. Manure from the first source, denominated as manure 1, was used in the AD1 experiment, and it was collected at a dairy farm where the animals were kept in a semi-intensive system (feeding both in the pasture and in the barn). Sampling was performed at dawn in three barns where the animals were kept confined at night. Portions were deposited in 5-L plastic bags and kept under refrigeration at 4 °C for a few hours until the moment of use in the experiment, which was setup on the same day of manure collection. Manure from the second source (manure 2) was used in the pilot-scale biodigester 2 Material and methods Parameters FW BM Moisture (%) pH Alkalinity (mg·L-1) Total solids (%) Volatile solids (%) Total carbon (%) Total nitrogen (%) C/N Hydrogen (%) Sulfur (%) COD (mgO2 L-1) Phosphorus (%) Potassium Calorific power (Jg-1) 70.8 4.8 128 29.2 92.5 46.8 4.04 11.6 5.6 1.5 116.13 0.096 0.30 18660 85 7.9 Nd 15 54 43.8 1.69 25.9 5.11 17123 Two experiments were performed at different scales. The first experiment consisted of batch tests conducted in 250-mL glass bottles at 36 °C, hereafter referred to as “AD1” (anaerobic digester 1). The second experiment was conducted on a pilotscale (8 m3) tubular model biodigester (referred to as “AD2”). 2.1 Collection and preparation of food waste and manure samples Food waste samples were collected at the restaurant of the Universidade Federal de Pernambuco (UFPE) in Recife, Pernambuco, Brazil, from January to December 2016. The food waste contained mainly rice, beans, meats, pasta, vegetables, fruits, cereals, and fats, together with wastes from vegetable preparation, such as peels, stems, and roots. The basic components of Table 1 Characterization of food waste (FW) collected at the restaurant and bovine manure (BM) used in anaerobic co-digestion Nd, not determined Biomass Conv. Bioref. (AD2) and was obtained at the experimental station of the Agronomic Institute of Pernambuco - IPA, in São Bento do Una municipality. The herd of dairy cattle is raised in an intensive system (fed exclusively in the barn). The manure samples were collected after pilling the feces deposited in the barn floor of the farm and homogenizing it with a hoe. The collected manure was stored in 200-L plastic barrels and transported immediately (3 h of travel by truck) to be added to the biodigester feed tank at UFPE. Before the beginning of both incubation experiments, the manure was diluted 1: 1 (v/v) with non-chlorinated water to decrease the concentration of solids in the substrate and to facilitate pumping. The compositions of the food waste and bovine manure samples are presented in Table 1. 2.2 Experimental setup of the batch tests (AD1) The small-scale anaerobic co-digestion experiment was performed using batch-type biodigesters, following the methodology adapted from Biochemical Methane Potential (BMP) and originally proposed by Owen et al. [16]. The biodigesters were 250-mL borosilicate flasks, with an effective volume of 150 mL. Each flask had a nylon cap with one valve for biogas collection, connected to one 1 kgf cm-2 manometer, with a 0.2 kgf cm-2 scale, and screwed with a ring rubber seal. The experiment was set up as a completely randomized design, with three replicates, and three treatments, corresponding to 0, 33, and 75 % of food waste and the complementary proportions of fresh manure (100, 67, and 25%) in the digester substrate (on a volatile solids basis, gvs). These proportions were based on the study by Zhang et al. [17] and corresponded to ratios of 0:1, 1:2, and 3:1 of food waste to manure. After feeding the digesters, the pH of the manure and food waste mixture was adjusted to approximately pH = 7.0 with calcium carbonate. Each digester was closed and, to ensure anaerobic conditions, N2 was circulated for 2 min while manually stirring in order to remove the oxygen present in the internal gas atmosphere and dissolved in the solution. Subsequently, each digester was wrapped in aluminum foil to prevent exposure of the substrate to the light, which could harm the microorganisms and alter the biodegradation process. The digesters were maintained in a bacteriological oven at 36 ± 2 °C for 65 days. Biogas production was checked daily by the displacement of the manometer pointer coupled to the cap of each reactor. The volume of biogas produced was calculated from the difference in daily pressure using the equation described by ElMashad and Zhang [18]: Vb ¼ P⋅V hs ⋅C R⋅T where Vb (mL) is the volume of daily biogas production; P (mbar) is the pressure difference; Vhs (mL) is the headspace volume; C is the molar volume (22.41 L mol-1); R is the perfect gas universal constant (83.14 L mbar K-1 mol-1); and T (°K) is the absolute temperature. 2.3 Experimental setup of the pilot-scale biodigester (AD2) The pilot-scale experiment was conducted in a continuous 8 m3 biodigester, tubular model, made with flexible PVC laminates, structured with black polyester fabric (Table 2). The study was carried out at the Experimental Biorefinery of Organic Solid Wastes of the Federal University of Pernambuco, Brazil. Since the restaurant produces, on average, 1000 kg of food waste daily, the anaerobic digestion system was defined to be continuous, with daily feeding. The digestion technique was single phase, anaerobic, wet, with a working temperature in the mesophilic stage of 36 ± 1 °C and a retention time of 27 days. The pilotscale biodigester was previously fed with bovine manure, diluted with non-chlorinated water in a proportion of 1: 1 (g:g), and operated for 30 days. After this period, the biodigester started to be fed with food waste. The pilot-scale biodigester operated with an organic load of 2.74 kgVS m-3 day-1. The stability of the system was evaluated for 30 days, performing weekly measurements of pH and VFA concentration in samples collected through the lateral flanges of the biodigester. The performance of the anaerobic digestion system was evaluated by quantifying the daily biogas production, with weekly determination of the biogas methane concentration, quantified collecting the gas through a gas line–coupled valve, and storing it in gas-impermeable plastic bags. 2.4 Analytical methods Total solids (TS) and volatile solids (VS) contents were determined according to the APHA [19]. The pH of the food waste samples was determined using a portable pH meter (Digimed) Table 2 Mass and volume of the substrates used to feed the pilot-scale biodigester (8 m3) Description Bovine manure1 Food waste2 Fresh substrate 1600 kg 70 kg day-1 Total solids 200 kg 11.9 kg day-1 Water Volume of substrate added Total volume 1.61 m3 1.68 m3 3.29 m3 0.07 m3 day-1 0.08 m3 day-1 0.15 m3 day-1 1 Single application at the beginning of the study; 2 Daily additions Biomass Conv. Bioref. after diluting them with distilled water in a ratio of 1: 2.5 (mass basis), under horizontal shaking for 10 min, and waiting for 30 min before reading. Elemental analysis (C, H, N, S) was performed using an EA300 Euro Vector elemental analyzer. Phosphorus was determined by colorimetry method in a spectrophotometer and potassium by flame photometry in a Digimed photometer, according to the methodology for plant and organic residue analysis [20]. Carbon and nitrogen were determined by combustion at 925 °C in a CHNS-O elemental analyzer, using BBOT, cysteine, sulfonyl amide, and methionine as standards. Characterization of the biogas (in terms of CO2 and CH4) was performed by gas chromatography, in GC-Master equipment with a thermal conductivity detector (TCD), using a Porapak “N” column, with an oven temperature of 60 °C and sample injection of 100 μL. The standard gas used for calibration consisted of 60% (v/v) CH4 and 40% CO2. Data of each analyzed variable were submitted to analysis of variance (ANOVA), and the averages were compared by the Tukey test at the 0.05 probability level. 3 Results and discussion 3.1 Small-scale reactors with batch feeding (AD1) The initial pH in the mixtures added to each reactor did not differ significantly in the treatments with the different proportions of food waste in the digester substrate (Fig. 1). In all treatments, the pH decreased during the digestion, but the reduction was less pronounced in the treatment without food Fig. 1 Initial and final pH in an anaerobic co-digestion systems of bovine manure (BM) and different proportions of food waste (FW) in batch feeding small-scale digesters (AD1). Means followed by the same letters are not significantly different at the 5% significance level by the Tukey test waste (FW 0%), which was still neutral (7.06) at the end of the experiment. In the treatment with the highest proportion of food waste (FW 75%), the pH became excessively acidic (4.16). Therefore, the co-digestion of manure and food residues is a viable alternative to alleviate the inhibition caused by excessive acidification, provided that the proportion of food residues is less than 50% [21]. Similar pH values have been reported by Zhai et al. [22], with pH values dropping to a minimum of 4.8 in anaerobic reactors with a sludge and manure ratio of 1:1, and by Khairuddin et al. [8], with a minimum pH of 4.5 with food waste and anaerobic sludge in a 2:1 ratio. They suggested that the buffering capacity of the stabilized inoculum increases with the increase in the incubation time of the substrate and residue mixture. The reduction of VS did not differ among treatments, reaching around 98% in all of them. The degradation of VS in anaerobic digestion systems varies widely [23], depending on the chemical composition of the food waste and the type of manure used, 9.01 and 8.21 g L-1 in the digesters containing food waste (FW 33% and FW 75%) (Table 3), while in the biodigester without food waste, the concentration was below 1 g L-1. The stability of the VFA concentration in the absence of food waste may have occurred because the C/N ratio was equal to 25.9, within the 20 to 30 range, suggested as ideal [24], contributing to the proper release of NH3 and providing the concentration of VFA was equilibrium of the system with the formation of acids as intermediates in the digestion of manure. Other studies have suggested that severe inhibition of methane production may occur due to the accumulation of VFA as long as the waste content in the biodigester exceeds Biomass Conv. Bioref. Table 3 Reduction of volatile solids (VS) and accumulation of volatile fatty acids in an anaerobic co-digestion systems of bovine manure (BM) and different proportions of food waste (FW) in batch feeding small-scale digesters (AD1) FW proportions VS reduction (%) VFA (g L-1) FW 33% FW 75% FW 0% 99.0 a ± 1.15 98.6 a ± 0.57 98.2 a ± 0.57 9.01 a ± 3.61 8.21 a ± 0.55 0.052 b ± 0.15 Means followed by the same letters are not significantly different at the 5% significance level by the Tukey test 50 % [21]. In the present study, only the treatment without food waste (FW 0%) had a VFA concentration (0.052 g L-1) in a range considered adequate for a stable anaerobic digestion system [25], which demonstrates that even a relatively low proportion (33%) of food waste inhibited the process in the small-scale batch feeding digesters. Therefore, we observed that the stabilization of the anaerobic digestion process, even with a food waste proportion lower than 50%, might require intervention to contribute to the buffering capacity. This intervention could be the addition of a base, co-digestion with higher ratios of an alkaline substrate, or maybe recirculation of the effluent. The accumulated biogas production was favored by the addition of food waste, compared with the treatment which received only manure (Fig. 2), and the production was higher in FW 33% than in FW 75% (273 and 232 mL gVS-1, respectively). The lower production was probably related to the pH decrease observed in FW 75%, due to the food waste proportion exceeding 50% [17]. The accumulated yields are lower than those reported with co-digestion of food waste and manure with the addition of anaerobic sludge: El-Mashad et al. Fig. 2 Accumulated biogas production in anaerobic codigestion systems of bovine manure (BM) and different proportions of food waste (FW) in batch feeding small-scale digesters (AD1) [18] reported productions of 570 to 621 mL gVS-1 and Marañón et al. [26] 644 to 880 L kgVS-1. Our lower yields could be due to the high acidification in the digesters with the addition of food waste and the absence of a stabilized inocula. The biogas production in all treatments peaked in the first 10 days of incubation. In FW 33%, about 60 mL gVS -1 biogas production peaks were observed until the fifth day, being reduced to 16.3 mL gVS-1 around the 15th day, and tending to stabilize from the third week until the end of the assay, with an average yield of 6.2 mL gVS-1. Biogas production in FW 75% reached 14.0 mL gVS-1 on the first 3 days of incubation, decreasing to an average of 7.5 mL gVS-1 around the sixth day, 6.2 mL gVS-1 on the sixteenth day, and tending to stabilize from the twentieth day on, with an average production rate of 5.3 mL gVS-1. The rapid reduction in the biogas production rate may indicate that the available organic matter was rapidly degraded and may have favored the accumulation of volatile fatty acids, jeopardizing methane production (Fig. 3). The behavior of the biogas production curve in the FW 75% treatment differed from those of the other treatments. Production peaked at 11.5 mL gVS-1 and then decreased to 66% of this average, increasing approximately 6% by the thirteenth day. The trend towards production stability occurred after about one and a half months, with averages between 5.6 and 4.2 mL gVS-1. These low values may be due to acidification during the first digestion stage, which is not compatible with the normal growth of methanogenic organisms [27]. These results corroborate those of Li et al. [28], who reported that the substrate/inoculum ratio was the main factor affecting anaerobic digestion during the initial phase of the process. The possible inhibition of methanogenesis due to acidification caused by the accumulation of VFA and low pH values was irreversible. Biomass Conv. Bioref. Fig. 3 Daily biogas production rate in an anaerobic co-digestion system of bovine manure (BM) and different proportions of food waste (FW) in batch feeding small-scale digesters (AD1) Accumulated biogas production stabilized at different moments for the different treatments, ranging from 57 days in FW 33% to 62 days in FW 75%. Anaerobic digestion with high concentrations of solids (> 15%), such as those in FW 33% and FW 75%, which operated with 15 and 20% of total solids, respectively, require inoculum application, due to the long time required for stabilization of biogas production [24]. Khairuddin et al. [8], co-digesting different ratios of food waste and bovine manure, with the application of inoculum and total solids concentration around 20%, reported 40 days for stabilization of biogas production. Zhai et al. [22] reported a period of 45 to 56 days to stabilize biogas production in digesters with 23% of total solids. However, establishing the ideal inoculum selection for anaerobic digestion of food residue is still a problem due to the heterogeneity of residues [10]. 3.2 Pilot-scale digester (AD2) In the first 2 weeks of digestion, the concentration of VFA was reduced from 1346 to 1214 g L-1, while the pH increased from 6.49 to 6.71 (Table 4). In the third week, the pH decreased to 6.54 and increased again in the last week of digestion, reaching the same value observed in the second week (6.71). Table 4 Weekly variation of pH and accumulation of volatile fatty acids (VFA) in a pilot-scale anaerobic continuous digestion of food wastes and bovine manure (AD2) Weeks pH VFA (mg L-1) 1 2 3 4 6.49 6.71 6.54 6.71 1346 1214 1526 1515 As a consequence, in the third week, the VFA concentration increased to 1526 g L-1 and decreased to 1515 g L-1 in the last week. The variation in pH mentioned above is within the optimal range to maintain a stable process of anaerobic digestion [29]. Similarly, the process of methanogenesis operates best at a pH range from 6 to 8, which favors the proliferation of slow-growing methanogenic arches [30]. The biogas yield ranged from 130 to 330 L kg VS-1, with the production peak on the 17th retention day, after which it oscillated to reach a new but lower peak (260 L kg VS-1 ) on the 20th retention day (Fig. 4). Since the biodigester was fed with food waste collected daily in the restaurant and its composition was slightly heterogeneous on a daily basis, we hypothesize that the volume and composition of the biogas formed may have varied in part as a response to the quality of the waste. The average biogas yield was 39 L kg -1 of the food waste, on a fresh weight basis, lower than those reported by Rapport et al. [31], in the range of 100 to 150 L kg -1, and by Grimberg et al. [30], with a mean yield of 120 L kg -1. The low yield may be related to the short retention time and or to the food waste composition. This result points out to the need to monitor, during the feeding of the biodigester, the proportion of the main types of components that make up the food waste, which may significantly affect its chemical composition. In the present study, we have a value of the average chemical composition of the food waste, based on composite samples taken during several days, but do not have data on the daily variation of food waste quality. The concentration of methane in the biogas of the pilot-scale biodigester varied between 58 - 60%, which is in the same range of other studies with similar experimental setup. However, the average methane yield in our pilot-scale Biomass Conv. Bioref. Volume (m3 kgVS-1) Fig. 4 Daily production of biogas and methane in a pilot-scale anaerobic continuous digestion system s of food waste (FW) and bovine manure (BM) biodigester was low, varying from 75.4 to 198 L kg VS-1, with peak production on the 17th retention day, after which it fluctuated to reach a new, but smaller peak (135 L kg VS-1) in the 20th day of retention (Fig. 4). The methane concentration in most similar studies reported in the literature generally varies between 60 and 64% (volume), with an average yield of around 220 L kgVS-1. Grimberg et al. [30] reported similar methane concentrations (from 59 to 60%) but higher yields, reaching up to 380 L kgVS day-1, in 5 m3 biodigesters, single-stage food treatment and a load of 3.79 kgVS m-3 day-1. This higher volumetric organic load suggests that the increase in the organic load may favor the increase in methane concentration. Zeshan et al. [32], working with pilot-scale biodigesters with organic loads of 2.60 and 10.65 kgVS m-3 day-1 and retention time of 54 and 13 days, observed a maximum methane yield of 327 L kgVS-1 and a minimum of 121 L kgVS-1. They pointed as possible reasons for the maximum yield the inversely proportional relation between volumetric organic load and retention time. Therefore, it can be inferred that the 30 days of the present study did not allow the maximum methane yield to be reached in this pilot-scale biodigester. excessive acidification and accumulation of VFA, which resulted in a methane yield of 220 L kgVS-1 during the 30-day trial. Therefore, food waste can be successfully co-digested with fresh bovine manure in continuous feed digesters, with an initial organic load rate of at least 2.74 kgVS m-3 day-1. Future studies should aim to test progressive increases in food waste load to identify the threshold for inhibition. 4 Conclusions 2. Co-digestion of food waste with fresh bovine manure in batch feeding systems did not provide sufficient buffering to the system to prevent excessive acidification and accumulation of VFA, resulting in methanogenesis inhibition with proportions of food waste of 33 and 75% in the digesters. In the pilotscale digester, the continuous feed system with an organic volumetric load of 2.74 kgVS m-3 day-1 was able to avoid Funding information The authors acknowledge the CNPq, for financial support CHAMADA UNIVERSAL– MCTI/CNPq N° 14/2014 (Processo 454423/2014-4) and the doctoral scholarship grant (Processo 141364/2014-9) to this research. This work is part of the National Observatory of Water and Carbon Dynamics in the Caatinga Biome NOWCDCB, supported by FACEPE (grants: APQ-0296-5.01/17; APQ-0498-3.07/17 ONDACBC; APQ-0532-5.01/14), CNPq (grants: 441305/2017-2; 465764/2014-2), and CAPES (grants: 88887.136369/ 2017-00).and FINEP (CT-Infra 01/2013 - REF 0648/13 - SUGERE). References 1. 3. 4. 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