Methane production from previously fermented cacao waste pod husks

Methane production from previously fermented cacao waste pod husks | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Methane production from previously fermented cacao waste pod husks Fabiany de Jesús Morgado-León, Simon Gonzalez-Martinez, German Dimitriv Jojoa-Unigarro This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5981919/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 May, 2025 Read the published version in Waste and Biomass Valorization → Version 1 posted 6 You are reading this latest preprint version Abstract The wastes from cacao processing represent approximately 80% of the fruit weight and are rich in readily biodegradable carbohydrates and cellulosic and lignocellulosic compounds. There is little information about the processing of these wastes, and few authors report the potential of these wastes to produce energy through anaerobic digestion. As an alternative to disposing of these wastes in the field, associated with negative consequences, such as soil pollution, this research deals with the fermentation of the cacao pod husks under different organic loading rates and/or solids retention times to enhance hydrolysis through the production of acids and alcohols. Ethanol, lactic, acetic, propionic, and butyric acids were identified in fermented cacao pod husks. The digestates from the fermentation were tested for methane production. From the main results, the following conclusions can be drawn. The fermentation efficiency depends on the solids retention time, and the hydrolysis rate is inversely proportional to the organic load and directly proportional to solids retention times. Metabolic displacements show sudden changes in the composition of the volatile acid due to solids retention time. Methane production did not tend to be associated with the organic load or solids retention times. The specific methane production increased with decreasing substrate concentration, indicating substrate inhibition. Agricultural waste cacao pod husk anaerobic digestion biogas methane production Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights Cacao pod husks contain significant amounts of readily degradable carbohydrates Ten tons of waste are produced for every ton of cacao seeds Fermentation of waste cacao pod husks can produce alcohols and acids Methane is more efficiently produced from previously fermented cacao waste Substrate inhibition is significant during methane production INTRODUCTION According to the Mexican Agriculture Ministry [ 1 ], cacao is Mexico's most estimated shade agricultural product after coffee. The International Cacao Organization (ICO) estimates that between 2015 and 2016, clean cacao grain production worldwide was 3.9 thousand tons, generating 16 thousand tons of waste biomass [ 2 ]. According to ICO, cacao production during 2022/2023 was 4,953 thousand tons. Africa reported the highest production with 73.4%, then the tropical regions of the American Continent, and finally, Asia and Oceania with 5.4%. Akinjokun et al [ 3 ] propose that 10 tons of waste are generated for every ton of cacao beans. The waste that originates during the separation of the cacao seed from the pods is abundant and renewable. Of the waste cacao pods, only 10% is processed for industrial purposes [ 4 ]; the rest corresponds to the mucilage and peel. In the cacao plantations, after hand-separating the grain from the pods, the waste biomass is left to rot in the field, causing soil deterioration, aquifer contamination, and, because of the fermentation processes, insects and other harmful organisms can develop. Notorious is the long-time anaerobic digestion producing methane and carbon dioxide released into the atmosphere [ 5 – 8 ]. Some strategies for using cacao waste have been as animal forage (fibers and carbohydrates) and for the food and pharma industries [ 2 ]. Solid-state fermentation of cocoa pod husk using Aspergillus niger can be used to produce citric acid [ 39 ]. Cocoa pod husks contain pectin, which can be extracted for biological, pharmacological, and food applications [ 26 ]. Besides anaerobic digestion for biogas production, cocoa pod husk can be used in other thermochemical processes such as direct combustion, gasification, and pyrolysis [ 40 ]. Allen et al. [ 10 ] calculated similar values for domestic solid wastes, garden trimmings from parks, seaweed, and corn silage for methane production. This assessment implies that the waste biomass from cacao production has the potential for methane production through anaerobic digestion. The composition of the pod husks indicates lower oil and grease contents but higher values of non-amylase polysaccharides such as cellulose and hemicellulose [ 2 , 4 , 8 , 11 , 12 ]. Lignin is an essential component of the peel. Fermentation with naturally occurring microorganisms, as a pretreatment of the pod husks at ambient temperature in tropical regions where cacao is cultivated, is considered an alternative before methane production. The fermentation of readily biodegradable substances and other fibers can contain metabolites that methanogens can easily transform into biogas. Some publications confirm that the fermentation of lignocellulosic biomasses enhances the hydrolysis of fibers and increases the subsequent methane production [ 13 – 15 ]. Jojoa-Unigarro and González-Martínez [ 15 ] conclude that the fermentation of solid organic waste should be oriented to ethanolic and acetic fermentations for further better methane production. The fermentation of different biomasses shows that ethanol fermentations are common at lower organic loading rates or higher solids retention times and that, at higher loading rates or lower solids retention times, acid fermentation promotes the formation of acetic, lactic, and butyric acids [ 15 ]. Antwi et al . [ 16 ] report that the whole cacao pod husk produces 922 L/kg VS as theoretical biogas potential with 51% methane. These same authors report lab analyses showing that cacao pod husks are not readily biodegradable as a whole, with a biogas production of 375 L/kg VS , representing this value only 47% of the theoretical value. The low biodegradability lies within the outer Shell of the cacao pod husks, which is rich in lignin. The softer internal parts are rich in low molecular weight carbohydrates and hemicellulose, which are readily biodegradable through fermentation [ 17 , 18 ]. This is why acid fermentation is required before methanization. Comparing the theoretical biomethane production of the cacao pod husks with other substrates, the waste pod husks produce similar amounts of methane as domestic organic solid waste, marine algae, maize silage, and garden and park wastes. The main conclusion is that waste cacao pod husks must be considered an adequate substrate for methane production through anaerobic digestion [ 10 ]. This research proposes evaluating the fermentation of cacao waste husk pods under different organic loading rates and/or solids retention times and determining the kinetics of methane production from the previously fermented waste husk pods. The main innovation of this work lies in the combination of controlled fermentation of cacao husks with the kinetic analysis of methane production. This research explores an intermediate fermentation stage as a pretreatment before methanization. This stage can potentially enhance the conversion of lignocellulosic compounds in cacao husks and optimize methane production in anaerobic processes. MATERIALS AND METHODS Preparation of the sample Twenty kilos of cacao fruit (pods) were bought from a producer in Pichucalco, Chiapas, Mexico. They were cut, and the grains were separated, together with the so-called "placenta," which is part of the fruit that binds the grains together (Fig. 1 ). The cacao pod husk includes the exocarp, mesocarp, and a layer of endocarp from the freshly harvested cacao fruit. The remaining husks and placenta were extruded through a 0.5 mm sieve and then ground with a plate mill to deliver particles approximately 0.5 mm in diameter. Following the proposal of Kreuger et al. [ 19 ], the mass resulting from this process was frozen in specialized plastic bags at -18°C to be used during the experimental procedure to ensure that the characteristics did not change with different deliveries. The parameters used for the characterization were chemical oxygen demand (COD), total and volatile solids (TS and VS, respectively), humidity, pH, carbohydrates (total, soluble, sugars, and fiber), fats and oils, cellulose, hemicellulose, and lignin. Fermentation of cacao pod husks under different organic loading rates Figure 2 describes the experimental setup for the fermentation of the cacao pod husks. For the fermentation of the cacao pod husks under different organic loading rates (OLRs), a jacketed glass reactor with a 4.5-litre volume was used. The reactor is provided with a mechanical stirrer (Bioprocess Control, Sweden). The pH was monitored daily using an electrode submerged in the reactor; however, no active control was applied. The working temperature was held constantly at 35°C. The reactor was operated semi-continuously and fed with a ground cacao pod husk suspension with 25 ± 2 g COD /L. The different organic loading rates were adjusted by adjusting the volume of the daily feed, resulting in different solids retention times (SRT). The OLRs tested were 1.3, 2.6, 3.7, and 4.9 g COD /L·d. Hydraulic retention time (HRT) and solids retention times were the same, resulting in values between 19 and 4.8 days. The runs needed to last at least twice the SRT to guarantee stable operation during the experimental runs under different OLRs. The fermentation was monitored using pH, COD, total and dissolved solids, volatile fatty acids (VFA), lactic acid, lower alcohols, and total and soluble carbohydrates with a frequency of 3 to 4 times per week, that is, on alternate days, even though the feeding was daily. The solubilization rate was calculated as the ratio of soluble COD to total COD times 100. Methanization of the fermented digestates The potential to produce methane from the fermented ground cacao pod husks was tested using an automatic methane potential test system (AMPTS II, Bioprocess Control, Sweden). The specific methane production of the fermented substrates was compared to that of fresh unfermented cacao pod husks. The test consists of placing different amounts of the substrate, as gram VS, together with an acclimated and "washed" inoculum, 100 mL of a phosphate buffer solution at pH 7, one mL of a micronutrient solution, and chlorine- and ozone-free tap water to complete 400 mL in every test flask [ 20 ]. Every flask has a stirrer adjusted at 110 RPM and submerged in a water bath at 35°C. Five flasks were used for every sample, adjusting between 0.3 and 2.0 gVS in the flasks to test different substrate concentrations and perform further kinetic analysis. Inoculum For the methane production tests, anaerobic granular sludge from the wastewater treatment plant of a large brewery in Mexico City was used as inoculum. The sludge originally developed at 35°C. The preparation consists of the following steps: 1) The granules are dispersed using an Ultraturrax T18 homogenized at 2,000 RPM for 15 seconds; 2) Tap water without chlorine or ozone is used to dilute the sample; 3) Centrifugation at 3,700 RPM 10 min and the supernatant is discarded; the procedure is repeated two more times. This procedure guarantees that the inoculum has no dissolved substances during the test. For the test, 8 g VS inoculums are added to every flask. Analytical determinations Dissolved COD was determined after filtering the sample through 0.45 µm membrane, and pH was determined according to Standard Methods [ 21 ]. Lactic acid was determined using the spectrophotometric method Borshchevskaya et al [ 22 ] proposed. Methanol, ethanol, and VFA (acetic, propionic, isobutyric, butyric, isovaleric, valeric, and hexanoic acids) were determined using a gas chromatograph (HP 5890 GC System) equipped with a flame ionization detector (FID), Stabilwax column - DA, with hydrogen as carrier. The sample was previously filtered using 0.22 µm cellulose filters. The biogas composition (CO 2 and CH 4 ) was determined using a gas chromatograph (SRI 8610c) equipped with a thermal conductivity detector, stainless steel column packed with silica gel (8600-PK1A), helium as carrier gas with a flow of 27 mL/min. The detector temperature was 150°C. RESULTS AND DISCUSSION Characterization of the cacao pod husks Characterization is vital as cacao fruit can change according to variety and origin, storage, and transport. Table 1 shows that the cacao pod husks have a pH of 5.4, which is lower than the value reported by Castillo et al . [ 4 ]; in the characterization, they omitted the endocarp, which belongs together with the mucilage, the acid component of cacao; humidity was 77% and total solids (TS) 23%; these authors report no COD value. From the TS, 91% correspond to volatile solids (VS). Antwi et al . [ 16 ] report similar results: Dry matter of 20%, from which 93% corresponds to volatile solids. The most thorough characterizations, such as the ones from Antwi et al. [ 16 ] and Castillo et al. [ 4 ], do not report COD as a parameter to be considered for biogas production. Using the organic fraction of municipal solid waste (OFMSW) for comparison, Ossa-Arias and González-Martínez [ 23 ] report two values, 1014 ± 47.2 g/kg TS and 1387 ± 9.5 g/kg TS corresponding to two different sampling periods. Castellón-Zelaya and González-Martínez [ 24 ] report 1,140 g/kg TS for municipal organic solid waste. This means the cacao husk pods, with a COD of 987 g/kg, can be considered viable biomass for methane production through anaerobic digestion. The bromatological characterization shows a shell (exocarp or epicarp) rich in lignin, cellulose, and hemicellulose. Antwi et al. [ 16 ] report values of 21% for lignin, 26% for cellulose, and 4.8% for hemicellulose, like the ones in Table 1 . The values reported by Ogunjobi and Lajide [ 25 ] and Vriesmann and Petkowicz [ 26 ] are also like the ones reported by Antwi et al. [ 16 ] and in this research. The values for grease and oil in Table 1 are lower than the ones reported by Castillo et al . [ 4 ]. Table 1 Physicochemical and bromatological characteristics of the cacao pod husk after grinding. In parentheses is the number of repetitions. pH 5.4 (3) - Humidity 77 ± 0.2 (3) % Total solids 234 ± 1.6 (3) g/kg Volatile solids 212 ± 2.0 (3) g/kg Fixed solids 21 ± 1.2 (3) g/kg VS/TS 91 % COD 987 ± 12.5 (5) g/kg TS Carbohydrates 279 ± 7.1 (5) g/kg TS Cellulose 260 ± 14 (4) g/kg TS Hemicellulose 81 ± 19.8 (4) g/kg TS Lignin 158 ± 21 (4) g/kg TS Fats and oils 16 ± 1.3 (3) g/kg TS Fermentation under different organic loading rates Fermentation in a semicontinuous reactor ran under different organic loading rates (OLRs) with only the indigenous microorganisms in the pod husks as inoculum. The system started under an OLR of 1.3 kg VS /m 3 ·d (solids retention time (SRT) of 19 days), and after 50 days, it was increased to 2.55 kg VS /m 3 ·d, and then again, after another 43 days, to 3.74 kg VS /m 3 ·d and, finally, to 4.9 kg VS /m 3 ·d (SRT of 4.9 days). The total experiment required 130 days (Fig. 3 ). At the end of the first OLR, pH was 4.7, and then it slightly increased to 5.01 at the end of the second OLR, 5.05 at the end of the third OLR, and finally 5.17 at the end of the fourth OLR, at the end of the experiment (Fig. 3 ). The identified metabolites, products of the fermentation, were the acids acetic, propionic, butyric, valeric, and lactic. Only ethanol was detected among the alcohols. Furthermore, traces of hexanoic and heptanoic acids were detected under the chromatograph's confidence limits. Organic loading rate of 1.3 g COD /L·d (SRT = 19 days) The total and volatile solids influent and effluent concentrations during this stage do not differ significantly from the other stages. The main differences can be observed during the initial and final days. From day 43 to 50, solids removal can be observed as metabolite production increases. The hydrolysis rate fluctuated more during the first stage than in the subsequent ones. The mean value was 20 ± 4%. Independent of the OLR, the hydrolysis rate remained around 20%, indicating that hydrolyzable substances depend on the substrate, not the process adjustments, and implying that the established microbial community indigenous to the cocoa husk, under controlled fermentation conditions, resulted in a consistent level of initial breakdown of the substrate. The subsequent changes in metabolites indicate adaptation at the level of fermentation pathways, but the initial hydrolysis, being the rate-limiting step for complex substrates like cocoa husk, remained relatively constant, suggesting an equilibrium in the hydrolytic enzyme activities within the adapted microbial community. This OLR corresponds to the start of the experiment. On the first fermentation day, all the metabolites had a COD of 2.8 g/L, where ethanol showed the highest relative concentration, followed by acetic acid. Ethanol concentration decreased steadily until day 26 when it completely disappeared, and therefore ethanol does not play a role in the methanogenesis of the first organic load. On day 26, lactic acid concentration was 40% of the total metabolites, followed by butyric acid with 27%: while the thermodynamic analysis suggests lactic acid could be beneficial for methanogenesis [ 15 ] found that it had the lowest specific methane production among the analyzed metabolites. This is attributed to the partial acetogenesis of lactic acid, which can lead to the formation of propionate and acetate. Importantly, lactic acid tends to transform into propionic acid at higher initial concentrations. The higher butyric acid concentration suggests the absence or depletion of readily degradable carbohydrates, forcing the microorganisms to consume more complex substances [ 24 ]. The acetogenesis of butyric acid was observed to be a limiting step in methanogenesis [ 15 ]. Zheng et al . [ 27 ] report similar results for the fermentation of fruits and vegetables; ethanol concentration decreases while lactic acid increases. They also report that higher ethanol concentrations can be achieved under higher OLR. The total metabolite concentration at the end of this stage was 3.2 ± 0.2 g DQO /L. The differences between the sum of all metabolites and COD correspond to unidentified substances and traces of hexanoic and heptanoic acids. The fermentation degree was calculated by dividing the COD of the identified metabolites among dissolved COD times 100. The average fermentation degree during this OLR was 57%. The lowest value was 37 and the highest was 88%. Organic loading rate of 2.55 g COD /L·d (SRT = 9.5 days) This OLR ran for 41 days after the OLR of 1.3 g COD /L·d (Fig. 3 ). Solids removal represented values between 15 and 20%. The same metabolites were produced in this stage as in the previous stage, except for ethanol, and in different concentrations. During the first 20 days, lactic fermentation predominated, accounting for 67% of all metabolites (as COD). The second most important metabolite was butyric acid, which comprised 20% of the total. The other 13% corresponds to the different metabolites combined. Between days 61 and 63, the highest metabolites could be observed with 3.73 g DQO /L. At the beginning of this OLR, acetic acid represented 7% of COD, and it did not change significantly until the lactic acid production decreased, when the value increased to 14%, as all other metabolites decreased. After day 71, the lactic acid fermentation decreased as the lactic acid concentration decreased dramatically after day 75. Acetic, propionic, and butyric acid concentrations increased after day 75, and the overall metabolite production decreased from 3.3 to 1.88 g COD /L. According to Bühlmann et al. [ 28 ] and Mäki-Arvela et al. [ 29 ], although lactic acid microorganisms grow under different pH values and temperatures, in this case, decreasing lactic acid concentrations can be related to decreasing solids retention times (SRT). From day 52 to 71, the SRT of 9.5 days took place twice, and the lactic communities resented the change, indicating that they preferred a longer SRT. For fermentation with only indigenous microorganisms, the degree of initial fermentation of cacao pod husks was 77%, not very discrepant from the 75 to 79% from the OFMSW values reported by Jojoa-Unigarro and González-Martínez [ 15 ]. With decreasing lactic acid production and a gradual increase in acetic acid, the degree of fermentation is drastically reduced by up to half. By the end of this stage, it is 35%. Organic loading rate of 3.7 and 4.9 g COD /L·d (SRT = 6.3, 4.75 days) Figure 3 shows that the removal of total and volatile solids in the feed and the effluent remains between 18 and 20%, as in the previous stage. On day 100, a sudden drop in metabolite production can be observed due to a change in the substrate batch. The values were adjusted on day 115, and the removal rates did not vary. For these two OLRs, the identified metabolites represent less than 50% of the dissolved COD, meaning that other non-identified substances constitute the difference. Besides, the fermentation rate was 33 ± 2% and 34 ± 1% for the lower OLR of 3.7 and 4.9 g COD /L·d, respectively. For the OLR of 3.7 g COD /L·d, on the first day, the total production of metabolites was 1.87 gCOD/L, where acetic acid represents the highest relative concentration, followed by propionic, lactic, and butyric acids. The high concentration of acetic acid can be highly beneficial for methanogenesis, as it is a key intermediate and a direct substrate for acetotrophic methanogens, which convert acetate directly into methane and carbon dioxide. According to [ 15 ], acetic acid showed the highest specific methane production, and complete consumption during methanization was observed without forming other byproducts. The opposite occurs during the methanization of propionic acid, where at higher initial concentrations, it undergoes incomplete acetogenesis, leading to the formation of isobutyric, butyric, isovaleric, and valeric acids. Furthermore, propionic acid is a well-known methanogenesis inhibitor, particularly affecting hydrogenotrophic methanogens. These concentrations remained constant during the stage and changed during the following stage of OLR 4.9 g COD /L·d. Under this last OLR, the metabolite production was less than 30% of the total dissolved COD; the total metabolite production was 1.06 g COD /L, where lactic, acetic, and propionic acids followed, in this order. Lactic acid production increased slightly compared to the previous OLR of 3.7 g COD /L·d. Overall, metabolite production decreases with an increasing organic loading rate. This can be explained as the solids retention time decreases with increasing OLR; naturally occurring microorganisms are exposed to lower times for their metabolic functions and reproduction. The hydrolysis rate remained constant for the 4.75 g COD /L·d organic load (SRT = 4.75 days), suggesting that the SRT was sufficient to hydrolyze the complex material susceptible to hydrolysis. Still, it is necessary to mention that the rates present throughout the experiment are low. According to Kandylis et al. [ 30 ], the hydrolytic processes are deficient under a pH of 5.5: The enzymatic activity is very low and inhibits microbial metabolism. The low hydrolysis rate throughout the fermentation process, even when high solids retention times are included, indicates that the hydrolysis of the substrate is not based on these times (as is the case of other substrates such as OFMSW) but depends on the population of microorganisms in the substrate and its distribution over time. Methane production from fermented cacao pod husks For the methane production calculation, the samples taken at the end of every organic load were used for methane production for 25 days to ensure complete methanization. Figure 4 shows the methane production curves from unfermented pod husks and the digestates produced during the cacao pod fermentation described in the previous chapter. The primary methane production reaction (CH 4 from an exogenous source) was considered finished when the curves approached the slope of the blank (dots in every curve in Fig. 4 ) according to VDI-4630 [ 31 ] and Holliger et al. [ 32 ]. The unfermented cacao pod husks rapidly produced methane during the first five days; the slopes continued parallel to each other, except for the one with the 0.63 g VS (1.58 g VS /L) initial substrate concentration. The methane production from the digestate with the OLR 1.3 g COD /L·d delivered confident values for the lower substrate concentration with reaction times of 5.5, 8, and 14 days. The curve for the highest substrate concentration delivered unreliable results. Three curves from the digestate obtained with the OLR 2.6 g COD /L·d were valid with reaction times of 6 and 8 days. All four curves from the digestate from OLR 3.7 g COD /L·d delivered reliable values with reaction times of 4, 5, and 6 days. All four curves from the OLR 4.9 g COD /L·d digestate were reliable, with 4.5-, 6-, 9-, and 10-day reaction times. The methodology proposed by Jojoa-Unigarro and González-Martínez [ 15 ] was used to determine the specific methane production (SMP), which consists of performing a linearization of the production of each curve vs. the initial substrate mass. Using the values in Fig. 4 , Fig. 5 was built, where the final methane production of every curve is plotted against the substrate (VS) initial concentration; the slope of every set of points represents the specific methane production (SMP) for every digestate and unfermented cacao pod husk. Table 2 shows the SMP, y-intercept, and correlation coefficients. The lowest SMP was for the unfermented cacao pod husks with 100 NmL/g VS . SMP values of 210, 144, and 115 NmL/g VS correspond to the digestates from the OLR of 1.3, 2.6, and 3.7 g COD /L·d, respectively, indicating that the SMP behaves inversely to the fermentation OLR. The SMP value for the digestate from the highest OLR was higher than expected, with 192 NmL/g VS (Table 2 ). The y-intercept is the case-selective value for the endogenous methane production (blank). This value is calculated as the blank curves (methane production from endogenous substrates) change with the inoculum type and initial substrate concentration. Table 2 shows that the endogenous methane production ranged from 39 to 66 NmL, with intermediate values of 51, 57, and 59 NmL. This indicates that the inoculum was reliable and not dependent on the initial substrate concentration. Table 2 also shows the correlation coefficients from Fig. 5 . Figure 6 shows the behavior of methane production against the supplied substrate quantity (concentration divided by 0.4 L). The CH 4 production increased with decreasing substrate concentration, indicating inhibition. Under 1.0 g VS (2.5 g VS /L), CH 4 production remains increased with decreasing substrate concentration, and above 1.0 g VS , the CH 4 production remains constant for every specific case. The specific methane production (Table 2 ) considers the variations due to substrate concentration. This behavior explains why the different CH 4 production values reported in the literature differ. Every author determined the methane production without specifying the initial substrate concentration in the test or reactor. For example, Acosta et al. [ 34 ] reported a COD recovery of 59 ± 4% as methane, corresponding to an average methane yield of 174 mL/g VS (wet) and 193 mL/g VS (dry) fed. These values were achieved under mesophilic conditions after approximately 10 days. The biogas yield was 307 ± 15 mL/g VS and 271 ± 70 mL/g VS for the two different inocula (BMP1 and BMP2, respectively), with corresponding methane yields of 175 ± 9 mL/g VS and 173 ± 45 mL/g VS . The methane content in the biogas was 57% for BMP1 and 64% for BMP2. Other studies have reported yields ranging from 163 and 382 mL CH4 /g VS for the mono-digestion of cocoa residues without pretreatment [ 35 ] [ 36 ] and values between 139 and up to 2,485 mL CH4 /g VS for co-digestion with other wastes such as anaerobic sludge or pig manure [ 37 ] [ 38 ]. Table 2 Specific methane production from unfermented and fermented cacao pod husks. The values were calculated using Fig. 5 . Specific CH 4 (NmL/g VS ) y-intercept (NmL) Corr. Coef. Unfermented cacao pod husks 100 66 0.9934 OLR 1.3 g VS /L·d (SRT = 19 d) 210 51 0.9992 OLR 2.6 g VS /L·d (SRT = 9.5 d) 144 39 0.9925 OLR 3.7 g VS /L·d (SRT = 6.3 d) 115 57 0.9956 OLR 4.9 g VS /L·d (SRT = 4.75 d) 192 59 0.9954 Kinetics of methanization The analysis of methanation kinetics was performed by calculating the initial slopes (initial methane production rates) from the methane production curves in Fig. 4 after the Michaelis and Menten model. This model provided clear methodological advantages for our research's specific conditions and objectives. Anaerobic digestion involves multiple simultaneous reactions, making it difficult to reliably monitor the concentration changes of a single substrate throughout the process [ 15 ]. In contrast, methane production can be continuously and accurately measured using systems such as AMPTS, making the methane production rate a practical and robust kinetic parameter within the Michaelis-Menten framework. This approach has been previously validated by Jojoa-Unigarro and González-Martínez [ 15 ], who successfully applied the model to both simple and complex substrates. Additionally, they demonstrated that the experimental methane production data showed a strong fit to the model, with high correlation coefficients. According to the Lineweaver and Burk linearization process (Fig. 6 ), the initial methane production rates' inverse values are plotted against the initial substrate concentrations' inverse values [ 15 , 33 ]. Table 3 shows the kinetic parameters. Vmax is the maximum possible reaction rate, and Km is the Michaelis-Menten proportionality constant. The latter can be interpreted as an indicator of the affinity between the enzymes involved and the substrate: Decreasing Km values indicate increasing affinity. The interpretation of Vmax and Km provides essential information for practical implementation, enabling the development of targeted optimization strategies, particularly in systems employing a two-stage approach. A higher Vmax indicates a faster substrate conversion to methane under optimal conditions. This suggests that if the fermentation process is optimized to generate higher concentrations of substrates with elevated Vmax, the subsequent methanization stage could become more efficient regarding reaction time and throughput. For substrates with high Vmax and low Km, a smaller reactor volume may be sufficient to achieve the desired methane yield within a specific retention time. Conversely, substrates with low Vmax or high Km may require larger reactor volumes or longer retention times. Understanding these parameters for the mixture of metabolites in a fermented digestate (such as those derived from OFMSW) is crucial for optimizing loading rates and hydraulic retention times in a full-scale digester [ 15 ]. Table 3 also shows the correlation coefficients from Fig. 6 . Table 3 Kinetic parameters were calculated using Lineweaver and Burk's linearization method. Organic load (g COD /L·d) Slope Intercept Vmax (NmL CH4 /d) Km (g/L) R 2 CH 4 Prod. ( Nml/g VS ) 1.3 0.0140 0.0099 101 1.409 0.914 210 2.6 0.0062 0.0071 140 0.869 0.970 144 3.7 0.0167 0.0093 108 1.806 0.966 115 4.9 0.0171 0.0109 92 1.573 0.956 192 Unfermented pod husks 0.0132 0.0059 171 2.251 0.999 100 The highest Vmax belongs to the raw, unfermented pod husks, implying that during the first hours of the reaction, the fermented digestates do not have high methane generation rates; however, the specific methane production of any of the fermented digestates is higher than the one of raw, unfermented pod husks. The organic loading rates of 1.3, 3.7, and 4.9 g COD /L·d presented the highest CH 4 generation rates and similar Km values. These results conclude that under these organic loading rates, CH 4 production rates are similar, and the affinity, as Km values, are also similar. The OLR of 2.6 g COD /L·d reports the highest Vmax and the lowest Km (highest affinity), consistent with the acetic acid concentration in this digestate. Figure 7 compares the adjusted CH 4 production values (dotted lines) with the experimental ones. The model fits the conditions and experimental data well, with more than 91% for all cases. The organic load does not considerably influence the kinetic constants. The model fits the experimental conditions and data well, with more than 91% for all cases. This model has been successfully used in previous research as it allows correlating substrate consumption with product formation through yield coefficients [ 15 , 24 , 33 ]. This is particularly important when substrate concentration over reaction time cannot be accurately measured. CONCLUSIONS The fermentation of cacao pod husks shows a significant increase in carbohydrate solubilization, which favors methane production; however, solubilization does not depend on the organic loading rate or the solids retention time. This process facilitates the conversion of organic waste into energy, contributing to sustainability in waste management. Finding an adequate organic loading rate in fermentation is crucial to maximizing methane production. The organic loading rates of 1.3 and 4.9 g COD /L·d were the most effective in promoting methanization, providing a benchmark for designing future, more efficient anaerobic processes. Independent of the organic load, methane production from the fermented pod husks exceeds the specific methane production of raw cacao wastes. Metabolites generated during fermentation may initially inhibit methanization, but methane production significantly increases over time. This behavior suggests system adaptation to these metabolites could enhance methanization efficiency in later stages. Methane production increased with decreasing substrate concentration, indicating substrate inhibition. The kinetic constants obtained with the Michaelis and Menten model and the excellent correlation coefficients confirm the quality of the experimental results. Future studies should focus on optimizing fermentation conditions to maximize subsequent methane yield. Further research could explore the long-term adaptation of microbial communities to various fermentation byproducts, the scaling-up of the process for industrial applications, and the potential use of other organic waste substrates for similar biotechnological purposes. Declarations Fabiany de J. Morgado-León provided the results while developing her MSc thesis. The design of the article, edition of figures and tables, and final version were developed by Simón González-Martínez and Fabiany de J. Morgado-León. Germán D. Jojoa-Unigarro provided the first raw version of the article and the initiative to write it. The authors have no conflict of interest. Acknowledgments This work was supported by the General Directorate for Academic Affairs (DGAPA) of the National University of Mexico (UNAM), projects IT101320 and IT100523. This research was performed at the Environmental Engineering Laboratory (LIA), Institute of Engineering, National University of Mexico. Thanks to CONAHCYT for an MSc scholarship. The authors acknowledge Dr. Francisco Rojo-Calleja for the analytical support at the Chemistry School at UNAM and Dr. Óscar González Barceló for the support in laboratory activities. Data availability All data is included in the article. The complete MSc thesis of Ms. Morgado-León can be consulted online (in Spanish) at http://132.248.9.195/ptd2023/agosto/0846538/Index.html . References SADER Ministry for Agricultural and Rural Development: Mexico (Secretaría de Agricultura y Desarrollo Rural) 14th (2021). https://www.gob.mx/agricultura/articulos/cacao-riqueza-del-campo-mexicano Vásquez, Z.S., de Carvalho-Neto, D.P., Pereira, G.V.M., Vandenberghe, L.P.S., de Oliveira, P.Z., Tiburcio, P.B., Rogez, H.L.G., Góes-Neto, A., Soccol, C.R.: Biotechnological approaches for cocoa waste management: A review. 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Chem. 71 , 755–758 (2016). https://doi.org/10.1134/S1061934816080037 Ossa-Arias, M.M., González-Martínez, S.: Methane Production from the Organic Fraction of Municipal Solid Waste Under Psychrophilic, Mesophilic, and Thermophilic Temperatures at Different Organic Loading Rates. Waste Biomass Valor. 12 , 4859–4871 (2021). https://doi.org/10.1007/s12649-021-01354-9 Castellón-Zelaya, M.F., González-Martínez, S.: Silage of the organic fraction of municipal solid waste to improve methane production. Wat Sci. Technol. 83 , 2536–2548 (2021). https://doi.org/10.2166/wst.2021.148 Ogunjobi, J.K., Lajide, L.: The potential of cocoa pods and plantain peels as renewable sources in Nigeria the potential of cocoa pods and plantain peels as renewable sources in Nigeria. Int. J. Green. Energy. 12 , 440–445 (2016). https://doi.org/10.1080/15435075.2013.848403 Vriesmann, L.C., Petkowicz, C.L.O.: Highly acetylated pectin from cacao pod husks ( Theobroma cacao L.) forms gel. Food Hydrocoll. 33 , 58–65 (2013). https://doi.org/10.1016/j.foodhyd.2013.02.010 Zheng, M., Zheng, M., Wu, Y., Ma, H., Wang, K.: Effect of pH on types of acidogenic fermentation of fruit and vegetable wastes. Biotechnol. Bioproc Eng. 20 , 298–303 (2015). https://doi.org/10.1007/s12257-014-0651-y Bühlmann, C., Mickan, B., Tait, S., Renton, M., Bahri, P.: Lactic acid from mixed food wastes at a commercial biogas facility: Effect of feedstock and process conditions. J. Clean. Prod. 284 , 125243 (2020). https://doi.org/10.1016/j.jclepro.2020.125243 Mäki-Arvela, P., Simakova, I.L., Salmi, T., Murzin, D.Y.: Production of lactic acid/lactates from biomass and their catalytic transformations to commodities. Chem. Rev. 114 (3), 1909–1971 (2014). https://doi.org/10.1021/cr400203v Kandylis, P., Bekatorou, A., Pissaridi, K., Lappa, K., Dima, A., Kanellaki, M., Koutinas, A.A.: Acidogenesis of cellulosic hydrolysates for new generation biofuels. Biomass Bioenerg. 91 , 210–216 (2016). https://doi.org/10.1016/j.biombioe.2016.05.006 VDI: Verein Deutscher Ingenieure (VDI 4630) Fermentation of organic materials. Characterisation of the substrate, sampling, collection of material data, fermentation tests. VDI-Handbuch Energietechnik (2016) Holliger, C., Astals, S., Fruteau de Laclos, H., Hafner, S.D., Koch, K., Weinrich, S.: Towards a standardization of biomethane potential tests: a commentary. Water Sci. Technol. 83 (1), 247–250 (2021). https://doi.org/10.2166/wst.2020.569 Fernández-Rodríguez, J., Pérez, M., Romero, L.I.: Comparison of mesophilic and thermophilic dry anaerobic digestion of OFMSW: Kinetic analysis. Chem. Eng. J. 232 , 59–64 (2013). https://doi.org/10.1016/j.cej.2013.07.066 Acosta, N., De Vrieze, J., Sandoval, V., Sinche, D., Wierinck, I., Rabaey, K.: Cocoa residues as viable biomass for renewable energy production through anaerobic digestion. Bioresour Technol. 265 , 568–572 (2018). https://doi.org/10.1016/j.biortech.2018.05.100 Suhartini, S., Hidayat, N., Hadi, M.W.R.: Co-digestion of cocoa pods and cocoa leaves : Effect of C / N ratio to biogas and energy potential Co-digestion of cocoa pods andcocoa leaves: Effect of C / N ratio to biogas and the energy potential. In IOP conference series: earth and environmental science paper (pp. 1–9). (2021). https://doi.org/10.1088/1755-1315/733/1/012139 Dahunsi, S.O., Osueke, C.O., Olayanju, T.M.A., Lawal, A.I.: Co-digestion of Theobroma cacao (Cocoa) pod husk and poultry manure for energy generation: Effects of pretreatment methods. Bioresour Technol. 283 , 229–241 (2019). https://doi.org/10.1016/j.biortech.2019.03.093 Vintila, T., Ionel, I., Tiegam, T., Fregue, R., Julean, C., Gabche, A.S.: Residual biomass from food processing industry in Cameroon as feedstock for second generation biofuels. Bioresources Com. 14 , 3731–3745 (2019) Rodríguez, A., 'Angel, J., Rivero, E., Acevedo, P., Santis, A.: Evaluation of the biochemical methane potential of pig manure, organic fraction of municipal solid waste and cocoa industry residues in Colombia. Chem. Eng. Trans. 57 , 55–60 (2017). https://doi.org/10.3303/CET1757010 Perwitasari, U., Agustina, N.T., Pangestu, R., Amanah, S., Saputra, H., Fahrurrozi, A.A., Juanssilfero, A.B., Thontowi, A., Widyaningsih, T.D., Eris, D.D., Amaniyah, M., Yopi, Habibi, M.S.: Cacao pod husk for citric acid production under solid state fermentation using response surface method. Biomass Convers. Biorefinery. (2021). https://doi.org/10.1007/s13399-021-01690-9 Syamsiro, M., Saptoadi, H., Tambunan, B.H., Pambudi, N.A.: Energy for Sustainable Development A preliminary study on use of cocoa pod husk as a renewable source of energy in Indonesia. Energy Sustain. Dev. 16 , 74–77 (2012). https://doi.org/10.1016/j.esd.2011.10.005 Supplementary Files GA.jpg Graphical abstract Cite Share Download PDF Status: Published Journal Publication published 30 May, 2025 Read the published version in Waste and Biomass Valorization → Version 1 posted Editorial decision: Accept 08 May, 2025 Reviewers agreed at journal 20 Apr, 2025 Reviewers invited by journal 20 Apr, 2025 Editor invited by journal 20 Apr, 2025 Editor assigned by journal 17 Apr, 2025 First submitted to journal 16 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5981919","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445282626,"identity":"242b7ab8-bf1e-420e-8d0a-935c03b06961","order_by":0,"name":"Fabiany de Jesús Morgado-León","email":"","orcid":"https://orcid.org/0000-0001-6394-869X","institution":"Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"prefix":"","firstName":"Fabiany","middleName":"de Jesús","lastName":"Morgado-León","suffix":""},{"id":445282627,"identity":"c2b563fe-1764-477a-b26a-b4d41484cc29","order_by":1,"name":"Simon Gonzalez-Martinez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYHACxgdQuuEAsVqYDUjWwiZBgpOAQL5/dVp1Yds2efkG5sYDjG135HUbuNPwGmJw4+222zPbbhtuOAB0GGPbM8NtB3g3G+DVInF2223ettuMGxjAWg4zArVsfIDXYTPObisGarGf3wDRYg/UsuEAXs+c793GDNSS2ABx2OFEgrYY3ODdLD3j3O3kDYeBWhLOHU7edpiAX+T7z278XFB223Z+e/vjDx/KDttuO967DX+wSyQwMIMZIDIBxsAL+A8QVjMKRsEoGAUjHAAAUDJVAWY8drwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0452-195X","institution":"Universidad Nacional Autonoma de Mexico","correspondingAuthor":true,"prefix":"","firstName":"Simon","middleName":"","lastName":"Gonzalez-Martinez","suffix":""},{"id":445282628,"identity":"f898d3f3-0402-4760-95fc-d6da49be7852","order_by":2,"name":"German Dimitriv Jojoa-Unigarro","email":"","orcid":"https://orcid.org/0000-0001-9256-6850","institution":"Universidad Nacional Autonoma de Mexico","correspondingAuthor":false,"prefix":"","firstName":"German","middleName":"Dimitriv","lastName":"Jojoa-Unigarro","suffix":""}],"badges":[],"createdAt":"2025-02-07 14:16:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5981919/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5981919/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12649-025-03115-4","type":"published","date":"2025-05-30T15:57:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81091888,"identity":"06836642-fe6e-4d0c-b122-9811a5e27de5","added_by":"auto","created_at":"2025-04-22 07:17:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":535438,"visible":true,"origin":"","legend":"\u003cp\u003eThe cacao pod husk includes the exocarp, mesocarp, and a layer of endocarp from the freshly harvested cacao fruit.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5981919/v1/5f33ab50d1ff9d5250dc3b79.png"},{"id":81091340,"identity":"3ccc90ff-4d5b-4296-b80e-288f8f35a5f5","added_by":"auto","created_at":"2025-04-22 07:09:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27614,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the experimental setup for the fermentation of cacao pod husks.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5981919/v1/75788c94487ba04b226114a8.jpg"},{"id":81091348,"identity":"dbfff513-a3a1-495c-9f49-bbc1b929d820","added_by":"auto","created_at":"2025-04-22 07:09:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66517,"visible":true,"origin":"","legend":"\u003cp\u003eSR, solids solubilization rate, CODdiss (soluble), and metabolites (including low-molecular-weight acids) under different organic loading rates. OLR, organic loading rate; SRT, solids retention time\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5981919/v1/88fc901cc4daaf14b953b033.jpg"},{"id":81091887,"identity":"a64d0632-7c4b-459b-a36f-14ed7550671f","added_by":"auto","created_at":"2025-04-22 07:17:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74717,"visible":true,"origin":"","legend":"\u003cp\u003eMethane production curves and values for final exogenous methane production\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5981919/v1/4c170c031e3837cc8ef5757f.jpg"},{"id":81092880,"identity":"79d60e33-72f5-49bf-bd39-f64de098db61","added_by":"auto","created_at":"2025-04-22 07:25:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":64999,"visible":true,"origin":"","legend":"\u003cp\u003eThe specific methane production was calculated using the end-of-production methane values for the previously fermented cacao pod husks. UNF, unfermented.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5981919/v1/0ebdd69bf512551cbe871180.jpg"},{"id":81091889,"identity":"1459b10f-7210-4f13-9e37-3f1c70d41e28","added_by":"auto","created_at":"2025-04-22 07:17:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":69369,"visible":true,"origin":"","legend":"\u003cp\u003eLineweaver-Burk plot.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5981919/v1/ff9d737a77d3ecfeca559846.jpg"},{"id":81091892,"identity":"01191c3c-7b19-4dbe-a9f5-2993fac867a7","added_by":"auto","created_at":"2025-04-22 07:17:43","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":50848,"visible":true,"origin":"","legend":"\u003cp\u003eMethane production rate, experimental (markers), and calculated values (dotted lines).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5981919/v1/a68db7c1cb201e7dee4447bf.jpg"},{"id":83782781,"identity":"861e3139-2d96-45a0-aeda-df38af23e39e","added_by":"auto","created_at":"2025-06-02 16:05:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1747812,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5981919/v1/1c21ca97-5fa1-49de-a265-8a7cfb6deced.pdf"},{"id":81091341,"identity":"27383355-dde3-4c57-ae01-e85aac074ec6","added_by":"auto","created_at":"2025-04-22 07:09:43","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":82869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5981919/v1/b49a3f61e347670925d17bd2.jpg"}],"financialInterests":"","formattedTitle":"Methane production from previously fermented cacao waste pod husks","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eCacao pod husks contain significant amounts of readily degradable carbohydrates\u003c/li\u003e\n \u003cli\u003eTen tons of waste are produced for every ton of cacao seeds\u003c/li\u003e\n \u003cli\u003eFermentation of waste cacao pod husks can produce alcohols and acids\u003c/li\u003e\n \u003cli\u003eMethane is more efficiently produced from previously fermented cacao waste\u003c/li\u003e\n \u003cli\u003eSubstrate inhibition is significant during methane production\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eAccording to the Mexican Agriculture Ministry [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], cacao is Mexico's most estimated shade agricultural product after coffee. The International Cacao Organization (ICO) estimates that between 2015 and 2016, clean cacao grain production worldwide was 3.9 thousand tons, generating 16 thousand tons of waste biomass [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. According to ICO, cacao production during 2022/2023 was 4,953 thousand tons. Africa reported the highest production with 73.4%, then the tropical regions of the American Continent, and finally, Asia and Oceania with 5.4%. Akinjokun \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] propose that 10 tons of waste are generated for every ton of cacao beans. The waste that originates during the separation of the cacao seed from the pods is abundant and renewable. Of the waste cacao pods, only 10% is processed for industrial purposes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]; the rest corresponds to the mucilage and peel. In the cacao plantations, after hand-separating the grain from the pods, the waste biomass is left to rot in the field, causing soil deterioration, aquifer contamination, and, because of the fermentation processes, insects and other harmful organisms can develop. Notorious is the long-time anaerobic digestion producing methane and carbon dioxide released into the atmosphere [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome strategies for using cacao waste have been as animal forage (fibers and carbohydrates) and for the food and pharma industries [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Solid-state fermentation of cocoa pod husk using \u003cem\u003eAspergillus niger\u003c/em\u003e can be used to produce citric acid [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Cocoa pod husks contain pectin, which can be extracted for biological, pharmacological, and food applications [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Besides anaerobic digestion for biogas production, cocoa pod husk can be used in other thermochemical processes such as direct combustion, gasification, and pyrolysis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Allen et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] calculated similar values for domestic solid wastes, garden trimmings from parks, seaweed, and corn silage for methane production. This assessment implies that the waste biomass from cacao production has the potential for methane production through anaerobic digestion. The composition of the pod husks indicates lower oil and grease contents but higher values of non-amylase polysaccharides such as cellulose and hemicellulose [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Lignin is an essential component of the peel.\u003c/p\u003e \u003cp\u003eFermentation with naturally occurring microorganisms, as a pretreatment of the pod husks at ambient temperature in tropical regions where cacao is cultivated, is considered an alternative before methane production. The fermentation of readily biodegradable substances and other fibers can contain metabolites that methanogens can easily transform into biogas. Some publications confirm that the fermentation of lignocellulosic biomasses enhances the hydrolysis of fibers and increases the subsequent methane production [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Jojoa-Unigarro and Gonz\u0026aacute;lez-Mart\u0026iacute;nez [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] conclude that the fermentation of solid organic waste should be oriented to ethanolic and acetic fermentations for further better methane production.\u003c/p\u003e \u003cp\u003eThe fermentation of different biomasses shows that ethanol fermentations are common at lower organic loading rates or higher solids retention times and that, at higher loading rates or lower solids retention times, acid fermentation promotes the formation of acetic, lactic, and butyric acids [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntwi \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] report that the whole cacao pod husk produces 922 L/kg\u003csub\u003eVS\u003c/sub\u003e as theoretical biogas potential with 51% methane. These same authors report lab analyses showing that cacao pod husks are not readily biodegradable as a whole, with a biogas production of 375 L/kg\u003csub\u003eVS\u003c/sub\u003e, representing this value only 47% of the theoretical value. The low biodegradability lies within the outer Shell of the cacao pod husks, which is rich in lignin. The softer internal parts are rich in low molecular weight carbohydrates and hemicellulose, which are readily biodegradable through fermentation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This is why acid fermentation is required before methanization. Comparing the theoretical biomethane production of the cacao pod husks with other substrates, the waste pod husks produce similar amounts of methane as domestic organic solid waste, marine algae, maize silage, and garden and park wastes. The main conclusion is that waste cacao pod husks must be considered an adequate substrate for methane production through anaerobic digestion [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis research proposes evaluating the fermentation of cacao waste husk pods under different organic loading rates and/or solids retention times and determining the kinetics of methane production from the previously fermented waste husk pods. The main innovation of this work lies in the combination of controlled fermentation of cacao husks with the kinetic analysis of methane production. This research explores an intermediate fermentation stage as a pretreatment before methanization. This stage can potentially enhance the conversion of lignocellulosic compounds in cacao husks and optimize methane production in anaerobic processes.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of the sample\u003c/h2\u003e \u003cp\u003eTwenty kilos of cacao fruit (pods) were bought from a producer in Pichucalco, Chiapas, Mexico. They were cut, and the grains were separated, together with the so-called \"placenta,\" which is part of the fruit that binds the grains together (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The cacao pod husk includes the exocarp, mesocarp, and a layer of endocarp from the freshly harvested cacao fruit. The remaining husks and placenta were extruded through a 0.5 mm sieve and then ground with a plate mill to deliver particles approximately 0.5 mm in diameter. Following the proposal of Kreuger \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], the mass resulting from this process was frozen in specialized plastic bags at -18\u0026deg;C to be used during the experimental procedure to ensure that the characteristics did not change with different deliveries.\u003c/p\u003e \u003cp\u003eThe parameters used for the characterization were chemical oxygen demand (COD), total and volatile solids (TS and VS, respectively), humidity, pH, carbohydrates (total, soluble, sugars, and fiber), fats and oils, cellulose, hemicellulose, and lignin.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFermentation of cacao pod husks under different organic loading rates\u003c/h3\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e describes the experimental setup for the fermentation of the cacao pod husks. For the fermentation of the cacao pod husks under different organic loading rates (OLRs), a jacketed glass reactor with a 4.5-litre volume was used. The reactor is provided with a mechanical stirrer (Bioprocess Control, Sweden). The pH was monitored daily using an electrode submerged in the reactor; however, no active control was applied. The working temperature was held constantly at 35\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe reactor was operated semi-continuously and fed with a ground cacao pod husk suspension with 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g\u003csub\u003eCOD\u003c/sub\u003e/L. The different organic loading rates were adjusted by adjusting the volume of the daily feed, resulting in different solids retention times (SRT). The OLRs tested were 1.3, 2.6, 3.7, and 4.9 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d. Hydraulic retention time (HRT) and solids retention times were the same, resulting in values between 19 and 4.8 days.\u003c/p\u003e \u003cp\u003eThe runs needed to last at least twice the SRT to guarantee stable operation during the experimental runs under different OLRs. The fermentation was monitored using pH, COD, total and dissolved solids, volatile fatty acids (VFA), lactic acid, lower alcohols, and total and soluble carbohydrates with a frequency of 3 to 4 times per week, that is, on alternate days, even though the feeding was daily. The solubilization rate was calculated as the ratio of soluble COD to total COD times 100.\u003c/p\u003e\n\u003ch3\u003eMethanization of the fermented digestates\u003c/h3\u003e\n\u003cp\u003eThe potential to produce methane from the fermented ground cacao pod husks was tested using an automatic methane potential test system (AMPTS II, Bioprocess Control, Sweden). The specific methane production of the fermented substrates was compared to that of fresh unfermented cacao pod husks. The test consists of placing different amounts of the substrate, as gram VS, together with an acclimated and \"washed\" inoculum, 100 mL of a phosphate buffer solution at pH 7, one mL of a micronutrient solution, and chlorine- and ozone-free tap water to complete 400 mL in every test flask [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Every flask has a stirrer adjusted at 110 RPM and submerged in a water bath at 35\u0026deg;C. Five flasks were used for every sample, adjusting between 0.3 and 2.0 gVS in the flasks to test different substrate concentrations and perform further kinetic analysis.\u003c/p\u003e\n\u003ch3\u003eInoculum\u003c/h3\u003e\n\u003cp\u003eFor the methane production tests, anaerobic granular sludge from the wastewater treatment plant of a large brewery in Mexico City was used as inoculum. The sludge originally developed at 35\u0026deg;C. The preparation consists of the following steps: 1) The granules are dispersed using an Ultraturrax T18 homogenized at 2,000 RPM for 15 seconds; 2) Tap water without chlorine or ozone is used to dilute the sample; 3) Centrifugation at 3,700 RPM 10 min and the supernatant is discarded; the procedure is repeated two more times. This procedure guarantees that the inoculum has no dissolved substances during the test. For the test, 8 g\u003csub\u003eVS\u003c/sub\u003e inoculums are added to every flask.\u003c/p\u003e\n\u003ch3\u003eAnalytical determinations\u003c/h3\u003e\n\u003cp\u003eDissolved COD was determined after filtering the sample through 0.45 \u0026micro;m membrane, and pH was determined according to Standard Methods [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Lactic acid was determined using the spectrophotometric method Borshchevskaya et al [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] proposed. Methanol, ethanol, and VFA (acetic, propionic, isobutyric, butyric, isovaleric, valeric, and hexanoic acids) were determined using a gas chromatograph (HP 5890 GC System) equipped with a flame ionization detector (FID), Stabilwax column - DA, with hydrogen as carrier. The sample was previously filtered using 0.22 \u0026micro;m cellulose filters.\u003c/p\u003e \u003cp\u003eThe biogas composition (CO\u003csub\u003e2\u003c/sub\u003e and CH\u003csub\u003e4\u003c/sub\u003e) was determined using a gas chromatograph (SRI 8610c) equipped with a thermal conductivity detector, stainless steel column packed with silica gel (8600-PK1A), helium as carrier gas with a flow of 27 mL/min. The detector temperature was 150\u0026deg;C.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of the cacao pod husks\u003c/h2\u003e \u003cp\u003eCharacterization is vital as cacao fruit can change according to variety and origin, storage, and transport. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows that the cacao pod husks have a pH of 5.4, which is lower than the value reported by Castillo \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]; in the characterization, they omitted the endocarp, which belongs together with the mucilage, the acid component of cacao; humidity was 77% and total solids (TS) 23%; these authors report no COD value. From the TS, 91% correspond to volatile solids (VS). Antwi \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] report similar results: Dry matter of 20%, from which 93% corresponds to volatile solids. The most thorough characterizations, such as the ones from Antwi \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and Castillo \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], do not report COD as a parameter to be considered for biogas production. Using the organic fraction of municipal solid waste (OFMSW) for comparison, Ossa-Arias and Gonz\u0026aacute;lez-Mart\u0026iacute;nez [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] report two values, 1014\u0026thinsp;\u0026plusmn;\u0026thinsp;47.2 g/kg\u003csub\u003eTS\u003c/sub\u003e and 1387\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5 g/kg\u003csub\u003eTS\u003c/sub\u003e corresponding to two different sampling periods. Castell\u0026oacute;n-Zelaya and Gonz\u0026aacute;lez-Mart\u0026iacute;nez [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] report 1,140 g/kg\u003csub\u003eTS\u003c/sub\u003e for municipal organic solid waste. This means the cacao husk pods, with a COD of 987 g/kg, can be considered viable biomass for methane production through anaerobic digestion.\u003c/p\u003e \u003cp\u003eThe bromatological characterization shows a shell (exocarp or epicarp) rich in lignin, cellulose, and hemicellulose. Antwi et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] report values of 21% for lignin, 26% for cellulose, and 4.8% for hemicellulose, like the ones in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The values reported by Ogunjobi and Lajide [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and Vriesmann and Petkowicz [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] are also like the ones reported by Antwi et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and in this research. The values for grease and oil in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e are lower than the ones reported by Castillo \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical and bromatological characteristics of the cacao pod husk after grinding. In parentheses is the number of repetitions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.4 (3)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHumidity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 (3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal solids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e234\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 (3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eg/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVolatile solids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e212\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 (3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eg/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFixed solids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 (3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eg/kg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVS/TS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e987\u0026thinsp;\u0026plusmn;\u0026thinsp;12.5 (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eg/kg\u003csub\u003eTS\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbohydrates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e279\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1 (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eg/kg\u003csub\u003eTS\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCellulose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e260\u0026thinsp;\u0026plusmn;\u0026thinsp;14 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eg/kg\u003csub\u003eTS\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemicellulose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81\u0026thinsp;\u0026plusmn;\u0026thinsp;19.8 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eg/kg\u003csub\u003eTS\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLignin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e158\u0026thinsp;\u0026plusmn;\u0026thinsp;21 (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eg/kg\u003csub\u003eTS\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFats and oils\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 (3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eg/kg\u003csub\u003eTS\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFermentation under different organic loading rates\u003c/h3\u003e\n\u003cp\u003eFermentation in a semicontinuous reactor ran under different organic loading rates (OLRs) with only the indigenous microorganisms in the pod husks as inoculum. The system started under an OLR of 1.3 kg\u003csub\u003eVS\u003c/sub\u003e/m\u003csup\u003e3\u003c/sup\u003e\u0026middot;d (solids retention time (SRT) of 19 days), and after 50 days, it was increased to 2.55 kg\u003csub\u003eVS\u003c/sub\u003e/m\u003csup\u003e3\u003c/sup\u003e\u0026middot;d, and then again, after another 43 days, to 3.74 kg\u003csub\u003eVS\u003c/sub\u003e/m\u003csup\u003e3\u003c/sup\u003e\u0026middot;d and, finally, to 4.9 kg\u003csub\u003eVS\u003c/sub\u003e/m\u003csup\u003e3\u003c/sup\u003e\u0026middot;d (SRT of 4.9 days). The total experiment required 130 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At the end of the first OLR, pH was 4.7, and then it slightly increased to 5.01 at the end of the second OLR, 5.05 at the end of the third OLR, and finally 5.17 at the end of the fourth OLR, at the end of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe identified metabolites, products of the fermentation, were the acids acetic, propionic, butyric, valeric, and lactic. Only ethanol was detected among the alcohols. Furthermore, traces of hexanoic and heptanoic acids were detected under the chromatograph's confidence limits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eOrganic loading rate of 1.3 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d (SRT\u0026thinsp;=\u0026thinsp;19 days)\u003c/h2\u003e \u003cp\u003eThe total and volatile solids influent and effluent concentrations during this stage do not differ significantly from the other stages. The main differences can be observed during the initial and final days. From day 43 to 50, solids removal can be observed as metabolite production increases. The hydrolysis rate fluctuated more during the first stage than in the subsequent ones. The mean value was 20\u0026thinsp;\u0026plusmn;\u0026thinsp;4%. Independent of the OLR, the hydrolysis rate remained around 20%, indicating that hydrolyzable substances depend on the substrate, not the process adjustments, and implying that the established microbial community indigenous to the cocoa husk, under controlled fermentation conditions, resulted in a consistent level of initial breakdown of the substrate. The subsequent changes in metabolites indicate adaptation at the level of fermentation pathways, but the initial hydrolysis, being the rate-limiting step for complex substrates like cocoa husk, remained relatively constant, suggesting an equilibrium in the hydrolytic enzyme activities within the adapted microbial community.\u003c/p\u003e \u003cp\u003eThis OLR corresponds to the start of the experiment. On the first fermentation day, all the metabolites had a COD of 2.8 g/L, where ethanol showed the highest relative concentration, followed by acetic acid. Ethanol concentration decreased steadily until day 26 when it completely disappeared, and therefore ethanol does not play a role in the methanogenesis of the first organic load. On day 26, lactic acid concentration was 40% of the total metabolites, followed by butyric acid with 27%: while the thermodynamic analysis suggests lactic acid could be beneficial for methanogenesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] found that it had the lowest specific methane production among the analyzed metabolites. This is attributed to the partial acetogenesis of lactic acid, which can lead to the formation of propionate and acetate. Importantly, lactic acid tends to transform into propionic acid at higher initial concentrations. The higher butyric acid concentration suggests the absence or depletion of readily degradable carbohydrates, forcing the microorganisms to consume more complex substances [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The acetogenesis of butyric acid was observed to be a limiting step in methanogenesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Zheng \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] report similar results for the fermentation of fruits and vegetables; ethanol concentration decreases while lactic acid increases. They also report that higher ethanol concentrations can be achieved under higher OLR.\u003c/p\u003e \u003cp\u003eThe total metabolite concentration at the end of this stage was 3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g\u003csub\u003eDQO\u003c/sub\u003e/L. The differences between the sum of all metabolites and COD correspond to unidentified substances and traces of hexanoic and heptanoic acids. The fermentation degree was calculated by dividing the COD of the identified metabolites among dissolved COD times 100. The average fermentation degree during this OLR was 57%. The lowest value was 37 and the highest was 88%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOrganic loading rate of 2.55 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d (SRT\u0026thinsp;=\u0026thinsp;9.5 days)\u003c/h2\u003e \u003cp\u003eThis OLR ran for 41 days after the OLR of 1.3 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Solids removal represented values between 15 and 20%. The same metabolites were produced in this stage as in the previous stage, except for ethanol, and in different concentrations. During the first 20 days, lactic fermentation predominated, accounting for 67% of all metabolites (as COD). The second most important metabolite was butyric acid, which comprised 20% of the total. The other 13% corresponds to the different metabolites combined. Between days 61 and 63, the highest metabolites could be observed with 3.73 g\u003csub\u003eDQO\u003c/sub\u003e/L. At the beginning of this OLR, acetic acid represented 7% of COD, and it did not change significantly until the lactic acid production decreased, when the value increased to 14%, as all other metabolites decreased.\u003c/p\u003e \u003cp\u003eAfter day 71, the lactic acid fermentation decreased as the lactic acid concentration decreased dramatically after day 75. Acetic, propionic, and butyric acid concentrations increased after day 75, and the overall metabolite production decreased from 3.3 to 1.88 g\u003csub\u003eCOD\u003c/sub\u003e/L. According to B\u0026uuml;hlmann et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and M\u0026auml;ki-Arvela et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], although lactic acid microorganisms grow under different pH values and temperatures, in this case, decreasing lactic acid concentrations can be related to decreasing solids retention times (SRT). From day 52 to 71, the SRT of 9.5 days took place twice, and the lactic communities resented the change, indicating that they preferred a longer SRT.\u003c/p\u003e \u003cp\u003eFor fermentation with only indigenous microorganisms, the degree of initial fermentation of cacao pod husks was 77%, not very discrepant from the 75 to 79% from the OFMSW values reported by Jojoa-Unigarro and Gonz\u0026aacute;lez-Mart\u0026iacute;nez [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. With decreasing lactic acid production and a gradual increase in acetic acid, the degree of fermentation is drastically reduced by up to half. By the end of this stage, it is 35%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOrganic loading rate of 3.7 and 4.9 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d (SRT\u0026thinsp;=\u0026thinsp;6.3, 4.75 days)\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the removal of total and volatile solids in the feed and the effluent remains between 18 and 20%, as in the previous stage. On day 100, a sudden drop in metabolite production can be observed due to a change in the substrate batch. The values were adjusted on day 115, and the removal rates did not vary. For these two OLRs, the identified metabolites represent less than 50% of the dissolved COD, meaning that other non-identified substances constitute the difference. Besides, the fermentation rate was 33\u0026thinsp;\u0026plusmn;\u0026thinsp;2% and 34\u0026thinsp;\u0026plusmn;\u0026thinsp;1% for the lower OLR of 3.7 and 4.9 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d, respectively.\u003c/p\u003e \u003cp\u003eFor the OLR of 3.7 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d, on the first day, the total production of metabolites was 1.87 gCOD/L, where acetic acid represents the highest relative concentration, followed by propionic, lactic, and butyric acids. The high concentration of acetic acid can be highly beneficial for methanogenesis, as it is a key intermediate and a direct substrate for acetotrophic methanogens, which convert acetate directly into methane and carbon dioxide. According to [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], acetic acid showed the highest specific methane production, and complete consumption during methanization was observed without forming other byproducts. The opposite occurs during the methanization of propionic acid, where at higher initial concentrations, it undergoes incomplete acetogenesis, leading to the formation of isobutyric, butyric, isovaleric, and valeric acids. Furthermore, propionic acid is a well-known methanogenesis inhibitor, particularly affecting hydrogenotrophic methanogens. These concentrations remained constant during the stage and changed during the following stage of OLR 4.9 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d. Under this last OLR, the metabolite production was less than 30% of the total dissolved COD; the total metabolite production was 1.06 g\u003csub\u003eCOD\u003c/sub\u003e/L, where lactic, acetic, and propionic acids followed, in this order. Lactic acid production increased slightly compared to the previous OLR of 3.7 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d.\u003c/p\u003e \u003cp\u003eOverall, metabolite production decreases with an increasing organic loading rate. This can be explained as the solids retention time decreases with increasing OLR; naturally occurring microorganisms are exposed to lower times for their metabolic functions and reproduction.\u003c/p\u003e \u003cp\u003eThe hydrolysis rate remained constant for the 4.75 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d organic load (SRT\u0026thinsp;=\u0026thinsp;4.75 days), suggesting that the SRT was sufficient to hydrolyze the complex material susceptible to hydrolysis. Still, it is necessary to mention that the rates present throughout the experiment are low. According to Kandylis et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], the hydrolytic processes are deficient under a pH of 5.5: The enzymatic activity is very low and inhibits microbial metabolism. The low hydrolysis rate throughout the fermentation process, even when high solids retention times are included, indicates that the hydrolysis of the substrate is not based on these times (as is the case of other substrates such as OFMSW) but depends on the population of microorganisms in the substrate and its distribution over time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMethane production from fermented cacao pod husks\u003c/h2\u003e \u003cp\u003eFor the methane production calculation, the samples taken at the end of every organic load were used for methane production for 25 days to ensure complete methanization. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the methane production curves from unfermented pod husks and the digestates produced during the cacao pod fermentation described in the previous chapter. The primary methane production reaction (CH\u003csub\u003e4\u003c/sub\u003e from an exogenous source) was considered finished when the curves approached the slope of the blank (dots in every curve in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) according to VDI-4630 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and Holliger et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe unfermented cacao pod husks rapidly produced methane during the first five days; the slopes continued parallel to each other, except for the one with the 0.63 g\u003csub\u003eVS\u003c/sub\u003e (1.58 g\u003csub\u003eVS\u003c/sub\u003e/L) initial substrate concentration. The methane production from the digestate with the OLR 1.3 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d delivered confident values for the lower substrate concentration with reaction times of 5.5, 8, and 14 days. The curve for the highest substrate concentration delivered unreliable results. Three curves from the digestate obtained with the OLR 2.6 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d were valid with reaction times of 6 and 8 days. All four curves from the digestate from OLR 3.7 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d delivered reliable values with reaction times of 4, 5, and 6 days. All four curves from the OLR 4.9 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d digestate were reliable, with 4.5-, 6-, 9-, and 10-day reaction times.\u003c/p\u003e \u003cp\u003eThe methodology proposed by Jojoa-Unigarro and Gonz\u0026aacute;lez-Mart\u0026iacute;nez [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] was used to determine the specific methane production (SMP), which consists of performing a linearization of the production of each curve vs. the initial substrate mass. Using the values in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e was built, where the final methane production of every curve is plotted against the substrate (VS) initial concentration; the slope of every set of points represents the specific methane production (SMP) for every digestate and unfermented cacao pod husk. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the SMP, y-intercept, and correlation coefficients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe lowest SMP was for the unfermented cacao pod husks with 100 NmL/g\u003csub\u003eVS\u003c/sub\u003e. SMP values of 210, 144, and 115 NmL/g\u003csub\u003eVS\u003c/sub\u003e correspond to the digestates from the OLR of 1.3, 2.6, and 3.7 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d, respectively, indicating that the SMP behaves inversely to the fermentation OLR. The SMP value for the digestate from the highest OLR was higher than expected, with 192 NmL/g\u003csub\u003eVS\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe y-intercept is the case-selective value for the endogenous methane production (blank). This value is calculated as the blank curves (methane production from endogenous substrates) change with the inoculum type and initial substrate concentration. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that the endogenous methane production ranged from 39 to 66 NmL, with intermediate values of 51, 57, and 59 NmL. This indicates that the inoculum was reliable and not dependent on the initial substrate concentration. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e also shows the correlation coefficients from Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the behavior of methane production against the supplied substrate quantity (concentration divided by 0.4 L). The CH\u003csub\u003e4\u003c/sub\u003e production increased with decreasing substrate concentration, indicating inhibition. Under 1.0 g\u003csub\u003eVS\u003c/sub\u003e (2.5 g\u003csub\u003eVS\u003c/sub\u003e/L), CH\u003csub\u003e4\u003c/sub\u003e production remains increased with decreasing substrate concentration, and above 1.0 g\u003csub\u003eVS\u003c/sub\u003e, the CH\u003csub\u003e4\u003c/sub\u003e production remains constant for every specific case. The specific methane production (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) considers the variations due to substrate concentration. This behavior explains why the different CH\u003csub\u003e4\u003c/sub\u003e production values reported in the literature differ. Every author determined the methane production without specifying the initial substrate concentration in the test or reactor. For example, Acosta et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] reported a COD recovery of 59\u0026thinsp;\u0026plusmn;\u0026thinsp;4% as methane, corresponding to an average methane yield of 174 mL/g\u003csub\u003eVS\u003c/sub\u003e (wet) and 193 mL/g\u003csub\u003eVS\u003c/sub\u003e (dry) fed. These values were achieved under mesophilic conditions after approximately 10 days. The biogas yield was 307\u0026thinsp;\u0026plusmn;\u0026thinsp;15 mL/g\u003csub\u003eVS\u003c/sub\u003e and 271\u0026thinsp;\u0026plusmn;\u0026thinsp;70 mL/g\u003csub\u003eVS\u003c/sub\u003e for the two different inocula (BMP1 and BMP2, respectively), with corresponding methane yields of 175\u0026thinsp;\u0026plusmn;\u0026thinsp;9 mL/g\u003csub\u003eVS\u003c/sub\u003e and 173\u0026thinsp;\u0026plusmn;\u0026thinsp;45 mL/g\u003csub\u003eVS\u003c/sub\u003e. The methane content in the biogas was 57% for BMP1 and 64% for BMP2. Other studies have reported yields ranging from 163 and 382 mL\u003csub\u003eCH4\u003c/sub\u003e/g\u003csub\u003eVS\u003c/sub\u003e for the mono-digestion of cocoa residues without pretreatment [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and values between 139 and up to 2,485 mL\u003csub\u003eCH4\u003c/sub\u003e/g\u003csub\u003eVS\u003c/sub\u003e for co-digestion with other wastes such as anaerobic sludge or pig manure [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpecific methane production from unfermented and fermented cacao pod husks. The values were calculated using Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecific CH\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(NmL/g\u003csub\u003eVS\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ey-intercept\u003c/p\u003e \u003cp\u003e(NmL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCorr. Coef.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnfermented cacao pod husks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9934\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOLR 1.3 g\u003csub\u003eVS\u003c/sub\u003e/L\u0026middot;d (SRT\u0026thinsp;=\u0026thinsp;19 d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9992\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOLR 2.6 g\u003csub\u003eVS\u003c/sub\u003e/L\u0026middot;d (SRT\u0026thinsp;=\u0026thinsp;9.5 d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9925\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOLR 3.7 g\u003csub\u003eVS\u003c/sub\u003e/L\u0026middot;d (SRT\u0026thinsp;=\u0026thinsp;6.3 d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9956\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOLR 4.9 g\u003csub\u003eVS\u003c/sub\u003e/L\u0026middot;d (SRT\u0026thinsp;=\u0026thinsp;4.75 d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9954\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eKinetics of methanization\u003c/h2\u003e \u003cp\u003eThe analysis of methanation kinetics was performed by calculating the initial slopes (initial methane production rates) from the methane production curves in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e after the Michaelis and Menten model. This model provided clear methodological advantages for our research's specific conditions and objectives. Anaerobic digestion involves multiple simultaneous reactions, making it difficult to reliably monitor the concentration changes of a single substrate throughout the process [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In contrast, methane production can be continuously and accurately measured using systems such as AMPTS, making the methane production rate a practical and robust kinetic parameter within the Michaelis-Menten framework. This approach has been previously validated by Jojoa-Unigarro and Gonz\u0026aacute;lez-Mart\u0026iacute;nez [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], who successfully applied the model to both simple and complex substrates. Additionally, they demonstrated that the experimental methane production data showed a strong fit to the model, with high correlation coefficients. According to the Lineweaver and Burk linearization process (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), the initial methane production rates' inverse values are plotted against the initial substrate concentrations' inverse values [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the kinetic parameters. Vmax is the maximum possible reaction rate, and Km is the Michaelis-Menten proportionality constant. The latter can be interpreted as an indicator of the affinity between the enzymes involved and the substrate: Decreasing Km values indicate increasing affinity. The interpretation of Vmax and Km provides essential information for practical implementation, enabling the development of targeted optimization strategies, particularly in systems employing a two-stage approach. A higher Vmax indicates a faster substrate conversion to methane under optimal conditions. This suggests that if the fermentation process is optimized to generate higher concentrations of substrates with elevated Vmax, the subsequent methanization stage could become more efficient regarding reaction time and throughput. For substrates with high Vmax and low Km, a smaller reactor volume may be sufficient to achieve the desired methane yield within a specific retention time. Conversely, substrates with low Vmax or high Km may require larger reactor volumes or longer retention times. Understanding these parameters for the mixture of metabolites in a fermented digestate (such as those derived from OFMSW) is crucial for optimizing loading rates and hydraulic retention times in a full-scale digester [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e also shows the correlation coefficients from Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKinetic parameters were calculated using Lineweaver and Burk's linearization method.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eOrganic load\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSlope\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIntercept\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVmax (NmL\u003csub\u003eCH4\u003c/sub\u003e/d)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKm (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCH\u003csub\u003e4\u003c/sub\u003e Prod.\u003c/p\u003e \u003cp\u003e(\u003cem\u003eNml/g\u003c/em\u003e\u003csub\u003e\u003cem\u003eVS\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e1.3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0099\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.914\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e2.6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0062\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0071\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.869\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.970\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e3.7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.806\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003e4.9\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.573\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.956\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eUnfermented pod husks\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe highest Vmax belongs to the raw, unfermented pod husks, implying that during the first hours of the reaction, the fermented digestates do not have high methane generation rates; however, the specific methane production of any of the fermented digestates is higher than the one of raw, unfermented pod husks. The organic loading rates of 1.3, 3.7, and 4.9 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d presented the highest CH\u003csub\u003e4\u003c/sub\u003e generation rates and similar Km values. These results conclude that under these organic loading rates, CH\u003csub\u003e4\u003c/sub\u003e production rates are similar, and the affinity, as Km values, are also similar. The OLR of 2.6 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d reports the highest Vmax and the lowest Km (highest affinity), consistent with the acetic acid concentration in this digestate.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e compares the adjusted CH\u003csub\u003e4\u003c/sub\u003e production values (dotted lines) with the experimental ones. The model fits the conditions and experimental data well, with more than 91% for all cases. The organic load does not considerably influence the kinetic constants. The model fits the experimental conditions and data well, with more than 91% for all cases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis model has been successfully used in previous research as it allows correlating substrate consumption with product formation through yield coefficients [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This is particularly important when substrate concentration over reaction time cannot be accurately measured.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eThe fermentation of cacao pod husks shows a significant increase in carbohydrate solubilization, which favors methane production; however, solubilization does not depend on the organic loading rate or the solids retention time. This process facilitates the conversion of organic waste into energy, contributing to sustainability in waste management.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eFinding an adequate organic loading rate in fermentation is crucial to maximizing methane production. The organic loading rates of 1.3 and 4.9 g\u003csub\u003eCOD\u003c/sub\u003e/L\u0026middot;d were the most effective in promoting methanization, providing a benchmark for designing future, more efficient anaerobic processes.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eIndependent of the organic load, methane production from the fermented pod husks exceeds the specific methane production of raw cacao wastes.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eMetabolites generated during fermentation may initially inhibit methanization, but methane production significantly increases over time. This behavior suggests system adaptation to these metabolites could enhance methanization efficiency in later stages.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eMethane production increased with decreasing substrate concentration, indicating substrate inhibition.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eThe kinetic constants obtained with the Michaelis and Menten model and the excellent correlation coefficients confirm the quality of the experimental results.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eFuture studies should focus on optimizing fermentation conditions to maximize subsequent methane yield. Further research could explore the long-term adaptation of microbial communities to various fermentation byproducts, the scaling-up of the process for industrial applications, and the potential use of other organic waste substrates for similar biotechnological purposes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFabiany de J. Morgado-Le\u0026oacute;n provided the results while developing her MSc thesis. The design of the article, edition of figures and tables, and final version were developed by Sim\u0026oacute;n Gonz\u0026aacute;lez-Mart\u0026iacute;nez and Fabiany de J. Morgado-Le\u0026oacute;n. Germ\u0026aacute;n D. Jojoa-Unigarro provided the first raw version of the article and the initiative to write it.\u003c/p\u003e \u003cp\u003eThe authors have no conflict of interest.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by the General Directorate for Academic Affairs (DGAPA) of the National University of Mexico (UNAM), projects IT101320 and IT100523. This research was performed at the Environmental Engineering Laboratory (LIA), Institute of Engineering, National University of Mexico. Thanks to CONAHCYT for an MSc scholarship. The authors acknowledge Dr. Francisco Rojo-Calleja for the analytical support at the Chemistry School at UNAM and Dr. \u0026Oacute;scar Gonz\u0026aacute;lez Barcel\u0026oacute; for the support in laboratory activities.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data is included in the article. 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Dev. \u003cb\u003e16\u003c/b\u003e, 74\u0026ndash;77 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.esd.2011.10.005\u003c/span\u003e\u003cspan address=\"10.1016/j.esd.2011.10.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Agricultural waste, cacao pod husk, anaerobic digestion, biogas, methane production","lastPublishedDoi":"10.21203/rs.3.rs-5981919/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5981919/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The wastes from cacao processing represent approximately 80% of the fruit weight and are rich in readily biodegradable carbohydrates and cellulosic and lignocellulosic compounds. There is little information about the processing of these wastes, and few authors report the potential of these wastes to produce energy through anaerobic digestion. As an alternative to disposing of these wastes in the field, associated with negative consequences, such as soil pollution, this research deals with the fermentation of the cacao pod husks under different organic loading rates and/or solids retention times to enhance hydrolysis through the production of acids and alcohols. Ethanol, lactic, acetic, propionic, and butyric acids were identified in fermented cacao pod husks. The digestates from the fermentation were tested for methane production. From the main results, the following conclusions can be drawn. The fermentation efficiency depends on the solids retention time, and the hydrolysis rate is inversely proportional to the organic load and directly proportional to solids retention times. Metabolic displacements show sudden changes in the composition of the volatile acid due to solids retention time. Methane production did not tend to be associated with the organic load or solids retention times. The specific methane production increased with decreasing substrate concentration, indicating substrate inhibition.","manuscriptTitle":"Methane production from previously fermented cacao waste pod husks","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 07:09:38","doi":"10.21203/rs.3.rs-5981919/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2025-05-08T16:01:09+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-20T07:31:16+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-20T06:45:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Waste and Biomass Valorization","date":"2025-04-20T06:38:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-17T17:06:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Waste and Biomass Valorization","date":"2025-04-16T21:54:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"736abef6-e720-46a1-8d84-75c61735c222","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T15:58:56+00:00","versionOfRecord":{"articleIdentity":"rs-5981919","link":"https://doi.org/10.1007/s12649-025-03115-4","journal":{"identity":"waste-and-biomass-valorization","isVorOnly":false,"title":"Waste and Biomass Valorization"},"publishedOn":"2025-05-30 15:57:00","publishedOnDateReadable":"May 30th, 2025"},"versionCreatedAt":"2025-04-22 07:09:38","video":"","vorDoi":"10.1007/s12649-025-03115-4","vorDoiUrl":"https://doi.org/10.1007/s12649-025-03115-4","workflowStages":[]},"version":"v1","identity":"rs-5981919","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5981919","identity":"rs-5981919","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}