Enhanced Biomethane Production from Olive Mill Wastewater via Co-Digestion with Cow Dung, Fruits, Vegetable, and Fish Wastes: An Experimental and Kinetic Study

Enhanced Biomethane Production from Olive Mill Wastewater via Co-Digestion with Cow Dung, Fruits, Vegetable, and Fish Wastes: An Experimental and Kinetic Study | 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 Enhanced Biomethane Production from Olive Mill Wastewater via Co-Digestion with Cow Dung, Fruits, Vegetable, and Fish Wastes: An Experimental and Kinetic Study Hassan Erraji, Essadek Abdessadek, Anas Tallou, Abdeslam Asehraou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5821546/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Apr, 2025 Read the published version in Waste and Biomass Valorization → Version 1 posted 5 You are reading this latest preprint version Abstract Olive mill wastewater (OMW) is the main effluent resulting in huge amounts from olive oil manufacturing. This effluent is mostly composed of organic matter and polyphenolic compounds, known for their antimicrobial activity and compromise their biological treatment. This work investigates the impact of the co-digestion of olive mill wastewater with fruits and vegetable waste (FVW), fish waste (FW), and cow dung (CD) under mesophilic conditions at two different inoculum-to-substrate ratios. The effect on biomethane yield, volatile solids reduction, and polyphenol removal efficiency were evaluated. Moreover, kinetic modeling was applied to describe biomethane production. The co-digestion of OMW with CD at an I/S ratio of 2:1, and a mixture consisting of 33% OMW, 33% FVW, and 33% FW at I/S ratio of 3:1 achieved biomethane yields of 155.00 NmLCH 4 gVS − 1 and 132.20 NmLCH 4 gVS − 1 , respectively after 49-day retention time at 37°C whereas the mono-digestion of OMW was completely inhibited. These treatments demonstrated strong performance in terms of volatile solids and polyphenol removal, achieving rates of 76%, 81%, and 95% and 84%, respectively. Similarly, the logistic function model provided a good fit for predicting biomethane production, with high R² values of 0.9941 and 0.9930, respectively. Anaerobic co-digestion Olive mill wastewater Fruits and vegetable waste Fish waste Cow dung Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Olive oil manufacturing in Mediterranean countries (Spain, Italy, Greece, Morocco, Tunisia, Palestine, etc.) represents a significant market share worldwide. This economic activity poses different threats to the ecosystem (soil contamination, water pollution and odor emissions, etc.) due to the mismanagement of its by-products [ 1 ]. One of these by-products is olive mill wastewater (OMW), generated during olive oil extraction. It contains high salt levels, organic matter, suspended and dissolved particles, and toxic compounds such as phenols, and it has a high biological and chemical oxygen demand (BOD and COD) [ 2 ]. The issue with OMW comes from its phenolic content, which is responsible of its dark color as well as its antibacterial and phytotoxic effects [ 3 ]. Moreover, polyphenols, fatty acids, and volatile compounds are toxic to the anaerobic bacterial community, limiting the biological treatment of OMW, specifically during the anaerobic digestion (AD) process. In addition, the high levels in BOD and COD are also limiting factors that make OMW difficult to treat or manage [ 4 , 5 ]. OMW components are known to negatively affect the physico-chemical and biological properties of soil, making it phytotoxic [ 3 ]. Land spreading and other treatment practices, such as evaporation ponds, could lead to groundwater pollution. The use of OMW without treatment in agriculture can affect the acidity, salinity, nitrogen immobilization, microbial community, leaching of nutrients, and concentration of lipids, organic acids, and phenolic compounds and can negatively impact plants development [ 6 ]. AD is one of the best alternative technologies for the valorization and treatment of diverse organic wastes [ 7 ]. However, the success of the mono-digestion process (i.e., AD using a single waste) is facing the serious challenges related to the characteristics of the substrates. Co-digestion of multiple feedstocks provides the potential to overcome these limitations [ 8 , 9 ]. In recent years, the potential of co-digestion has been studied and documented. For instance, it has been reported that anaerobic co-digestion enhances the digestibility of co-substrates, improves the process stability, and produces a digestate of high agronomic value [ 10 , 11 ]. This positive effect is attributed to the diversity and improved nutrient balance in the substrates, particularly an optimum C/N ratio, the synergetic effect of the microorganisms provided by the co-substrates, the dilution of inhibitory and toxic compounds for AD, and the buffering capacity of the AD mixture. To achieve this positive effect, we suggest in this study the co-digestion of OMW with cow dung (CD), fruits and vegetable waste (FVW) and fish waste (FW). Large quantities of FW with low economic value (fish head, scale, viscera, fins, tail, and backbones) are produced during fish processing. The sustainable management of FW has become a serious challenge nowadays [ 12 , 13 ]. FW is known to contain a high concentration of biodegradable organic compounds, mainly proteins and lipids, making it favorable for anaerobic co-digestion [ 14 ]. Furthermore, another promising organic waste with a promising value and high potential as a substrate for AD is FVW, characterized by high moisture content, low total solids (TS), and high volatile solids (VS), making it suitable for AD [ 15 , 16 ]. The main characteristic of CD, FW and FVW is that they are available throughout the year, whereas OMW is seasonally available. Therefore, incorporating these substrates into a co-digestion process is highly relevant. Indeed, the C/N ratio of OMW is high [ 17 ], whereas the other two substrates, particularly FW, are known to be rich in nitrogen, making them good candidates for co-digestion with OMW. According to the literature, many efforts have been made to improve the AD of OMW either by applying pretreatments to reduce the inhibitory effect of polyphenols and the high organic load of OMW or by optimizing digestion parameters including inoculum/substrate ratios (I/S) or selecting suitable co-substrates to compensate for the C/N imbalance of OMW. Several studies have investigated the effect of co-digestion of OMW with other substrates [ 17 – 21 ]. For instance, Sounni et al. [ 18 ] examined the co-digestion of OMW with poultry manure, cheese whey, grass, and slaughterhouse wastewater with substrate/OMW ratios of 80:20, 20:80, 20:80, and 50:50, respectively. The authors concluded that co-digestion of OMW with slaughterhouse wastewater (ratio 50:50) achieved the highest biomethane yield in batch mode. Another study conducted by Mouftahi et al. [ 20 ] reported an average net specific methane production of 384 NmLCH 4 gVS − 1 in the case of mixture composed of 4.4% organic fraction of municipal solid wastes, 2.2% chicken dropping, 4.4% OMW and 89% inoculum. While previous studies have explored the co-digestion of OMW with multiple organic substrates, to the best of our knowledge, no study has specifically investigated its co-digestion with cow dung, fruits and vegetable waste, and fish waste in batch system. Furthermore, this study applied kinetic modeling to describe the biomethane production potential of the aforementioned co-substrates, providing deeper insights into process performance and optimization. The aim of the present study is to investigate the effect of co-digestion of liquid OMW with CD, FVW and FW using two I/S ratios of 2:1 and 3:1 under mesophilic conditions (37°C) during a 49-day incubation period on biomethane production and bioprocess performance in terms of volatile solids and phenols removal. Biomethane modelling was also conducted using three models: Modified Gompertz Model, Transference Function Model, and Logistic Function Model to describe the kinetic mechanisms of biomethane production in different treatments. Material and methods Selection of co-substrates and experimental procedure The liquid OMW tested in this study as the main substrate was composed of wastewater mixed with olive cake, which was rich in fragments of olive stones. It was collected directly from the storage pond of a two-phase olive oil mill in Oujda, Morocco. Fruits and vegetable waste (FVW) as well as fish waste (FW) were taken from the IFMEREE training institute canteen while the cow dung (CD) was obtained from a dairy cattle farm in the Oujda region. The CD was used as both an inoculum and co-substrate after a 15-day incubation period under mesophilic (37°C) and anaerobic conditions to stimulate microbial activity. FVW and FW were separately grinded in the laboratory and stored at -4°C until their use (Fig. 1 ). To investigate the effect of co-digestion of OMW with CD, FVW and FW, the following experimental procedure was followed (Table 1 ). In this study, a mixture of OMW, FVW, and FW (labelled as Mx) was prepared in equal proportions, with each waste representing 33% of the mixture. This mixture was anaerobically digested using two I/S ratios of 2:1 and 3:1 based on volatile solids content (VS) of the inoculum (CD) and mixture. These I/S ratios have been recognized in previous studies as suitable for anaerobic digestion process, and a higher I/S ratio is recommended when potential substrate inhibition is expected [ 22 , 23 ]. At the same time, the OMW was digested with inoculum (CD) while respecting the two I/S ratio of 2:1 and 3:1. As a control, OMW was digested alone to evaluate biomethane production without the influence of any additional co-substrates. Table 1 Substrates mixtures and experimental procedure used in the study Reactor number Substrates ratio (fresh weight based) I/S ratio Treatment code Reactor 1 to 3 33% olive mill wastewater + 33% fruits and vegetable waste + 33% fish waste + cow dung 2:1 (CD-Mx) R 2:1 Reactor 4 to 6 Olive mill wastewater + cow dung 2:1 (CD-OMW) R 2:1 Reactor 7 to 9 33% olive mill wastewater + 33% fruits and vegetable waste + 33% fish waste + cow dung 3:1 (CD-Mx) R 3:1 Reactor 10 to 12 Olive mill wastewater + cow dung 3:1 (CD-OMW) R 3:1 Reactor 13 to 15 Olive mill wastewater NA OMW CD : Cow dung, OMW : Olive mill wastewater, Mx : Mixture of fruits and vegetable waste and fish waste, I/S ratio ( R 2:1 and R 3:1 ): Inoculum to substrate ratio Physico-chemical characterization of feedstocks The characterization of feedstocks before and after anaerobic digestion included classical analyses, and results are shown in Table 2 . All analyses were determined according to the standard methods [ 24 ]. Volatile solids (VS), dry matter (DM), and pH were determined directly from the homogenised samples. DM was determined after drying samples at 105°C for 24 hours. VS was obtained by the loss-on-ignition method at 550°C in Nabertherm muffle furnace. pH of raw substrates, mixtures and digestates was measured using liquid samples and TOLEDO pH meter. Chemical oxygen demand (COD) was analysed according to the Open reflux method [ 24 ]. After digestion of samples with potassium dichromate (K 2 Cr 2 O 7 ) for 2 hours at 150°C, the COD was determined by titration using ferrous sulfate (FeSO 4 ) according to the Eq. (1) and expressed as gO 2 /L. COD= [(FS Bl -FS Sample )×C FS ×8]V Sample (1) Where FS Bl is the volume of FeSO 4 used in titration of the blank sample (mL), FS Sample is the volume of FeSO 4 used in titration of sample, V Sample is the volume of sample (mL), C FS is the concentration of reducing agent (N), and COD is expressed in gO 2 /L of sample. Polyphenols were analyzed following Folin-Ciocalteu method [ 25 ]. Polyphenols extraction was performed using method described by Leouifoudi et al. [ 26 ]. The pH of OMW samples was first adjusted to 2 to maximise the recovery of polyphenols [ 27 ]. Then OMW samples were subjected to a defatting process using hexane (95%) (1:1, (v/v)) followed by clarification through centrifugation (4000 rpm, 15 min). The phenolic compounds contained in the defatted and clarified OMW samples were subjected to two liquid-liquid extractions using ethyl acetate (95%) (1:1, v/v) and centrifugation at 4000 rpm for 10 min. Subsequently, the ethyl acetate phase was evaporated using a rotary evaporator (BUCHI Rotavapor R-114) at 40°C. The dried residues were then dissolved in 10 mL of methanol and used to determine phenols content. Fifty µL of the phenolic extract was mixed with 1.35 mL of distilled water and 200 µL of Folin ciocalteaux solution. The solution was incubated for 3 min in the dark. Then 400 µL of sodium carbonate (Na 2 CO 3 20%) was added to the solution. The development of a blue colour was obtained after incubation in the water bath for 20 min at 40°C. The absorbance was measured at 760 nm. The polyphenol content was expressed in g of gallic acid equivalent per g of dry matter (g GA/gDM). A calibration curve was used and obtained by 6 samples with different gallic acid concentrations (15.62, 31.25, 62.50, 125.00, and 250.00 mg/L) previously prepared. All analysis were performed in triplicate. Table 2 Chemical characteristics of substrates and their mixtures before anaerobic digestion Treatment CD OMW FW FVW (CD-Mx) R 2:1 (CD-OMW) R 2:1 (CD-Mx) R 3:1 (CD-OMW) R 3:1 pH 7.00 ± 0.08 5.80 ± 0.02 6.98 ± 0.08 4.90 ± 0.07 6.24 ± 0.01 6.90 ± 0.02 6.83 ± 0.01 6.58 ± 0.02 DM (% FM) 17.00 ± 1.30 10.52 ± 0.12 17.10 ± 0.13 8.44 ± 0.14 9.18 ± 1.05 5.39 ± 0.54 7.40 ± 0.84 4.00 ± 0.25 VS (% FM) 13.23 ± 0.15 6.70 ± 0.25 15.30 ± 0.19 7.37 ± 0.08 13.61 ± 3.08 8.30 ± 0.01 10.72 ± 3.13 10.64 ± 0.05 COD (gO 2 /L) 42.40 ± 3.67 67.20 ± 2.80 ND ND 20.80 ± 3.67 32.00 ± 3.67 48.00 ± 2.40 21.60 ± 2.40 Polyphenols (gGA/gDM) 1.15 ± 0.04 8.97 ± 0.15 ND ND 1.72 ± 0.05 4.46 ± 0.11 2.55 ± 0.07 2.85 ± 0.06 CD : Cow dung, OMW : Olive mill wastewater, FW : Fish waste, FVW : Fruits and vegetable waste, Mx : Mixture of fruits, vegetable and fish waste, I/S ratio ( R 2:1 and R 3:1 ): Inoculum to substrate ratio, FM : Fresh matter, DM , Dry matter, VS : Volatile solids, COD : Chemical oxygen demand, GA : Gallic acid Anaerobic digestion set-up and biochemical methane potential tests A set of 15 batch reactors (Glass bottles) with a total volume of 600 mL and a working volume of 400 mL were used in this experiment. Digesters were prepared using the quantities of substrates and inoculum shown in Table 3 , while maintaining the mixtures and I/S ratios as described in the first section (Table 1 ). All treatments were performed in triplicate to guaranty the reliability and accuracy of measurements and conclusions. After feeding batch digesters, the pH of slurries was immediately measured, and the digesters were then sealed using rubber stoppers with Tygon® tubing for extracting the biogas. Then all digesters were purged for 45 seconds with biogas from a fixed dome digester with an average CH 4 and CO 2 contents of 75% and 23%, respectively, to ensure anaerobic conditions and rapid start-up of process. Table 3 Amounts of substrates used in this experiment Digesters 1 to 3 4 to 6 7 to 9 10 to 12 13 to 15 Treatment (CD-Mx) R 2:1 (CD-OMW) R 2:1 (CD-Mx) R 3:1 (CD-OMW) R 3:1 OMW Substrate amount (Mx or OMW) [g FM] 161.19 198.72 124.14 158.78 400.00 Inoculum amount (CD) [g FM] 238.81 201.28 275.86 241.22 0.00 Total amount [g FM] 400.00 400.00 400.00 400.00 400.00 CD : Cow dung, OMW : Olive mill wastewater, Mx : Mixture of fruits and vegetable waste and fish waste, FM : Fresh matter The experiment was conducted during a total incubation period of 49 days, and the temperature was maintained at 37°C using thermostatic water bath (Precision 0.2°C). Mechanical mixing of slurries in the bioreactors was ensured by stepper motors operating at a frequency of 150 rpm during 5 min, followed by an off period of 60 minutes. To measure the pure biomethane, the produced biogas was passed continuously through 80mL bottles containing 3M NaOH solution to remove any potential CO 2 and H 2 S (Fig. 2 ). Few drops of thymolphthalein were added to NaOH solution for pH monitoring. The volume of CH 4 was automatically and continuously measured by liquid displacement and buoyancy method using a gas volume measuring device as shown in Fig. 2 [ 28 ]. The obtained CH 4 was continuously normalised by the automatic system (1.0 standard atmospheric pressure, 0°C and zero moisture content). The experiments were terminated when CH 4 generation stopped completely in some digesters or was significantly reduced in others. The cumulative biomethane yield was calculated using the following Eq. (2) [ 29 ]. Results are presented as means and standard deviations (SD) of three independent digesters for each treatment. NmLCH 4 gVS − 1 =[Cumulative volume of CH 4 (mL)] /[ Mass of VS content (g VS)] (2) Determination of phenols, COD and VS reduction At the end of digestion, total phenols, COD and VS were determined and their respective reduction was calculated according to the method adapted from Matjuda et al. [ 30 ] (Eq. 3): RD (%)= [(RS-DS) / RS]×100% (3) Where: RD: rate of degradation of the parameter RS: concentration or parameter content in the raw substrate DS: concentration or parameter content in the digested substrate Kinetic study Biogas production from AD of organic substrates can be described using kinetic models and several mathematical models have been proposed to predict biogas production and describe the biological process behaviour in digesters. Models such as Modified Gompertz model (MGP) (Eq. 4), Transference Function Model (TFM) (Eq. 5) and Logistic Function Model (LFM) (Eq. 6) are commonly used for this purpose [ 31 – 33 ]. This three models were used in this study and fitted with the experimental cumulative biomethane yields obtained from digesters. Y = Y m exp{-exp[( R m e) / Y m (λ-t) + 1]} (4) Y = Y m {1-exp[- R m ×(t-λ) / Y m ]} (5) Y = Y m / {1 + exp[4 R m ×((λ-t) / Y m ) + 2]} (6) Where Y is the cumulative biomethane production (NmLCH 4 gVS − 1 ) as a function of time t (days), Y m is the ultimate potential biomethane production (NmLCH 4 gVS − 1 ), R m is the maximum production rate (NmLCH 4 gVS day − 1 ), λ is the lag phase time (days), and e is a constant equivalent to 2.718282. The parameters Y m , R m , and λ were determined using non-linear regression by the solver ToolPack of Microsoft Excel 2016 [ 34 ]. Then a comparison was made among the three models to find the suitable one which describes better the biological process by validation of model parameters prediction. The validation methods used in this study are Root Mean Square Error (RMSE) and the coefficient of determination (R 2 ) that were calculated using the Excel Microsoft 2016 tool. Results and discussion Performance of digesters during anaerobic digestion The performance of the process in bioreactors can be assessed using various indicators including biogas production and the control of physicochemical parameters such as pH changes, VS, COD and polyphenol removal after anaerobic digestion. The results regarding pH evolution and the removal rates of VS, COD and polyphenol are shown in Table 4 and Fig. 3 , respectively. According to the literature, the pH of the slurry in digesters is one of the most important parameters significantly influencing the stability of AD [ 35 , 36 ]. For instance, if the AD process is efficient, the pH of the digestates generally increases toward alkalinity by the end of the AD process, as ammonia is released from the digested feedstock [ 37 ]. Given that no adjustments of pH were made in this study, initial pH of treatments (CD-Mx) R2:1 , (CD-Mx) R3:1 , (CD-OMW) R2:1 and (CD-OMW) R3:1 was 6.24, 6.83, 6.90 and 6.58, respectively. The pH values corresponding to the treatments (CD-Mx) R3:1 and (CD-OMW) R2:1 fell within the recommended AD pH range of 6.8 to 7.5 while the pH of the two other trials was slightly below this range [ 38 ]. The treatment (CD-Mx) R2:1 showed the lowest process stability, as the pH did not increase significantly during AD, changing only from 6.24 to 6.27. This was likely due to the acidic pH of the FVW and crude OMW (pH 4.90 and pH 5.80, respectively) and the inoculum ratio of 2:1 did not allow sufficient dilution of acidic compounds such as volatile fatty acids (VFA) in the mixture. In contrast, the treatment (CD-OMW) R3:1 demonstrated acceptable stability of the process as the pH tended to increase by the end of the experiment (pH increased from 6.58 to 7.47). The two treatments (CD-Mx) R3:1 and (CD-OMW) R2:1 demonstrated the best biological process performance as indicated by the pH measured in their respective digestates, biomethane yields, and the removal efficiency of organic matter, as will be discussed in the following sections. The (CD-Mx) R3:1 treatment initially consisted of a mixture of OMW, FVW, and FW, in addition to the inoculum (CD). These substrates had varying pH values (5.80, 4.90, 6.98, and 7.00, respectively), but when mixed, the feed reached a pH close to neutral (6.83), which was suitable for methanogenic archaea to initiate fermentation. As for treatment (CD-OMW) R2:1 , the co-digestion of OMW with CD at a 2:1 ratio balanced the feed pH to a suitable value of 6.90. Although OMW is known to be limiting for AD due to its low pH, phenolic inhibition, and limited nitrogen content, co-digestion with either a mixture of FVW and FW or with CD alone could create a synergistic effect among these substrates. Table 4 pH changes in different treatments before and after anaerobic digestion Parameters (CD-Mx) R2:1 (CD-OMW) R2:1 (CD-Mx) R3:1 (CD-OMW) R3:1 pH i 6.24 6.90 6.83 6.58 pH f 6.27 7.46 7.68 7.47 pH i : initial pH, pH f : final pH, VS : volatile solids, COD : chemical oxygen demand, CD : Cow dung, OMW : Olive mill wastewater, Mx : Mixture of fruits and vegetable waste and fish waste Solids and polyphenols removal efficiency The removal efficiency of solids refers to the degradation rate of VS and COD in the organic substrate throughout AD by microorganisms. Figure 3 illustrates the degradation of VS, COD and polyphenols in the digestate samples. As for VS removal, the highest degradation was observed in the (CD-Mx) R3:1 treatment (81%) followed by (CD-OMW) R2:1 (76%) while the other two treatments ((CD-Mx) R2:1 and (CD-OMW) R3:1 ) recorded the lowest percentage (56% and 53%, respectively). In a different context, Sounni et al. [ 18 ] reported a similar organic matter removal rate value of about 70% in the co-digestion of OMW with various agro-industrial wastes, using different organic loading rates and I/S ratios. Furthermore, Rubio et al. [ 21 ] observed in their study on co-digestion of OMW with cattle manure that increasing the proportion of OMW up to 75% in the mixture resulted in higher VS removal. A similar trend was observed for COD removal. The highest percentages of COD degradation were recorded for the treatments (CD-OMW) R2:1 and (CD-Mx) R3:1 with 40% and 30%, respectively while the other treatments showed lower degradation, with values below 15%. It should be noted that the maximum COD removal rate observed for (CD-OMW) R2:1 and (CD-Mx) R3:1 treatments did not exceed 50%, possibly because the microorganisms were unable to fully decompose the residual solubilized organic matter [ 39 ]. These COD removal values are consistent with those observed by Fragoso et al. [ 19 ] in their study, which aimed to enhance biomethane production and bioprocess stability by co-digesting dephenolised two-phase olive pomace with mixed sewage sludge, achieving a 40% COD degradation. Regarding polyphenol removal, the highest performance was observed in treatments (CD-OMW) R2:1 and (CD-Mx) R3:1 , achieving 95% and 84% phenol degradation, respectively, after anaerobic digestion. Fragoso et al. [ 19 ] obtained a polyphenol removal rate of 86% from the co-digestion of dephenolised two-phase olive pomace with mixed sewage sludge, which is lower than the rate obtained from (CD-Mx) R3:1 in this study. Another study that investigated the co-digestion of OMW with activated sludge reported a lower polyphenol removal rate than our results, achieving 42.69%, 60.45%, and 63.51% when OMW was aerated for 2, 4, and 6 days, respectively [ 40 ]. In conclusion, the co-digestion of OMW with a mixture of FVW and FW, or with CD solely, can contribute to both process stability and the efficient removal of organic matter in terms of VS, COD and polyphenols. However, this performance was observed only when an I/S ratio of 3:1 and 2:1 was adopted for co-digestion with FVW-FW, and CD, respectively. This was also supported by the biomethane yield best performance obtained for these two treatments, as will be discussed in the following sections. Impact of the co-digestion of OMW with FVW, FW and CD on biomethane yield The biomethane production from the co-digested 2-phase OMW was measured automatically and continuously for 49 days by the system. The results of daily (NmL day − 1 ) and specific biomethane production (NmLCH 4 gVS − 1 ) are shown in Fig. 4 a and Fig. 4 b, respectively. The daily CH 4 production during the AD process can vary widely due to the interference of several factors, including the type and quantity of substrate, the microbial community, the temperature of the digester, the fluctuation in pH values, the presence or absence of the inhibiting compounds and the efficiency of the system. Across all treatments, biogas production began after 3 days of incubation time, indicating a short lag phase (Fig. 4 a). This portion of biogas production was likely attributed to the rapid utilization of easily biodegradable compounds, such as carbohydrates and proteins, which are typically present in higher concentrations in fruits and vegetable waste that constituted the two treatments (CD-Mx) R2:1 and (CD-Mx) R3:1 [ 15 , 41 ]. Another factor that may explain these earlier biogas peaks is that the inoculum was rich in microbial methanogens and contained residual biodegradable organic matter, as indicated by its biogas kinetics from inoculum trials (results not shown). As it can be seen in Fig. 4 a, the maximum daily biomethane peaks for (CD-OMW) R3:1 , (CD-Mx) R2:1 , (CD-OMW) R2:1 , and (CD-Mx) R3:1 were recorded on days 4 (25.80 ± 0.86 NmL), 3 (48.56 ± 1.04 NmL), 23 (63.63 ± 1.85 NmL) and 27 (85.23 ± 2.89 NmL), respectively. Fluctuations in daily biomethane production were clearly observed in the two treatments, (CD-OMW) R2:1 , and (CD-Mx) R3:1 , with maximum production occurring between weeks 2 and 5. The substrates (CD-OMW) R2:1 and (CD-Mx) R3:1 were progressively biodegraded and leading to biogas production until the completion of the 49-day incubation period. Apparently, the process in these two treatments was not significantly affected, or was less affected, by the addition of phenolic compounds in OMW, and the microbial consortium was able to adapt to the potentially inhibitory substrate. In contrast, the treatment (CD-Mx) R2:1 ceased biomethane production on day 5, likely due to complete inhibition caused by phenols and or acidity of FVW and OMW (pH 4.90 and 5.80, respectively) as discussed earlier (Fig. 4 a). This was supported by the fact that, for the treatment (CD-Mx) R2:1 , the polyphenol removal was 0% (Fig. 3 ), and the pH remained below the optimum level recommended for AD (Table 4 ). In the treatment (CD-OMW) R3:1 , the biogas production stopped and entered a steady phase from day 7, with only a small amount of biogas production observed by the end of the experiment on day 46. In terms of specific biomethane production, Fig. 4 b shows that the two treatments (CD-OMW) R2:1 , and (CD-Mx) R3:1 exhibited similar behaviours regarding the shape of the plotted curves, indicating good quality of the biomethane potential test, as suggested by Koch et al. [ 42 ]. Both treatments showed a short lag phase of 3 days, followed by continuous biogas production until plateauing on day 47. Unexpectedly, the co-digested OMW with FVW and FW at I/S ratio of 2:1 ((CD-Mx) R2:1 )) resulted in the lowest biomethane yield (2.02 NmLCH 4 gVS − 1 ). It is worth noting that the FVW used in this experiment had been stored for one year at 6°C, and it is likely that the accumulation of VFAs and the pH reduction were caused by the fermentation of carbohydrates in the substrate and the long storage time, leading to its acidity, as shown in Table 2 (pH 4.90). The second lowest biomethane yield was recorded in the treatment of OMW with CD at ratio of 3:1 ((CD-OMW) R3:1 ) with only 6.70 NmLCH 4 gVS − 1 . The likely reason for this low performance is the low organic loading in the digesters, as the treatment demonstrated good results in terms of pH increase (up to 7.47), VS and polyphenol removal by the end of the experiment, as discussed earlier. The best performance in terms of total methane yield was achieved by the co-digestion of OMW with CD at I/S ratio of 2:1 (CD-OMW) R2:1 (155.00 NmLCH 4 gVS − 1 ) followed by the co-digestion of OMW with FVW and FW at I/S ratio of 3:1 (CD-Mx) R3:1 which obtained 132.20 NmLCH 4 gVS − 1 (The difference is statistically significant). These achievements in terms of biomethane yield are significantly higher compared to the mono-digestion of OMW, which suffered from complete inhibition at the start of the experiment (results not shown in this study). When comparing the results of this study to the literature, there is a wide variation in biomethane yield depending on the type of OMW (2-phase or 3-phase OMW), the I/S ratio, the type of co-substrate, and whether the OMW undergoes pretreatment. For example, Rubio et al. [ 21 ] reported a biomethane yield of 112.40 NmLCH 4 gVS − 1 from the co-digestion of two-phase olive mill waste (2POMW) with cattle manure in a 75:25 mixture. Another study revealed that the digestion of three-phase olive mill waste (3POMW) with digested wastewater sludge as inoculum resulted in a biomethane yield of 209.5 NmLCH 4 gVS − 1 , which was considered the control in this study [ 43 ]. The authors obtained a higher biomethane yield from 3POMW treated simultaneously with a mixture of enzymes and by mechanically removing olive cake stones (252.3 NmLCH 4 gVS − 1 ). Our results regarding the co-digestion of FVW and FW with OMW cannot be directly compared, as we have not found any studies in the literature using similar co-substrates. However, one study that is somewhat comparable treated 3POMW with FVW and waste-activated sludge, reporting a biomethane yield of 340 NmLCH 4 gVS − 1 using single-stage anaerobic sequencing batch reactors, which is significantly higher than the biomethane yield reported in our study for the treatment (CD-Mx) R3:1 [ 41 ]. The results of this study highlight that the co-digestion of 2POMW with animal slurries (cow dung) can help overcome the challenges associated with the mono-digestion of OMW and enhance biomethane production. The same conclusion was reported by Lenzuni et al. [ 44 ]. Furthermore, the addition of fruits, vegetable, and fish waste did not result in a significant change in terms of biomethane yield compared to the co-digestion with cow dung. However, the overall biomethane yield obtained was slightly lower than expected. The main reason for this lower performance may be related to the presence of olive stone fragments, lignocellulosic compounds, and polyphenols, which are commonly found in two-phase olive mills, as reported by other studies [ 19 , 21 , 45 ]. Kinetic study To predict biomethane yield and kinetic parameters, kinetic modelling was applied only to the two treatments that showed relatively good performance in terms of biomethane yield: the co-digestion of OMW with a mixture of fruits, vegetable, and fish waste (CD-Mx) at a 3:1 I/S ratio, and the co-digestion of OMW with cow dung (CD-OMW) at a 2:1 I/S ratio. The calculated biomethane kinetic parameters ( Y m , R m and λ ) and statistical indicators (R² and RMSE) from fitting the observed biomethane data to the models are shown in Tables 5 and 6 . Both the cumulative experimental data and the biomethane values predicted by the models are shown in Fig. 5 . Root Mean Square Error (RMSE) highlights the difference (%) in the parameter Y m between the observed and predicted biomethane production values. The lower the RMSE, the greater the accuracy of the model, indicating a better degree of model fitting and biogas production predictability. A visual examination of the curves presented in Fig. 5 shows that the model with the best linear relationship between predicted and experimental biomethane values is the LFM for both treatments (CD-Mx) R3:1 and (CD-OMW) R2:1 . For the treatment (CD-Mx) R3:1 , the LFM exhibited the best fit to observed biomethane values, with a difference of 4.8% and a high R² value of 0.9930, highlighting its reliability for the obtained results. In contrast, both the MGM and the TFM showed poor fits, with difference between observed and predicted values of 16% and 14%, respectively. These values exceeded the 10% limit for acceptable accuracy [ 31 , 46 ]. For treatment (CD-OMW) R2:1 , both the LFM (difference = 0.2%, R² = 0.9941) and MGM (difference of 5.2%, R² of 0.9902) displayed better model fits, with LFM again best describing the biomethane yield from samples (CD-OMW) R2:1 . However, the TFM model achieved a weaker fit with a 57% difference from the observed values, significantly more than 10% threshold. These findings revealed that the LFM model consistently provided the most accurate predictions among both treatments, highlighting its robustness and reliability in predicting the biomethane production kinetics from OMW under varied co-digestion conditions. Table 5 Estimated kinetic parameters of biomethane production from the co-digestion of OMW with mixture of FVW and FW (CD-Mx) R3:1 Kinetic model Y m (NmLCH 4 gVS − 1 ) R m (NmLCH 4 gVSday − 1 ) λ ( day ) RMSE R 2 MGM 154.24 5.09 10.69 6.28 0.9835 TFM 150.64 3.50 4.64 10.69 0.9543 LFM 138.46 5.77 12.70 4.09 0.9930 Table 6 Estimated kinetic parameters of biomethane production from the co-digestion of OMW with cow dung (CD-OMW) R2:1 Kinetic model Y m (NmLCH 4 gVS − 1 ) R m (NmLCH 4 gVSday − 1 ) λ (day) RMSE R 2 MGM 163.15 6.39 6.08 5.37 0.9902 TFM 244.63 6.35 3.01 10.31 0.9660 LFM 154.67 6.85 7.53 4.29 0.9941 Conclusion This study aimed to investigate the effect of co-digestion of two-phase olive mill wastewater with fruits and vegetable waste, fish waste, and cow dung to enhance biomethane yield. The results indicate that these co-substrates significantly enhance biomethane production, as well as solids and polyphenol removal efficiency, compared to the mono-digestion of OMW after a 49-day incubation period at 37°C. Specifically, the co-digestion of OMW with cow dung at an I/S ratio of 2:1 (CD-OMW) R2:1 and a mixture of fruits, vegetable, and fish waste at an I/S ratio of 3:1 (CD-Mx) R3:1 achieved VS and polyphenol removal rates of approximately 76%, 81%, and 95% and 84%, respectively. The same treatments produced 155.00 NmLCH 4 gVS − 1 and 132.20 NmLCH 4 gVS − 1 of biomethane, respectively, indicating that the co-digestion option of OMW offers a sustainable strategy for biogas generation from olive mill waste, while addressing the environmental impact of current management practices. The logistic function model (LFM) provided a good fit for biomethane production from the two treatments. However, this study was limited by the composition variability of OMW, fruits, vegetable, and fish waste used, which may have an impact on result consistency. Further research should investigate additional co-substrate options and assess system scalability to optimize biogas yields. Declarations Competing interests The authors have no relevant financial or non-financial interests to disclose. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author contributions H. Erraji designed and performed the experiments, processed the data, and wrote the manuscript. A. Essadek performed the chemical analyses. A. Asehraou wrote and revised the manuscript. Tallou contributed to the writing of the manuscript. Acknowledgement The authors gratefully acknowledge the administrative staff of the Renewable Energy and Energy Efficiency Training Institute, Oujda, Morocco. 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Applied Sciences. 11 , 6069 (2021). https://doi.org/10.3390/app11136069 Supplementary Files GRAPHICALABSTRACT.jpg Cite Share Download PDF Status: Published Journal Publication published 24 Apr, 2025 Read the published version in Waste and Biomass Valorization → Version 1 posted Reviewers agreed at journal 21 Mar, 2025 Reviewers invited by journal 21 Mar, 2025 Editor invited by journal 21 Mar, 2025 Editor assigned by journal 20 Mar, 2025 First submitted to journal 19 Mar, 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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wastewater)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5821546/v1/795292afb5411dcb18662fc6.png"},{"id":79320871,"identity":"67b5d5d3-c904-4403-b4d7-ce3f5ba158b0","added_by":"auto","created_at":"2025-03-27 04:25:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44520,"visible":true,"origin":"","legend":"\u003cp\u003eAutomatic methane potential test system used\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5821546/v1/cdc629f17a5ce8e26224e9e4.png"},{"id":79319508,"identity":"c2783562-999b-4ff4-a96d-8db6c4ea8914","added_by":"auto","created_at":"2025-03-27 04:17:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12315,"visible":true,"origin":"","legend":"\u003cp\u003eSolids and polyphenols removal efficiency\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5821546/v1/21596f406caa784d122bf813.png"},{"id":79319510,"identity":"d193676c-4a65-4153-a9d3-32c7613c3ccf","added_by":"auto","created_at":"2025-03-27 04:17:28","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":464911,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e): Daily biomethane production (NmL/day), (\u003cstrong\u003eb\u003c/strong\u003e): specific biomethane production (NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e -1\u003c/sup\u003e), (CD-OMW): olive mill wastewater + cow dung, (CD-Mx): mixture of 33% olive mill wastewater +33% fruits and vegetable waste + 33% fish waste + cow dung\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5821546/v1/36db20f38d4546fc3eef4e7e.jpeg"},{"id":79319513,"identity":"34bfbbe7-d2dd-4d56-84ad-ef735930be9e","added_by":"auto","created_at":"2025-03-27 04:17:28","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":180921,"visible":true,"origin":"","legend":"\u003cp\u003eObserved and predicted biomethane production in the treatments (CD-Mx) \u003csub\u003eR3:1 \u003c/sub\u003eand (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e: MGM: Modified Gompertz model, LFM: Logistic Function Model, TFM: Transference Function Model\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5821546/v1/412d907b643ff2b8178f2b6d.jpg"},{"id":81569597,"identity":"ffac18ec-ba61-4deb-89ed-e43a3fab346a","added_by":"auto","created_at":"2025-04-28 16:07:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2010540,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5821546/v1/4476f6bb-8b1e-4032-afe0-0de185979590.pdf"},{"id":79319515,"identity":"b598389a-b167-4443-8ebb-3bd7b9631117","added_by":"auto","created_at":"2025-03-27 04:17:29","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":745518,"visible":true,"origin":"","legend":"","description":"","filename":"GRAPHICALABSTRACT.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5821546/v1/d3df1029c9703e13eaee14d6.jpg"}],"financialInterests":"","formattedTitle":"Enhanced Biomethane Production from Olive Mill Wastewater via Co-Digestion with Cow Dung, Fruits, Vegetable, and Fish Wastes: An Experimental and Kinetic Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOlive oil manufacturing in Mediterranean countries (Spain, Italy, Greece, Morocco, Tunisia, Palestine, etc.) represents a significant market share worldwide. This economic activity poses different threats to the ecosystem (soil contamination, water pollution and odor emissions, etc.) due to the mismanagement of its by-products [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. One of these by-products is olive mill wastewater (OMW), generated during olive oil extraction. It contains high salt levels, organic matter, suspended and dissolved particles, and toxic compounds such as phenols, and it has a high biological and chemical oxygen demand (BOD and COD) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The issue with OMW comes from its phenolic content, which is responsible of its dark color as well as its antibacterial and phytotoxic effects [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Moreover, polyphenols, fatty acids, and volatile compounds are toxic to the anaerobic bacterial community, limiting the biological treatment of OMW, specifically during the anaerobic digestion (AD) process. In addition, the high levels in BOD and COD are also limiting factors that make OMW difficult to treat or manage [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOMW components are known to negatively affect the physico-chemical and biological properties of soil, making it phytotoxic [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Land spreading and other treatment practices, such as evaporation ponds, could lead to groundwater pollution. The use of OMW without treatment in agriculture can affect the acidity, salinity, nitrogen immobilization, microbial community, leaching of nutrients, and concentration of lipids, organic acids, and phenolic compounds and can negatively impact plants development [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAD is one of the best alternative technologies for the valorization and treatment of diverse organic wastes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the success of the mono-digestion process (i.e., AD using a single waste) is facing the serious challenges related to the characteristics of the substrates. Co-digestion of multiple feedstocks provides the potential to overcome these limitations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In recent years, the potential of co-digestion has been studied and documented. For instance, it has been reported that anaerobic co-digestion enhances the digestibility of co-substrates, improves the process stability, and produces a digestate of high agronomic value [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This positive effect is attributed to the diversity and improved nutrient balance in the substrates, particularly an optimum C/N ratio, the synergetic effect of the microorganisms provided by the co-substrates, the dilution of inhibitory and toxic compounds for AD, and the buffering capacity of the AD mixture. To achieve this positive effect, we suggest in this study the co-digestion of OMW with cow dung (CD), fruits and vegetable waste (FVW) and fish waste (FW).\u003c/p\u003e \u003cp\u003eLarge quantities of FW with low economic value (fish head, scale, viscera, fins, tail, and backbones) are produced during fish processing. The sustainable management of FW has become a serious challenge nowadays [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. FW is known to contain a high concentration of biodegradable organic compounds, mainly proteins and lipids, making it favorable for anaerobic co-digestion [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, another promising organic waste with a promising value and high potential as a substrate for AD is FVW, characterized by high moisture content, low total solids (TS), and high volatile solids (VS), making it suitable for AD [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The main characteristic of CD, FW and FVW is that they are available throughout the year, whereas OMW is seasonally available. Therefore, incorporating these substrates into a co-digestion process is highly relevant. Indeed, the C/N ratio of OMW is high [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], whereas the other two substrates, particularly FW, are known to be rich in nitrogen, making them good candidates for co-digestion with OMW.\u003c/p\u003e \u003cp\u003eAccording to the literature, many efforts have been made to improve the AD of OMW either by applying pretreatments to reduce the inhibitory effect of polyphenols and the high organic load of OMW or by optimizing digestion parameters including inoculum/substrate ratios (I/S) or selecting suitable co-substrates to compensate for the C/N imbalance of OMW. Several studies have investigated the effect of co-digestion of OMW with other substrates [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. For instance, Sounni et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] examined the co-digestion of OMW with poultry manure, cheese whey, grass, and slaughterhouse wastewater with substrate/OMW ratios of 80:20, 20:80, 20:80, and 50:50, respectively. The authors concluded that co-digestion of OMW with slaughterhouse wastewater (ratio 50:50) achieved the highest biomethane yield in batch mode. Another study conducted by Mouftahi et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] reported an average net specific methane production of 384 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the case of mixture composed of 4.4% organic fraction of municipal solid wastes, 2.2% chicken dropping, 4.4% OMW and 89% inoculum. While previous studies have explored the co-digestion of OMW with multiple organic substrates, to the best of our knowledge, no study has specifically investigated its co-digestion with cow dung, fruits and vegetable waste, and fish waste in batch system. Furthermore, this study applied kinetic modeling to describe the biomethane production potential of the aforementioned co-substrates, providing deeper insights into process performance and optimization.\u003c/p\u003e \u003cp\u003eThe aim of the present study is to investigate the effect of co-digestion of liquid OMW with CD, FVW and FW using two I/S ratios of 2:1 and 3:1 under mesophilic conditions (37\u0026deg;C) during a 49-day incubation period on biomethane production and bioprocess performance in terms of volatile solids and phenols removal. Biomethane modelling was also conducted using three models: Modified Gompertz Model, Transference Function Model, and Logistic Function Model to describe the kinetic mechanisms of biomethane production in different treatments.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSelection of co-substrates and experimental procedure\u003c/h2\u003e \u003cp\u003eThe liquid OMW tested in this study as the main substrate was composed of wastewater mixed with olive cake, which was rich in fragments of olive stones. It was collected directly from the storage pond of a two-phase olive oil mill in Oujda, Morocco. Fruits and vegetable waste (FVW) as well as fish waste (FW) were taken from the IFMEREE training institute canteen while the cow dung (CD) was obtained from a dairy cattle farm in the Oujda region. The CD was used as both an inoculum and co-substrate after a 15-day incubation period under mesophilic (37\u0026deg;C) and anaerobic conditions to stimulate microbial activity. FVW and FW were separately grinded in the laboratory and stored at -4\u0026deg;C until their use (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the effect of co-digestion of OMW with CD, FVW and FW, the following experimental procedure was followed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In this study, a mixture of OMW, FVW, and FW (labelled as Mx) was prepared in equal proportions, with each waste representing 33% of the mixture. This mixture was anaerobically digested using two I/S ratios of 2:1 and 3:1 based on volatile solids content (VS) of the inoculum (CD) and mixture. These I/S ratios have been recognized in previous studies as suitable for anaerobic digestion process, and a higher I/S ratio is recommended when potential substrate inhibition is expected [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. At the same time, the OMW was digested with inoculum (CD) while respecting the two I/S ratio of 2:1 and 3:1. As a control, OMW was digested alone to evaluate biomethane production without the influence of any additional co-substrates.\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\u003eSubstrates mixtures and experimental procedure used in the study\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=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReactor number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubstrates ratio (fresh weight based)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI/S ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTreatment code\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReactor 1 to 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33% olive mill wastewater\u0026thinsp;+\u0026thinsp;33% fruits and vegetable waste\u0026thinsp;+\u0026thinsp;33% fish waste\u0026thinsp;+\u0026thinsp;cow dung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(CD-Mx) \u003csub\u003eR 2:1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReactor 4 to 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOlive mill wastewater\u0026thinsp;+\u0026thinsp;cow dung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(CD-OMW) \u003csub\u003eR 2:1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReactor 7 to 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33% olive mill wastewater\u0026thinsp;+\u0026thinsp;33% fruits and vegetable waste\u0026thinsp;+\u0026thinsp;33% fish waste\u0026thinsp;+\u0026thinsp;cow dung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(CD-Mx) \u003csub\u003eR 3:1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReactor 10 to 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOlive mill wastewater\u0026thinsp;+\u0026thinsp;cow dung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(CD-OMW) \u003csub\u003eR 3:1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReactor 13 to 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOlive mill wastewater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOMW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003cem\u003eCD\u003c/em\u003e: Cow dung, \u003cem\u003eOMW\u003c/em\u003e: Olive mill wastewater, \u003cem\u003eMx\u003c/em\u003e: Mixture of fruits and vegetable waste and fish waste, I/S\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eratio (\u003csub\u003eR 2:1\u003c/sub\u003e and \u003csub\u003eR 3:1\u003c/sub\u003e): Inoculum to substrate ratio\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhysico-chemical characterization of feedstocks\u003c/h3\u003e\n\u003cp\u003eThe characterization of feedstocks before and after anaerobic digestion included classical analyses, and results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. All analyses were determined according to the standard methods [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Volatile solids (VS), dry matter (DM), and pH were determined directly from the homogenised samples. DM was determined after drying samples at 105\u0026deg;C for 24 hours. VS was obtained by the loss-on-ignition method at 550\u0026deg;C in Nabertherm muffle furnace. pH of raw substrates, mixtures and digestates was measured using liquid samples and TOLEDO pH meter. Chemical oxygen demand (COD) was analysed according to the Open reflux method [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. After digestion of samples with potassium dichromate (K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e) for 2 hours at 150\u0026deg;C, the COD was determined by titration using ferrous sulfate (FeSO\u003csub\u003e4\u003c/sub\u003e) according to the Eq.\u0026nbsp;(1) and expressed as gO\u003csub\u003e2\u003c/sub\u003e/L.\u003c/p\u003e \u003cp\u003eCOD= [(FS\u003csub\u003eBl\u003c/sub\u003e-FS\u003csub\u003eSample\u003c/sub\u003e)\u0026times;C\u003csub\u003eFS\u003c/sub\u003e\u0026times;8]V\u003csub\u003eSample\u003c/sub\u003e (1)\u003c/p\u003e \u003cp\u003eWhere FS\u003csub\u003eBl\u003c/sub\u003e is the volume of FeSO\u003csub\u003e4\u003c/sub\u003e used in titration of the blank sample (mL), FS\u003csub\u003eSample\u003c/sub\u003e is the volume of FeSO\u003csub\u003e4\u003c/sub\u003e used in titration of sample, V\u003csub\u003eSample\u003c/sub\u003e is the volume of sample (mL), C\u003csub\u003eFS\u003c/sub\u003e is the concentration of reducing agent (N), and COD is expressed in gO\u003csub\u003e2\u003c/sub\u003e/L of sample.\u003c/p\u003e \u003cp\u003ePolyphenols were analyzed following Folin-Ciocalteu method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Polyphenols extraction was performed using method described by Leouifoudi et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The pH of OMW samples was first adjusted to 2 to maximise the recovery of polyphenols [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Then OMW samples were subjected to a defatting process using hexane (95%) (1:1, (v/v)) followed by clarification through centrifugation (4000 rpm, 15 min). The phenolic compounds contained in the defatted and clarified OMW samples were subjected to two liquid-liquid extractions using ethyl acetate (95%) (1:1, v/v) and centrifugation at 4000 rpm for 10 min. Subsequently, the ethyl acetate phase was evaporated using a rotary evaporator (BUCHI Rotavapor R-114) at 40\u0026deg;C. The dried residues were then dissolved in 10 mL of methanol and used to determine phenols content. Fifty \u0026micro;L of the phenolic extract was mixed with 1.35 mL of distilled water and 200 \u0026micro;L of Folin ciocalteaux solution. The solution was incubated for 3 min in the dark. Then 400 \u0026micro;L of sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e 20%) was added to the solution. The development of a blue colour was obtained after incubation in the water bath for 20 min at 40\u0026deg;C. The absorbance was measured at 760 nm. The polyphenol content was expressed in g of gallic acid equivalent per g of dry matter (g GA/gDM). A calibration curve was used and obtained by 6 samples with different gallic acid concentrations (15.62, 31.25, 62.50, 125.00, and 250.00 mg/L) previously prepared. All analysis were performed in triplicate.\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\u003eChemical characteristics of substrates and their mixtures before anaerobic digestion\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOMW\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFW\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFVW\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(CD-Mx) \u003csub\u003eR 2:1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e(CD-OMW) \u003csub\u003eR 2:1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e(CD-Mx) \u003csub\u003eR 3:1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e(CD-OMW) \u003csub\u003eR 3:1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e6.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e6.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e6.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM (% FM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e17.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e10.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e9.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e5.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e7.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVS (% FM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e13.61\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e8.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e10.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e10.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOD (gO\u003csub\u003e2\u003c/sub\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e42.40\u0026thinsp;\u0026plusmn;\u0026thinsp;3.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e67.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e20.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e32.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e48.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e21.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolyphenols\u003c/p\u003e \u003cp\u003e(gGA/gDM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e4.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e2.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e2.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e\u003cem\u003eCD\u003c/em\u003e: Cow dung, \u003cem\u003eOMW\u003c/em\u003e: Olive mill wastewater, \u003cem\u003eFW\u003c/em\u003e: Fish waste, \u003cem\u003eFVW\u003c/em\u003e: Fruits and vegetable waste, \u003cem\u003eMx\u003c/em\u003e: Mixture of fruits, vegetable and fish waste, I/S ratio (\u003csub\u003eR 2:1\u003c/sub\u003e and \u003csub\u003eR 3:1\u003c/sub\u003e): Inoculum to substrate ratio, \u003cem\u003eFM\u003c/em\u003e: Fresh matter, \u003cem\u003eDM\u003c/em\u003e, Dry matter, \u003cem\u003eVS\u003c/em\u003e: Volatile solids, \u003cem\u003eCOD\u003c/em\u003e: Chemical oxygen demand, \u003cem\u003eGA\u003c/em\u003e: Gallic acid\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eAnaerobic digestion set-up and biochemical methane potential tests\u003c/h3\u003e\n\u003cp\u003eA set of 15 batch reactors (Glass bottles) with a total volume of 600 mL and a working volume of 400 mL were used in this experiment. Digesters were prepared using the quantities of substrates and inoculum shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, while maintaining the mixtures and I/S ratios as described in the first section (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All treatments were performed in triplicate to guaranty the reliability and accuracy of measurements and conclusions. After feeding batch digesters, the pH of slurries was immediately measured, and the digesters were then sealed using rubber stoppers with Tygon\u0026reg; tubing for extracting the biogas. Then all digesters were purged for 45 seconds with biogas from a fixed dome digester with an average CH\u003csub\u003e4\u003c/sub\u003e and CO\u003csub\u003e2\u003c/sub\u003e contents of 75% and 23%, respectively, to ensure anaerobic conditions and rapid start-up of process.\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\u003eAmounts of substrates used in this experiment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDigesters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 to 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 to 6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 to 9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10 to 12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13 to 15\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(CD-Mx) \u003csub\u003eR 2:1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CD-OMW) \u003csub\u003eR 2:1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(CD-Mx) \u003csub\u003eR 3:1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(CD-OMW) \u003csub\u003eR 3:1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOMW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrate amount (Mx or OMW) [g FM]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e161.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e198.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e124.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e158.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e400.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInoculum amount (CD) [g FM]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e238.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e201.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e275.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e241.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal amount [g FM]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e400.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e400.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e400.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e400.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e400.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eCD\u003c/em\u003e: Cow dung, \u003cem\u003eOMW\u003c/em\u003e: Olive mill wastewater, \u003cem\u003eMx\u003c/em\u003e: Mixture of fruits and vegetable waste and fish waste, \u003cem\u003eFM\u003c/em\u003e: Fresh matter\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe experiment was conducted during a total incubation period of 49 days, and the temperature was maintained at 37\u0026deg;C using thermostatic water bath (Precision 0.2\u0026deg;C). Mechanical mixing of slurries in the bioreactors was ensured by stepper motors operating at a frequency of 150 rpm during 5 min, followed by an off period of 60 minutes. To measure the pure biomethane, the produced biogas was passed continuously through 80mL bottles containing 3M NaOH solution to remove any potential CO\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Few drops of thymolphthalein were added to NaOH solution for pH monitoring. The volume of CH\u003csub\u003e4\u003c/sub\u003e was automatically and continuously measured by liquid displacement and buoyancy method using a gas volume measuring device as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe obtained CH\u003csub\u003e4\u003c/sub\u003e was continuously normalised by the automatic system (1.0 standard atmospheric pressure, 0\u0026deg;C and zero moisture content). The experiments were terminated when CH\u003csub\u003e4\u003c/sub\u003e generation stopped completely in some digesters or was significantly reduced in others. The cumulative biomethane yield was calculated using the following Eq.\u0026nbsp;(2) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Results are presented as means and standard deviations (SD) of three independent digesters for each treatment.\u003c/p\u003e \u003cp\u003eNmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e =[Cumulative volume of CH\u003csub\u003e4\u003c/sub\u003e(mL)]\u003cb\u003e/[\u003c/b\u003eMass of VS content (g VS)] (2)\u003c/p\u003e\n\u003ch3\u003eDetermination of phenols, COD and VS reduction\u003c/h3\u003e\n\u003cp\u003eAt the end of digestion, total phenols, COD and VS were determined and their respective reduction was calculated according to the method adapted from Matjuda et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] (Eq.\u0026nbsp;3):\u003c/p\u003e \u003cp\u003eRD (%)= [(RS-DS)\u003cb\u003e/\u003c/b\u003eRS]\u0026times;100% (3)\u003c/p\u003e \u003cp\u003eWhere:\u003c/p\u003e \u003cp\u003eRD: rate of degradation of the parameter\u003c/p\u003e \u003cp\u003eRS: concentration or parameter content in the raw substrate\u003c/p\u003e \u003cp\u003eDS: concentration or parameter content in the digested substrate\u003c/p\u003e\n\u003ch3\u003eKinetic study\u003c/h3\u003e\n\u003cp\u003eBiogas production from AD of organic substrates can be described using kinetic models and several mathematical models have been proposed to predict biogas production and describe the biological process behaviour in digesters. Models such as Modified Gompertz model (MGP) (Eq.\u0026nbsp;4), Transference Function Model (TFM) (Eq.\u0026nbsp;5) and Logistic Function Model (LFM) (Eq.\u0026nbsp;6) are commonly used for this purpose [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This three models were used in this study and fitted with the experimental cumulative biomethane yields obtained from digesters.\u003c/p\u003e \u003cp\u003e \u003cem\u003eY\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003eexp{-exp[(\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003ee)\u003cb\u003e/\u003c/b\u003e\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e(λ-t)\u0026thinsp;+\u0026thinsp;1]} (4)\u003c/p\u003e \u003cp\u003e \u003cem\u003eY\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e{1-exp[-\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e\u0026times;(t-λ)\u003cb\u003e/\u003c/b\u003e\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e]} (5)\u003c/p\u003e \u003cp\u003e \u003cem\u003eY\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e\u003cb\u003e/\u003c/b\u003e{1\u0026thinsp;+\u0026thinsp;exp[4\u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e\u0026times;((λ-t)\u003cb\u003e/\u003c/b\u003e\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e)\u0026thinsp;+\u0026thinsp;2]} (6)\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003eY\u003c/em\u003e is the cumulative biomethane production (NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as a function of time t (days), \u003cem\u003eY\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e is the ultimate potential biomethane production (NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), \u003cem\u003eR\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e is the maximum production rate (NmLCH\u003csub\u003e4\u003c/sub\u003e gVS day \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), λ is the lag phase time (days), and \u003cem\u003ee\u003c/em\u003e is a constant equivalent to 2.718282. The parameters \u003cem\u003eY\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, \u003cem\u003eR\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, and λ were determined using non-linear regression by the solver ToolPack of Microsoft Excel 2016 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Then a comparison was made among the three models to find the suitable one which describes better the biological process by validation of model parameters prediction. The validation methods used in this study are Root Mean Square Error (RMSE) and the coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e) that were calculated using the Excel Microsoft 2016 tool.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003ch3\u003ePerformance of digesters during anaerobic digestion\u003c/h3\u003e\n\u003cp\u003eThe performance of the process in bioreactors can be assessed using various indicators including biogas production and the control of physicochemical parameters such as pH changes, VS, COD and polyphenol removal after anaerobic digestion. The results regarding pH evolution and the removal rates of VS, COD and polyphenol are shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, respectively.\u003c/p\u003e \u003cp\u003eAccording to the literature, the pH of the slurry in digesters is one of the most important parameters significantly influencing the stability of AD [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For instance, if the AD process is efficient, the pH of the digestates generally increases toward alkalinity by the end of the AD process, as ammonia is released from the digested feedstock [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Given that no adjustments of pH were made in this study, initial pH of treatments (CD-Mx) \u003csub\u003eR2:1\u003c/sub\u003e, (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e, (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e and (CD-OMW) \u003csub\u003eR3:1\u003c/sub\u003e was 6.24, 6.83, 6.90 and 6.58, respectively. The pH values corresponding to the treatments (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e and (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e fell within the recommended AD pH range of 6.8 to 7.5 while the pH of the two other trials was slightly below this range [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe treatment (CD-Mx) \u003csub\u003eR2:1\u003c/sub\u003e showed the lowest process stability, as the pH did not increase significantly during AD, changing only from 6.24 to 6.27. This was likely due to the acidic pH of the FVW and crude OMW (pH 4.90 and pH 5.80, respectively) and the inoculum ratio of 2:1 did not allow sufficient dilution of acidic compounds such as volatile fatty acids (VFA) in the mixture. In contrast, the treatment (CD-OMW) \u003csub\u003eR3:1\u003c/sub\u003e demonstrated acceptable stability of the process as the pH tended to increase by the end of the experiment (pH increased from 6.58 to 7.47). The two treatments (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e and (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e demonstrated the best biological process performance as indicated by the pH measured in their respective digestates, biomethane yields, and the removal efficiency of organic matter, as will be discussed in the following sections. The (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e treatment initially consisted of a mixture of OMW, FVW, and FW, in addition to the inoculum (CD). These substrates had varying pH values (5.80, 4.90, 6.98, and 7.00, respectively), but when mixed, the feed reached a pH close to neutral (6.83), which was suitable for methanogenic archaea to initiate fermentation. As for treatment (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e, the co-digestion of OMW with CD at a 2:1 ratio balanced the feed pH to a suitable value of 6.90. Although OMW is known to be limiting for AD due to its low pH, phenolic inhibition, and limited nitrogen content, co-digestion with either a mixture of FVW and FW or with CD alone could create a synergistic effect among these substrates.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003epH changes in different treatments before and after anaerobic digestion\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(CD-Mx) \u003csub\u003eR2:1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(CD-OMW) \u003csub\u003eR3:1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003csub\u003ei\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003csub\u003ef\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003epH\u003csub\u003ei\u003c/sub\u003e: initial pH, pH\u003csub\u003ef\u003c/sub\u003e: final pH, \u003cem\u003eVS\u003c/em\u003e: volatile solids, \u003cem\u003eCOD\u003c/em\u003e: chemical oxygen demand, \u003cem\u003eCD\u003c/em\u003e: Cow dung,\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cem\u003eOMW\u003c/em\u003e: Olive mill wastewater, \u003cem\u003eMx\u003c/em\u003e: Mixture of fruits and vegetable waste and fish waste\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eSolids and polyphenols removal efficiency\u003c/h3\u003e\n\u003cp\u003eThe removal efficiency of solids refers to the degradation rate of VS and COD in the organic substrate throughout AD by microorganisms. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the degradation of VS, COD and polyphenols in the digestate samples. As for VS removal, the highest degradation was observed in the (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e treatment (81%) followed by (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e (76%) while the other two treatments ((CD-Mx) \u003csub\u003eR2:1\u003c/sub\u003e and (CD-OMW) \u003csub\u003eR3:1\u003c/sub\u003e) recorded the lowest percentage (56% and 53%, respectively). In a different context, Sounni et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] reported a similar organic matter removal rate value of about 70% in the co-digestion of OMW with various agro-industrial wastes, using different organic loading rates and I/S ratios. Furthermore, Rubio et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] observed in their study on co-digestion of OMW with cattle manure that increasing the proportion of OMW up to 75% in the mixture resulted in higher VS removal.\u003c/p\u003e \u003cp\u003eA similar trend was observed for COD removal. The highest percentages of COD degradation were recorded for the treatments (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e and (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e with 40% and 30%, respectively while the other treatments showed lower degradation, with values below 15%. It should be noted that the maximum COD removal rate observed for (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e and (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e treatments did not exceed 50%, possibly because the microorganisms were unable to fully decompose the residual solubilized organic matter [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. These COD removal values are consistent with those observed by Fragoso et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] in their study, which aimed to enhance biomethane production and bioprocess stability by co-digesting dephenolised two-phase olive pomace with mixed sewage sludge, achieving a 40% COD degradation.\u003c/p\u003e \u003cp\u003eRegarding polyphenol removal, the highest performance was observed in treatments (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e and (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e, achieving 95% and 84% phenol degradation, respectively, after anaerobic digestion. Fragoso et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] obtained a polyphenol removal rate of 86% from the co-digestion of dephenolised two-phase olive pomace with mixed sewage sludge, which is lower than the rate obtained from (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e in this study. Another study that investigated the co-digestion of OMW with activated sludge reported a lower polyphenol removal rate than our results, achieving 42.69%, 60.45%, and 63.51% when OMW was aerated for 2, 4, and 6 days, respectively [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, the co-digestion of OMW with a mixture of FVW and FW, or with CD solely, can contribute to both process stability and the efficient removal of organic matter in terms of VS, COD and polyphenols. However, this performance was observed only when an I/S ratio of 3:1 and 2:1 was adopted for co-digestion with FVW-FW, and CD, respectively. This was also supported by the biomethane yield best performance obtained for these two treatments, as will be discussed in the following sections.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImpact of the co-digestion of OMW with FVW, FW and CD on biomethane yield\u003c/h2\u003e \u003cp\u003eThe biomethane production from the co-digested 2-phase OMW was measured automatically and continuously for 49 days by the system. The results of daily (NmL day\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and specific biomethane production (NmLCH\u003csub\u003e4\u003c/sub\u003e gVS \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, respectively. The daily CH\u003csub\u003e4\u003c/sub\u003e production during the AD process can vary widely due to the interference of several factors, including the type and quantity of substrate, the microbial community, the temperature of the digester, the fluctuation in pH values, the presence or absence of the inhibiting compounds and the efficiency of the system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAcross all treatments, biogas production began after 3 days of incubation time, indicating a short lag phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). This portion of biogas production was likely attributed to the rapid utilization of easily biodegradable compounds, such as carbohydrates and proteins, which are typically present in higher concentrations in fruits and vegetable waste that constituted the two treatments (CD-Mx) \u003csub\u003eR2:1\u003c/sub\u003e and (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Another factor that may explain these earlier biogas peaks is that the inoculum was rich in microbial methanogens and contained residual biodegradable organic matter, as indicated by its biogas kinetics from inoculum trials (results not shown).\u003c/p\u003e \u003cp\u003eAs it can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the maximum daily biomethane peaks for (CD-OMW) \u003csub\u003eR3:1\u003c/sub\u003e, (CD-Mx) \u003csub\u003eR2:1\u003c/sub\u003e, (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e, and (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e were recorded on days 4 (25.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 NmL), 3 (48.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04 NmL), 23 (63.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85 NmL) and 27 (85.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89 NmL), respectively. Fluctuations in daily biomethane production were clearly observed in the two treatments, (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e, and (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e, with maximum production occurring between weeks 2 and 5. The substrates (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e and (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e were progressively biodegraded and leading to biogas production until the completion of the 49-day incubation period. Apparently, the process in these two treatments was not significantly affected, or was less affected, by the addition of phenolic compounds in OMW, and the microbial consortium was able to adapt to the potentially inhibitory substrate. In contrast, the treatment (CD-Mx) \u003csub\u003eR2:1\u003c/sub\u003e ceased biomethane production on day 5, likely due to complete inhibition caused by phenols and or acidity of FVW and OMW (pH 4.90 and 5.80, respectively) as discussed earlier (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). This was supported by the fact that, for the treatment (CD-Mx) \u003csub\u003eR2:1\u003c/sub\u003e, the polyphenol removal was 0% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), and the pH remained below the optimum level recommended for AD (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the treatment (CD-OMW) \u003csub\u003eR3:1\u003c/sub\u003e, the biogas production stopped and entered a steady phase from day 7, with only a small amount of biogas production observed by the end of the experiment on day 46.\u003c/p\u003e \u003cp\u003eIn terms of specific biomethane production, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb shows that the two treatments (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e, and (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e exhibited similar behaviours regarding the shape of the plotted curves, indicating good quality of the biomethane potential test, as suggested by Koch et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Both treatments showed a short lag phase of 3 days, followed by continuous biogas production until plateauing on day 47. Unexpectedly, the co-digested OMW with FVW and FW at I/S ratio of 2:1 ((CD-Mx) \u003csub\u003eR2:1\u003c/sub\u003e)) resulted in the lowest biomethane yield (2.02 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). It is worth noting that the FVW used in this experiment had been stored for one year at 6\u0026deg;C, and it is likely that the accumulation of VFAs and the pH reduction were caused by the fermentation of carbohydrates in the substrate and the long storage time, leading to its acidity, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (pH 4.90). The second lowest biomethane yield was recorded in the treatment of OMW with CD at ratio of 3:1 ((CD-OMW) \u003csub\u003eR3:1\u003c/sub\u003e) with only 6.70 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The likely reason for this low performance is the low organic loading in the digesters, as the treatment demonstrated good results in terms of pH increase (up to 7.47), VS and polyphenol removal by the end of the experiment, as discussed earlier. The best performance in terms of total methane yield was achieved by the co-digestion of OMW with CD at I/S ratio of 2:1 (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e (155.00 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) followed by the co-digestion of OMW with FVW and FW at I/S ratio of 3:1 (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e which obtained 132.20 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (The difference is statistically significant). These achievements in terms of biomethane yield are significantly higher compared to the mono-digestion of OMW, which suffered from complete inhibition at the start of the experiment (results not shown in this study). When comparing the results of this study to the literature, there is a wide variation in biomethane yield depending on the type of OMW (2-phase or 3-phase OMW), the I/S ratio, the type of co-substrate, and whether the OMW undergoes pretreatment. For example, Rubio et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] reported a biomethane yield of 112.40 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e from the co-digestion of two-phase olive mill waste (2POMW) with cattle manure in a 75:25 mixture. Another study revealed that the digestion of three-phase olive mill waste (3POMW) with digested wastewater sludge as inoculum resulted in a biomethane yield of 209.5 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was considered the control in this study [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The authors obtained a higher biomethane yield from 3POMW treated simultaneously with a mixture of enzymes and by mechanically removing olive cake stones (252.3 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Our results regarding the co-digestion of FVW and FW with OMW cannot be directly compared, as we have not found any studies in the literature using similar co-substrates. However, one study that is somewhat comparable treated 3POMW with FVW and waste-activated sludge, reporting a biomethane yield of 340 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using single-stage anaerobic sequencing batch reactors, which is significantly higher than the biomethane yield reported in our study for the treatment (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of this study highlight that the co-digestion of 2POMW with animal slurries (cow dung) can help overcome the challenges associated with the mono-digestion of OMW and enhance biomethane production. The same conclusion was reported by Lenzuni et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Furthermore, the addition of fruits, vegetable, and fish waste did not result in a significant change in terms of biomethane yield compared to the co-digestion with cow dung. However, the overall biomethane yield obtained was slightly lower than expected. The main reason for this lower performance may be related to the presence of olive stone fragments, lignocellulosic compounds, and polyphenols, which are commonly found in two-phase olive mills, as reported by other studies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eKinetic study\u003c/h2\u003e \u003cp\u003eTo predict biomethane yield and kinetic parameters, kinetic modelling was applied only to the two treatments that showed relatively good performance in terms of biomethane yield: the co-digestion of OMW with a mixture of fruits, vegetable, and fish waste (CD-Mx) at a 3:1 I/S ratio, and the co-digestion of OMW with cow dung (CD-OMW) at a 2:1 I/S ratio. The calculated biomethane kinetic parameters (\u003cem\u003eY\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, \u003cem\u003eR\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003eλ\u003c/em\u003e) and statistical indicators (R\u0026sup2; and RMSE) from fitting the observed biomethane data to the models are shown in Tables\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Both the cumulative experimental data and the biomethane values predicted by the models are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Root Mean Square Error (RMSE) highlights the difference (%) in the parameter \u003cem\u003eY\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e between the observed and predicted biomethane production values. The lower the RMSE, the greater the accuracy of the model, indicating a better degree of model fitting and biogas production predictability. A visual examination of the curves presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows that the model with the best linear relationship between predicted and experimental biomethane values is the LFM for both treatments (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e and (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e. For the treatment (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e, the LFM exhibited the best fit to observed biomethane values, with a difference of 4.8% and a high R\u0026sup2; value of 0.9930, highlighting its reliability for the obtained results. In contrast, both the MGM and the TFM showed poor fits, with difference between observed and predicted values of 16% and 14%, respectively. These values exceeded the 10% limit for acceptable accuracy [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. For treatment (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e, both the LFM (difference\u0026thinsp;=\u0026thinsp;0.2%, R\u0026sup2; = 0.9941) and MGM (difference of 5.2%, R\u0026sup2; of 0.9902) displayed better model fits, with LFM again best describing the biomethane yield from samples (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e. However, the TFM model achieved a weaker fit with a 57% difference from the observed values, significantly more than 10% threshold. These findings revealed that the LFM model consistently provided the most accurate predictions among both treatments, highlighting its robustness and reliability in predicting the biomethane production kinetics from OMW under varied co-digestion conditions.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEstimated kinetic parameters of biomethane production from the co-digestion of OMW with mixture of FVW and FW (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKinetic model\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eY\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eR\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (NmLCH\u003csub\u003e4\u003c/sub\u003e gVSday\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eλ\u003c/em\u003e (\u003cem\u003eday\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRMSE\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 \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMGM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e154.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9835\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTFM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e150.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9543\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLFM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e138.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9930\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\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEstimated kinetic parameters of biomethane production from the co-digestion of OMW with cow dung (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKinetic model\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eY\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eR\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (NmLCH\u003csub\u003e4\u003c/sub\u003e gVSday\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eλ\u003c/em\u003e (day)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eRMSE\u003c/em\u003e\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 \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMGM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e163.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9902\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTFM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e244.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9660\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLFM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e154.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.9941\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\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study aimed to investigate the effect of co-digestion of two-phase olive mill wastewater with fruits and vegetable waste, fish waste, and cow dung to enhance biomethane yield. The results indicate that these co-substrates significantly enhance biomethane production, as well as solids and polyphenol removal efficiency, compared to the mono-digestion of OMW after a 49-day incubation period at 37\u0026deg;C. Specifically, the co-digestion of OMW with cow dung at an I/S ratio of 2:1 (CD-OMW) \u003csub\u003eR2:1\u003c/sub\u003e and a mixture of fruits, vegetable, and fish waste at an I/S ratio of 3:1 (CD-Mx) \u003csub\u003eR3:1\u003c/sub\u003e achieved VS and polyphenol removal rates of approximately 76%, 81%, and 95% and 84%, respectively. The same treatments produced 155.00 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 132.20 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of biomethane, respectively, indicating that the co-digestion option of OMW offers a sustainable strategy for biogas generation from olive mill waste, while addressing the environmental impact of current management practices. The logistic function model (LFM) provided a good fit for biomethane production from the two treatments. However, this study was limited by the composition variability of OMW, fruits, vegetable, and fish waste used, which may have an impact on result consistency. Further research should investigate additional co-substrate options and assess system scalability to optimize biogas yields.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eH. Erraji designed and performed the experiments, processed the data, and wrote the manuscript. A. Essadek performed the chemical analyses. A. Asehraou wrote and revised the manuscript. Tallou contributed to the writing of the manuscript.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the administrative staff of the Renewable Energy and Energy Efficiency Training Institute, Oujda, Morocco.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShabir, S., Ilyas, N., Saeed, M., Bibi, F., Sayyed, R.Z., Almalki, W.H.: Treatment technologies for olive mill wastewater with impacts on plants. Environ. Res. \u003cstrong\u003e216\u003c/strong\u003e, 114399 (2023). https://doi.org/10.1016/j.envres.2022.114399\u003c/li\u003e\n\u003cli\u003eAl Bawab, A., Ghannam, N., Abu-Mallouh, S., Bozeya, A., Abu-Zurayk, R.A., Al-Ajlouni, Y.A., Alshawawreh, F., Odeh, F., Abu-Dalo, M.A.: Olive mill wastewater treatment in Jordan: A Review. IOP Conf. Ser. Mater. Sci. 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Applied Sciences. \u003cstrong\u003e11\u003c/strong\u003e, 6069 (2021). https://doi.org/10.3390/app11136069\u003c/li\u003e\n\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":"Anaerobic co-digestion, Olive mill wastewater, Fruits and vegetable waste, Fish waste, Cow dung","lastPublishedDoi":"10.21203/rs.3.rs-5821546/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5821546/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOlive mill wastewater (OMW) is the main effluent resulting in huge amounts from olive oil manufacturing. This effluent is mostly composed of organic matter and polyphenolic compounds, known for their antimicrobial activity and compromise their biological treatment. This work investigates the impact of the co-digestion of olive mill wastewater with fruits and vegetable waste (FVW), fish waste (FW), and cow dung (CD) under mesophilic conditions at two different inoculum-to-substrate ratios. The effect on biomethane yield, volatile solids reduction, and polyphenol removal efficiency were evaluated. Moreover, kinetic modeling was applied to describe biomethane production. The co-digestion of OMW with CD at an I/S ratio of 2:1, and a mixture consisting of 33% OMW, 33% FVW, and 33% FW at I/S ratio of 3:1 achieved biomethane yields of 155.00 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 132.20 NmLCH\u003csub\u003e4\u003c/sub\u003e gVS\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively after 49-day retention time at 37\u0026deg;C whereas the mono-digestion of OMW was completely inhibited. These treatments demonstrated strong performance in terms of volatile solids and polyphenol removal, achieving rates of 76%, 81%, and 95% and 84%, respectively. Similarly, the logistic function model provided a good fit for predicting biomethane production, with high R\u0026sup2; values of 0.9941 and 0.9930, respectively.\u003c/p\u003e","manuscriptTitle":"Enhanced Biomethane Production from Olive Mill Wastewater via Co-Digestion with Cow Dung, Fruits, Vegetable, and Fish Wastes: An Experimental and Kinetic Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 04:17:24","doi":"10.21203/rs.3.rs-5821546/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-21T15:16:05+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-21T14:03:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Waste and Biomass Valorization","date":"2025-03-21T13:23:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T18:03:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Waste and Biomass Valorization","date":"2025-03-19T15:48:00+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":"e57c9578-dda7-4c98-9cb3-3c8b9f00a347","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-28T16:00:26+00:00","versionOfRecord":{"articleIdentity":"rs-5821546","link":"https://doi.org/10.1007/s12649-025-03079-5","journal":{"identity":"waste-and-biomass-valorization","isVorOnly":false,"title":"Waste and Biomass Valorization"},"publishedOn":"2025-04-24 15:57:15","publishedOnDateReadable":"April 24th, 2025"},"versionCreatedAt":"2025-03-27 04:17:24","video":"","vorDoi":"10.1007/s12649-025-03079-5","vorDoiUrl":"https://doi.org/10.1007/s12649-025-03079-5","workflowStages":[]},"version":"v1","identity":"rs-5821546","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5821546","identity":"rs-5821546","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}