Valorization of Green Market Waste as a Renewable Energy Source

Valorization of Green Market Waste as a Renewable Energy Source | 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 Valorization of Green Market Waste as a Renewable Energy Source Serpil Ozmihci, İlknur Hacıoğlu, İlgi Karapınar, Meltem Küs This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4713736/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract High organic carbon wastes can be used for biofuel production for reducing raw material costs. Green market wastes (GMW) are one of the raw material candidates to generate biofuel by fermentation with low-cost production and high yield of fuel formation. Biohydrogen and biomethane are potential gaseous energy sources that can be obtained through sequential fermentation of GMW. In this study, the ground and hydrolyzed GWM (a mixture of lettuce, parsley, spring onion, and dill) solid-liquid mixture was first valorized for biohydrogen, then the organic acid-rich residue of biohydrogen fermentation was subjected to biomethane production. Biohydrogen and biomethane was produced at mesophilic (37°C) and thermophilic (55°C) temperatures. The initial GWM amount varied between 1000-5000 g wb at mesophilic biohydrogen production. The highest cumulative hydrogen formation (CHF) and the rate were obtained at 5000 g wb as CHF=8.9 L and 916 mL H 2 /L day, respectively. Thermophilic biohydrogen fermentation was conducted at 1000 and 5000 g wb and CHF reached to 14.2 L at 5000 g wb GMW. The yield of hydrogen formation was 1.6 times higher at thermophilic than that of mesophilic conditions. The residues from two fermentation temperatures of 5000 g wb GMW were used to produce biomethane under both fermentation conditions. Mesophilic and thermophilic conditions resulted in cumulative biomethane formations (CMFs) of 8.3 L and 5.8 L, and biomethane production yields of 0.17 mL CH 4 /g VFA and 0.13 mL CH 4 /g VFA, respectively. The findings revealed that GMW is a suitable substrate for efficient biohydrogen and biomethane production. Biohydrogen Biomethane Biohythane Green Market Waste Thermophilic Mesophilic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights Large amounts of green market wastes can be valorized to biohydrogen. Thermophilic fermentation of GMW produced more biohydrogen than mesophilic one. High organic acid contents obtained from biohydrogen fermentation can be valorized to biomethane. Mesophilic conditions resulted in higher biomethane volumes than thermophilic one. Sequential biohydrogen and biomethane processes provides complete waste valorization into biofuel. Introduction As a renewable energy source, biomass energy stands out with various advantages. It is suitable for energy production at all scales. It is storable, and sustainable, can be generated from organic wastes, leads to less greenhouse effect compared to fossil fuels and helps socioeconomic development, especially, in rural areas [ 1 – 3 ]. On the other hand, biomass achieved from energy crops is questioned for the fuel versus food debate regarding the use of agricultural areas for this purpose instead of human nutrition. Moreover, sustainability has emerged as a concern in the production of biofuels, even when it comes to non-food plant cultivation. Therefore, the production of biofuels from non-competitive biomass has been on the energy research agenda as a solution to fuel versus food debate [ 4 ]. It is clear that biofuel production requires a large amount of organic matter to build an efficient process. As non-competitiveness is an issue, one of the readily available organic matter rich raw material sources, without occupying agricultural land, is domestic solid waste. Studies have shown that the organic fraction of domestic solid waste is about 50%, and 70% of this fraction is disposed of in landfills [ 5 – 6 ]. This substantial amounts of organic matters are already in use for biogas production with low energy input through anaerobic digestion (AD) employing waste-to-energy technology for food waste treatment [ 7 – 8 ]. The approach to biofuel production has evolved towards a more holistic approach that considers new biomass energies in addition to biogas. In order to achieve this, it is essential to include other renewable, inexpensive, and abundant non-food resources that can be diverted to on-site biofuel production. The best candidates are agricultural residues and edible plant residues such as Green Market Wastes (GMW) which are rich in organic matter and can be processed directly without the need for pre-sorting, as is the case with domestic solid wastes. With these characteristics, they have a significant economic value and enormous potential to contribute to sustainable renewable energy such as biohydrogen, bioethanol, and biogas production [ 9 – 25 ]. Bioethanol and biomethane are the main and well-known biofuels produced from various organic materials [ 10 , 24 ]. Biohydrogen production researches and applications raised in the last decades which is known as a clean energy carrier and the technology for storage and transport is constantly developing [ 12 – 20 ]. Biohydrogen from biomass by fermentation has some limitations such as the need for pre-treatment, low production rate and yield, and a residual organic acid fraction that requires further processing either for valorization or treatment before discharge [ 12 – 20 ]. In the last decade, biohythane production gained attention for utilizing waste biomass. The main aim of these studies was biohythane (a blend of 10 to 30% biohydrogen (v/v) and 70 to 90% of biomethane (v/v)) production including sequential dark fermentation (DF, biohydrogen) and anaerobic digestion (AD, biomethane) processes [ 26 – 27 ]. Studies on relatively new technologies point to sequential hydrogen and methane production as a respectable solution to the listed problems encountered in single-stage biohydrogen or biomethane production [ 28 – 33 ]. The presence of 10–30% biohydrogen in the biomethane mixture results in enhanced thermal efficiency and reduced greenhouse gas emissions to achieve carbon neutrality for global warming effects [ 8 ]. Furthermore; this approach helps with biohydrogen transport and storage issues. It is particularly useful for on-site processes where the hydrogen can be used directly without storage. Additionally, the organic acids released from biohydrogen fermentation are not wasted or discharged, but are used for biomethane production, providing a completely carbon neutral process. In this study; partially hydrolyzed GMW, as a lignocellulosic organic waste, was valorized in a sequential DF and AD process for biohythane production. Among the biological hydrogen production methods, DF is the most stable and efficient one and some of the important parameters are pH, temperature, gas partial pressure, Oxidation Reduction Potential (ORP), nutrients, the organic acid types produced, substrate and biomass concentration, and microorganism culture [ 23 , 34 – 35 ]. Many studies focused on hydrogen gas production from lignocellulosic waste biomass by DF with different operational modes and temperature [ 34 – 39 ]. Substrates such as sweetsorghum, sugar cane pulp, animal manure, waste paper towels, domastic wastes etc. were valorized giving hydrogen production yields between 0.927–2.55 mol H 2 /mol glucose in the literature [ 37 – 39 ]. The hydrogen production potential from lignocellulosic wastes depend on the pretreatment applied, the type of biomass, and the microbial culture that enhance biohydrogen production yields and rates [ 12 , 16 – 18 , 20 , 39 ]. Physical, chemical, or enzymatic pre-treatments are needed before fermentation which unfortunately, increase the overall production cost [ 11 , 14 – 15 , 20 – 22 , 40 ]. Therefore, partial hydrolysis such as thermal treatment in moderate temperatures or consolidated fermentation could be key answers for reducing the operating cost [ 41 – 42 ]. Fermentation temperature positively affects the process such as thermophilic conditions help to eliminate hydrogen-consuming bacteria and efficient conversion of simple sugars to hydrogen [ 12 , 16 , 19 ] which may provide higher biohydrogen production yields and productivities [ 43 – 44 ]. Anaerobic digestion (AD) as the second step in our process is a well-known process, where agricultural wastes, some industrial wastes, domestic solid wastes, etc. can be valorized for biomethane production [ 24 , 45 – 46 ]. The success of methane gas production in the composition of the produced biogas depends on fermentation temperature, type and amount of raw material, alkalinity (pH), substrate particle size, fermentation time, carbon to nitrogen ratio (C/N), type of bioprocess and amount of dry matter [ 47 – 48 ]. Different C/N ratios contained in wastes, such as wheat straw (C/N: 87/1), corn straw (C/N: 53/1), animal feces (C/N: 29/1) need careful operations by maintaining the ideal ratio for biogas production in the mixed feedstock [ 49 ]. Therefore; preferring hydrogen production before methane formation can alter the need of specific conditions for biomethane production. Besides, the use of the effluent of DF, with rich organic acid content, for AD will shorten the fermentation time, decrease the reactor volume. One of the questions is the fermentation and digestion temperature. Dong et al, [ 50 ] revealed that thermophilic (50-65 o C) conditions gave higher biohydrogen and biomethane yields when compared with mesophilic ones. In different studies ranges of 17–292 mL H 2 / g VS and 150–570 mL CH 4 /g VS gases were achieved [ 31 – 32 , 50 – 52 ]. Green market wastes (GMW), which are produced in large quantities in bazaars and fruit and vegetable markets, can be utilized as raw material in energy sector, such as biohydrogen and biomethane, offering certain advantages in terms of producing high value-added products at low cost. By considering these facts, the aim of this study is to valorize the green market waste (GMW) by sequential biohydrogen (DF) and biomethane (AD) production under different temperatures using mixed culture. For this purpose, biohydrogen production potential at mesophilic conditions (37 o C) at different initial substrate concentrations varying between 1000–5000 g wet basis (wb) was evaluated and then, the selected substrate concentrations were fermented under thermophilic conditions to compare the effects of temperature on biohydrogen production. Finally, the organic acid-rich effluents of biohydrogen production was subjected to methanogenic organisms to evaluate the biomethane production potentials from these effluents. The novelty of the study is to modify the consolidated fermentation process by adding a simple pretreatment and then achieving an efficient biohydrogen-biomethane production from the solid-liquid mixture instead of using pretreatment supernatant in a conventional manner. Materials and Methods Substrate and Pre-treatment of the Substrate Green Market Wastes (GMW) were consisted of lettuce, parsley, dill, arugula, and spring onions. The wastes were collected from the markets in the province of İzmir/ Turkey. GMW was first shredded to reduce the particle size and then, blended to get a homogeneous feedstock. A viscous liquid-like mixture was obtained from the wet GMW containing 94% moisture. Thermal pre-treatment (partial treatment) was applied to the mixture at 100 o C for 1 hour. The chemical composition of 1 kg of GMW mixture is given in Table 1 . The organic carbon content of the raw GMW, without grinding or blending, was 439.4 g/ kg dw (dry weight). After grinding and blending processes partial liquefaction occurred. The TOC concentrations in the dried solid and liquid phases of the mixture were 491 g/ kg dw and 10.8 g/L, respectively. TOC concentration increased to 11.1 g/ L after thermal treatment in the liquid phase. Table 1 Characterization of Green Market Wastes after Blending (1 kg) Parameter Concentration Total Nitrogen (mg/L) 2940 Total Phosphorus (mg/L) 298.5 NH 4 -N (mg/L) 190.4 NO 3 -N (mg/L) 808.38 TOC in the liquid phase (g/L) 10.8 TOC in the solid phase (g/kg) 491 Total Sugar in the liquid phase (g/L) 10.8 Solid Content % 6 Microorganisms Biohydrogen-Producing Microorganisms Pretreated anaerobic sludge (thermally treated) was used as bacterial inoculum in biohydrogen production. Anaerobic sludge was obtained from the acidogenic phase of the treatment plant of Pakmaya Industry, İzmir, Turkey. The sludge was thermally treated at pH 5 and 100 o C for 1 hour under a fume hood, to eliminate methanogenic bacteria in the anaerobic sludge consortium. The pretreated anaerobic sludge was then cultivated at 37 o C and 55 o C to activate biohydrogen-producing Clostridium species. The growth media was composed of 10 g/ L peptone, 10 g/ L glucose, 3.9 g/ L KH 2 PO 4 , 2.8 g/ L K 2 HPO 4 , 0.25 g/ L MgSO 4 , 0.6 g/ L yeast extract, and 0.1 g/ L L-cysteine. Anaerobic conditions were accomplished by sparging nitrogen gas through the fermentation reactors for 10 minutes. The initial pH was adjusted to pH 7.2. Organisms that reached to the exponential growth phase in 48 hours were used as inoculum in biohydrogen production experiments. Biomethane-Producing Microorganisms Anaerobic sludge was obtained from the methanogenic reactor of the wastewater treatment plant of Pakmaya Industry, İzmir, Turkey. The anaerobic sludge was cultivated in nutrient-rich media for 48 hours and then used as inoculum for biomethane production experiments. The growth media consisted of 0.6 g/ L yeast extract, 10 g/ L peptone, 10 g/ L glucose, 3.9 g/ L KH 2 PO 4 , 2.8 g/ L K 2 HPO 4 , 0.25 g/ L MgSO 4 , and 0.1 g/ L L-cysteine. Anaerobic conditions were provided by passing N 2 gas through the headspace of the reactors. The pH of the fermentation media was adjusted to pH 7.5 when required. The fermentation temperatures were 37 o C and 55 o C depending on the experiment. Experimental Procedure Biohydrogen Production The experiments were carried out at 37 o C and 55 o C in a temperature-controlled incubator over a roller shaker with 20 rpm mixing speed. The substrate concentrations were 1000, 1200, 2500, and 5000 g under mesophilic condition and 1000 and 5000 g under thermophilic one. 1 kg of wet GMW resulted in approximately 1 L of mixed solid-liquid volume after blending. Therefore, the initial reaction volume varied depending on the amount of wet GMW used (reactor volumes varied between 2–10 L). The initial amount of microorganisms was 10% of the reactor volume. Silicone stoppers and caps were used to prevent gas leakage from the bottles. The initial pH was set to 7.2 at the beginning and adjusted to around 6.5 when needed with dilute NaOH solution. Anaerobic conditions were preserved by transferring nitrogen gas from the headspace of the bottles for 20–40 min. Maintaining anaerobic conditions was checked with negative (<-250 mV) oxidation-reduction potential (ORP). The pH, total sugar, organic acid, biohydrogen percentage, and total gas volume were monitored in daily samples. Biomethane Production For biogas production, the 1000 g and 5000 g GMW effluents were used, in which biohydrogen was produced. The methanogenic culture was directly inoculated to reactors after biohydrogen production ceased. The fermentation was conducted at 37 o C and 55°C. Experimental studies were terminated when the cumulative biomethane production volume reached a constant value. Experiments were carried out on rotating rollers, placed in an incubator, at 20 rpm. Analytic Methods Samples taken daily (10 mL) were centrifuged at 8000 rpm for 15 minutes and the solid phase was separated. Total sugar, acetic, butyric, propionic, and lactic acids concentrations were determined in the liquid phase. Total sugar concentration was determined by the Dubois method [ 53 ]. Organic acid concentrations were measured by HPLC (Agilent 1100) with a UV detector (220 nm) on Aminex HPX-87H column (9 µm x 300 x 7.8 mm), using 5mM sulfuric acid mobile phase with a flow rate of 0.6 mL/ min [ 54 ]. Biohydrogen and biomethane gas samples were taken daily from the headspace of the experimental bottles with a gas-tight glass syringe. The concentrations of the gases were determined by using a gas chromatograph (Agilent 6890 N-GC) and the column was Alltech, Hayesep D 80/100 6 in. x 1/8 in. x 0.85 in. Nitrogen gas was used as a carrier gas with a flow rate of 30 mL/ min. The temperatures of the oven, injection, detector, and filament were 35°C, 120°C, 120°C, and 140°C, respectively. The volume of total gas produced was measured by the water displacement method using a solution containing 2% H 2 SO 4 and 10% NaCl. Triplicate analysis of the samples showed a standard deviation lower than 5% [ 54 ]. Some parameters were used in characterization of the substrate. Total organic carbon concentration was carried out in Schimadzu TOC analyzer (SM 5310 B). Total Nitrogen (ASL.SCP.087.Rev.00), Total Phosphorus (SM 4500-P B SM 4500-P D), NO 3 -N, NH 4 -N concentrations were conducted according to APHA standard methods [ 55 ]. The yield, cumulative gas volume, and rate calculations were adapted from Ozmihci and Kargi [ 54 ] and Conde and Kaparaju [ 56 ]. Results and Discussion Biohydrogen Production under Mesophilic Conditions Different GMW substrate concentrations (1000, 1200, 2500, 5000 g wb) were tested under mesophilic batch dark fermentation. Figure 1a depicts the maximum cumulative hydrogen volumes and liquid phase initial and final total sugar concentrations in fermentation media at different substrate concentrations. Cumulative biohydrogen production increased up to around 1.1 L and 1.3 L for 1000 and 1200 g wb substrate amounts. The volume substantially increased to 5 L with 2500 g wb GMW and then reached the maximum volume of 9 L at 5000 g wb substrate. The total sugar in the fermentation media was measured in the liquid phase. Its concentration was nearly 1% of the GMW at 1000 and 1200 g wb GMW, while it was only around 0.53% for 2500 and 5000 g wb experiments. That solubilized total sugar concentration was sufficiently high to support the growth of the organism and to initiate biohydrogen production. However, there was a substantial variation in total sugar concentration in the liquid phase due to the hydrolysis of the solid phase to simple sugars during fermentation. The sugar consumption was fast for the first 24–48 hours of fermentation time at 1000 and 1200 g wb GMW (data not shown) resulting in a rapid biohydrogen generation phase. However, there was a delay in the production due to the required adaptation period of the microorganism at higher GMW amounts (data not shown). The final sugar concentrations in the reactors at the liquid phase were around 1–3 g/ L and the sugar consumption efficiency in the liquid phase was nearly constant around 91 ± 3%. The results indicated that both liquid and solid-phase sugars were used for biohydrogen production. The microbial processes can be employed to reduce costs and time. One such process is the consolidated bioprocess (CBP). In the production of biofuels, separate or simultaneous saccharification and fermentation are typically employed. However, CBP offers a distinct approach, utilizing a microbial consortium capable of producing both cellulolytic enzymes and hydrogen in the dark fermentation process. Clostridia species are the predominant microorganisms responsible for this function, and mixed cultures in which different species can coexist have been demonstrated to be successful in CBP [ 41 , 57 ]. In our study, this phenomenon also existed where biohydrogen production was accomplished through consolidated fermentation (CBP) where the microorganisms hydrolyzed GMW into simple sugars, which was then used for biohydrogen production [ 41 , 57 ]. The pH of the fermentation media is a good indicator of the organic acid production in dark fermentation. As seen in Fig. 1b, the variation in pH for all GMW amounts used showed that the 48 hours were critical for rich organic acid formation. pH decreased from 7.2 to around 5.1–5.4 within the first 48 hours for all tested GMW amounts. Later, pH values reached a steady state for 1000 and 1200 g wb GMW indicating that organic acid production was terminated. This result was in parallel with biohydrogen production and sugar consumption which were at high rates for the first 48 h of fermentation. However, the pH fluctuation for 2500 and 5000 g wb occurred during the fermentation period. The fluctuation in pH may be attributed to the gradual release of sugar from the solid phase through biological hydrolysis, which is then converted into organic acids. Figure 1c depicts the organic acid concentrations produced at the end of the fermentation period at different substrate amounts. The main organic acid types were acetic acid and butyric acids with low concentrations of propionic and lactic acid at high GMW amounts. The acetic acid concentration reached to 19.7 g/ L for 1200 g wb substrate and to around 16.55 g/L for 1000 g wb. The butyric acid concentrations were low in these substrate amounts (around 2 g/ L). On the other hand, biohydrogen-generating organic acids were almost equally produced at 2500 g wb and 5000 g wb GMW resulting in around 15 g/L total organic acid concentrations. The lower organic acid amounts in higher substrate concentrations may be due to slower simultaneous hydrolysis and fermentations. (a) (b) (c) Figure 1. The Variation of a. Cumulative Biohydrogen Gas Formations, Initial and Final Total Sugar Concentrations, b. pH, c. Organic Acid Types and Concentrations at the End of the Fermentation Time with GMW Amounts, Under Mesophilic Conditions. Biohydrogen production yields and rates with different GMW amounts are shown in Fig. 2 . The biohydrogen production yield and formation rate increased with increasing GMW amount up to 2500 g wb in the fermentation media. The maximum yield and rate were 35 mL/ g dw and 413 mL H 2 / L day. The yield was determined based on the dry organic matter of GMW consumed during fermentation. It is clear that although maximum cumulative biohydrogen formation (CHF, Fig. 1) was obtained at 5000 g GMW, the reaction was slow mainly due to the limitations in the simultaneous microbial hydrolysis of the GMW during fermentation and the long adaptation period as mentioned above. The biohydrogen production yield at 5000 g wb GMW was the lowest compared to the other initial GMWs. In other words, the microbial hydrolysis of the GWM during fermentation was not sufficient under mesophilic fermentation temperature. Thermophilic fermentation could help enhancing the hydrolysis of the substrate, more fermentable sugars can be generated and the efficiency of the process can be improved. Comparison of biohydrogen production under mesophilic and thermophilic conditions The effect of thermophilic fermentation on substrate utilization, hydrolysis of GMW, and biohydrogen production was investigated. The experiments were conducted at the lowest (1000 g wb) and highest (5000 g wb) GMW tested for biohydrogen production. The results were given as comparison of mesophilic and thermophilic conditions to understand the effects of temperature. As seen in Fig. 3a, the biohydrogen production terminated after 48 h of the fermentation period at 1000 g wb GMW under mesophilic and thermophilic conditions resulting in 1.96 L and 2.2 L CHF, respectively. The biohydrogen production at 5000 g GMW was substantially higher compared to 1000 g GMW. Thermophilic conditions enhanced the biohydrogen production which continued along the fermentation period. The maximum cumulative volumes reached to 14.2 L under thermophilic condition and to 8.8 L for mesophilic one. Thermophilic fermentation resulted in almost 1.61 times more biohydrogen volume at 5000 g wb GMW. (a) (b) Figure 3. Time-course of a. Cumulative Biohydrogen Formation b. Total Sugar Concentration under Thermophilic and Mesophilic Conditions for Different GMW Amounts. Figure 3b shows variation of the total sugar concentrations with time at different substrate concentrations and fermentation temperatures. The total sugar that was measured from the liquid phase of the fermentation time shows the available sugar to be converted into end products. The initial total sugar concentration in the liquid phase depended on the efficiency of partial hydrolysis. The soluble sugars increased from time to time in the fermentation media by biological hydrolysis of solid GMW particles in the media. This was observed clearly at 5000 g wb GMW tested in mesophilic conditions. Total soluble sugar in the fermentation media steadily decreased untill 192 hours and then, a sharp increase in the sugar concentration to 7.8 g/L occurred indicating that hydrolysis of the substrate took place. At the end of the fermentation period, the total sugar concentration was 2.6 g/L. The fluctuation of total sugar is due to consolidated bioprocessing where simultaneous hydrolysis and fermentation took place. The pH in the fermentation media decreased to around 5-5.5 within 24 hours due to rapid sugar consumption for all fermentation conditions, then they were nearly constant at around 6-6.5 until the end of fermentation period (data not presented). The total organic acids in mesophilic conditions consisted of acetic and butyric acid at 1000 g wb and 5000 g wb GMW were 18.53 g/ L and 15.249 g/L, respectively. In thermophilic conditions, the main organic acid was acetic acid with a concentration of 19.2 g/L in 1000 g wb GMW and 14.4 g/L in 5000 g wb GMW (data not presented). There was not a substantial increase in butyric acid production under thermophilic conditions. It is being understood from the organic acids that temperature rises also changes the end products due to dominated acid type of fermentation. The same trend, as in different substrate concentration trials at mesophilic conditions, was seen with 1000 g and 5000 g wb GMW. The biohydrogen production yields and rates for the mesophilic and thermophilic conditions with 1000 g wb and 5000 g wb GMW are given in Fig. 4 . The yields increased with increasing substrates and temperatures. The highest yield was obtained as 47.5 mL H 2 / g dw with 5000 g wb substrate under thermophilic conditions, but the maximum rate was 916 mL H 2 / L day at mesophilic conditions. High GMW concentrations such as 5000 g wb is suitable to use under thermophilic conditions to get high volumes and yields of biohydrogen production [ 55 ]. Biomethane Production under Different Fermentation Temperatures Converting organic acid-rich effluent of biohydrogen-producing reactor to biomethane as a secondary product provides an economic advantage in both waste management and energy generation. Therefore, there is a need to develop methods that will enable the direct use of the biohydrogen produced. In the two-stage split system, differences in operating and production conditions, rate-limiting biological reactions, and inhibitions are more easily controlled. As previously mentioned there is no need for specific operation conditions that biogas systems need. Thus, an increase in reaction rates and productivity and a reduction in retention time can be achieved [ 54 , 56 – 57 ]. The effluents of 5000 g wb GMW obtained from thermophilic fermentation conditions with high organic acids content were subjected to mesophilic and thermophilic biomethane formation. Figure 5a shows the volumes of biomethane formation under mesophilic and thermophilic conditions. The cumulative biomethane volumes were 8.3 L and 5.8 L under mesophilic and thermophilic conditions, respectively. The biogas was composed of 55% of CH 4 , 1% of H 2 , and 44% CO 2 under mesophilic condition. The percentage of biohydrogen in total gas slightly increased to 3% for thermophilic condition, but biomethane percentage was the same as mesophilic one. (a) (b) (c) Figure 5. Variations of (a) Cumulative Biomethane Formation (b) Total Sugar Concentration (c) Total Organic Acid Concentration with Time under Mesophilic and Thermophilic Conditions at GMW 5000 g wb Figure 5b-c depicts variation of total sugar and total organic acid concentrations with time, respectively. The fermentable sugar concentration in the effluent of mesophilic biohydrogen fermentation was already limited between 750–850 mg/ L. The sugar consumption profile under mesophilic methanogenesis was straight but it was varying under thermophilic one. The reason for the variation could be the progress of organic fraction hydrolysis in GMW to sugar monomers under the methanogenic phase (Fig. 5b). Similarly, total volatile fatty acid (TVFA) consumption varies in parallel to that of sugar concentrations. While TVFA was consumed for biomethane generation, the hydrolyzed sugars were converted into organic acid, which was then used for biomethane formation. This continuing hydrolysis and TVFA formation processes extend the biomethane production period with the increase in formation volume (Fig. 5c). Figure 6 depicts the biomethane production yields and rates under mesophilic and thermophilic conditions. The biomethane formation yield (0.17 mL CH 4 / g TVFA) and rate (502 mL CH 4 / g TVFA day) were higher in mesophilic conditions than in thermophilic conditions (0.13 mL CH 4 / g TVFA and 150 mL CH 4 / g TVFA day). The main reason for the slow rate at thermophilic fermentation is due to simultaneous hydrolysis, TVFA production, and biomethane generation. ■Biomethane Formation Rate, ■ Biomethane Formation Yield The study showed that GMW is a suitable substrate for sequential biohydrogen and biomethane production. There are limited thermophilic sequential biohydrogen and biogas production studies in the literature. The one reported by Sillero et al [ 51 ] was a thermophilic, two-stage anaerobic co-digestion system (55-70 o C). A mixture of sewage sludge and wine vinasse with a co-substrate of poultry manure was used. The results indicated that the addition of poultry manure to the media in thermophilic temperature (55°C) enhanced biohydrogen generation, yielding 27.1 mL H 2 /g VS and 59 mL CH 4 / g VS. They concluded that hyperthermophilic temperatures also favor CH 4 production [ 51 ]. Lovato et al [ 52 ] tried mesophilic (34 o C) and thermophilic (55 o C) conditions with food waste. They found higher biohydrogen yields in mesophilic and higher biomethane yields in thermophilic conditions, which was the opposite what we found. Table 2 summarizes the sequential biohydrogen and biomethane production from different organic waste materials under different environmental conditions. In our study, it is clear that thermophilic conditions improved biohydrogen production. Elevated temperature increased the hydrolysis rate, which was reflected in biohydrogen production (CHF: 14.2 L). The rate when compared with mesophilic conditions was low due to the time spend in hydrolysis stage of the substrate. However, the biohydrogen was lower when compared with the literature probably due to high organic carbon content of the substrate that was valorized under consolidated fermentation conditions which needed time. The biohydrogen and biomethane production rate was comparable in both conditions with the literature except the ones using continuous fermentation mode [ 32 , 50 ]. The results showed that respectable amounts of biohydrogen and biomethane, which can be blended to biohythane, could be produced with GMW. Most of the sequential processes were carried out at mesophilic conditions as given in Table 2 . Our results are comparable with these studies. Pretreatment methods, side products such as furfurals and hydroxymethyl furfurals-5 from acid treatment of biomass and the type of sugars in the soluble form affect the biohydrogen and organic acid formation. In our study, only thermal treatment was applied for partial hydrolysis allowing organisms to reach the substrate during initial phase of consolidated fermentation. It was shown that efficient biofuel production under mesophilic conditions can be achieved with basic heat thermal treatment application and consolidated fermentation. It should be taken into consideration that consolidated fermentation processes are slower, while simultaneous hydrolysis and fermentation occurs [ 39 , 53 ]. However at the end the cumulative biohydrogen and biomethane gas formations were comparable with the literature. The sequential biohydrogen and biogas production under mesophilic conditions enhanced biogas production having a high biomethane yield when compared with some studies in Table 2 . Moreover, from the studies it can be concluded that sequential biohydrogen and biomethane fermentation with GMW can be adapted to continuous fermentations with high gas production potentials. Table 2 Summary of sequential biohydrogen and biomethane production results from waste materials under different fermentation conditions. Substrate Organism Conditions Biohydrogen Production Rate Biomethane Production Rate Reference Food Waste Mixed culture 34 o C, H 2 :pH 5.5, CH 4 : pH 7 55°C, H 2 :pH 5.5, CH 4 : pH 7 11.17 mL H 2 / h 0.42 mL H 2 /L h 29.28 mL CH 4 / d 63.6 mL CH 4 / d [ 26 ] Sugarcane straw Mixed culture pH 5.50, 35°C 1.7 mLH 2 /.h 43.33 mL CH 4 / d [ 30 ] Vegatable waste (Onion) Mixed culture pH 7 and 35°C 2.52 mL H 2 /h 66.67 mL CH 4 / d [ 31 ] Molasses Dominated by H 2 : Clostridium butyricum ; CH 4 : Methanobacterium beijingense, Methanothrix soehngenii H 2 :pH 5.5, 35 o C; HRT: 6h CH 4 : pH 7, 35 o C; HRT: 6d 291.67 mL H 2 / h 3700 mL CH 4 / d [ 32 ] Rice Straw Mixed culture 60°C, (Mixture: 1.7% H 2 , 58.7% CH 4 ), continous 1062.55 mLH 2 / h 1528.72 mL CH 4 / d [ 50 ] Green market waste Mixed culture 37 o C, pH 6.5 55 o C, pH 6.5 85.85 mL H 2 / h 37.6 mL H 2 / h 488.86 mL CH 4 / d 260.1 mL CH 4 / d This study Conclusions The sequential biohydrogen and biomethane generation of GMW by dark batch fermentation was evaluated under different conditions. High substrate concentrations were suitable for biohydrogen production under mesophilic conditions. 5000 g wb GMW produced 8.9 L CHF and 916 mL H 2 /L day. Thermophilic conditions favored biohydrogen production (CHF = 14.2 L, Y P/S : 47.5 mL H 2 / g dw), enhanced conversion of sugars into VFA's, and resulted in 1.6 times higher yield than that of mesophilic conditions at GMW = 5000 g wb, but a lower rate of formation was obtained compared to mesophilic fermentation. The effluent of biohydrogen fermentation was rich in organic acid with TVFA (total volatile fatty acid) concentrations between 14–20 g/L in both conditions. Thermophilic fermentation helped enhancing the hydrolysis of the substrate. Higher amounts of fermentable sugars were achieved and the efficiency of the process improved resulting with higher biohydrogen production potential. Mesophilic and thermophilic biomethane fermentation of some of these effluents resulted in 8.3 L and 5.7 L biomethane volume, 0.17 and 0.13 mL CH 4 / g VFA yield, and biomethane production rates of 503 and 150 mL CH 4 / g VFA day, respectively. Elevated temperature increased the hydrolysis yield rate, which was reflected in biohydrogen production. However, the biogas was lower when compared with the literature probably due to high organic acid concentrations causing substrate (organic acid) inhibition and causing a decrease in biomethane yield. The study showed that GMW was biologically hydrolyzed during fermentation, the sugar content of the organic waste was released to the fermentation media and they were further used for biofuel production. These results are promising and can be improved to eliminate cost-intensive pretreatment methods applied for the hydrolysis of lignocellulosic wastes. A process pathway of thermophilic biohydrogen and mesophilic biogas production can be used for sequential biohydrogen and biomethane production to achieve improved valuable end products such as biohythane. Declarations Acknowledgment: This study was financially supported by GKE Energy in Turkey. The experiments were performed in Bioprocess Laboratory of Dokuz Eylül University, Environmental Engineering Department. Part of this research was presented in World Hydrogen Energy Congress-26-30 June 2022, İstanbul, Turkey. Competing Interests and Funding: Serpil Özmıhçı and İlgi Karapınarhas served on advisory board and received research funding from GKE Energy. İlknur Hacıoğlu has received scholarship support from GKE Energy. Meltem Küs was an employee of GKE Energy. Author Contributions: Serpil Özmıhçı and İlgi Karapınar contributed to the study conception and design. Material preparation, data collection and analysis were performed by İlknur Hacıoğlu. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4713736","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":341402705,"identity":"45ca6989-fd2b-473f-a904-af508b811de0","order_by":0,"name":"Serpil Ozmihci","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYLCChAIQydjGwGBgw9gAYvMQ1GIA15IG1yKBXw9YCwMbEB8mrEW+vffZgwcGDHLy0w63PfhRcF62XyKB8cHbNoY68wYc5p85bm4AdJgx4+zEdsMeg9vGM2ckMBvObWOQkDmAQ4tEGpsEUEtis3RimzSDwe3EDTcS2KR5gVpwuUx+/jOIljaIlnOJ+28ksP/Gp4XhBhtESw9Ey4HEDRIJbMz4tBicSWMH+kXCWAKoRbLHINl4xpmHzZJzzklIzsAZYsfYHv6osJGTn53+TOLHHzvZ/vbkgx/elNnw44kYUHSgSIOjBm9MsuGTHAWjYBSMglHAwAAA6v5Ol58iyIUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4817-7916","institution":"Dokuz Eylul Universitesi Muhendislik Fakultesi","correspondingAuthor":true,"prefix":"","firstName":"Serpil","middleName":"","lastName":"Ozmihci","suffix":""},{"id":341402706,"identity":"3cd62b8b-1b93-4aba-9c7c-bc4064848875","order_by":1,"name":"İlknur Hacıoğlu","email":"","orcid":"","institution":"Dokuz Eylül University The Graduate School of Natural and Applied Sciences: Dokuz Eylul Universitesi Fen Bilimleri Enstitusu","correspondingAuthor":false,"prefix":"","firstName":"İlknur","middleName":"","lastName":"Hacıoğlu","suffix":""},{"id":341402707,"identity":"fd206288-fdfd-496e-9996-9237040d6a61","order_by":2,"name":"İlgi Karapınar","email":"","orcid":"","institution":"Dokuz Eylul University Faculty of Engineering: Dokuz Eylul Universitesi Muhendislik Fakultesi","correspondingAuthor":false,"prefix":"","firstName":"İlgi","middleName":"","lastName":"Karapınar","suffix":""},{"id":341402708,"identity":"f4b9c1a5-082f-4178-a7e1-779e19f7bffd","order_by":3,"name":"Meltem Küs","email":"","orcid":"","institution":"Dokuz Eylul University: Dokuz Eylul Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Meltem","middleName":"","lastName":"Küs","suffix":""}],"badges":[],"createdAt":"2024-07-09 17:23:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4713736/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4713736/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64632691,"identity":"b4cd8184-4edf-4e4f-ae89-1f24a08104fe","added_by":"auto","created_at":"2024-09-16 21:07:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":47899,"visible":true,"origin":"","legend":"\u003cp\u003eThe Variation of \u003cstrong\u003ea.\u003c/strong\u003e Cumulative Biohydrogen Gas Formations, Initial and Final Total Sugar Concentrations, \u003cstrong\u003eb.\u003c/strong\u003e pH, \u003cstrong\u003ec.\u003c/strong\u003e Organic Acid Types and Concentrations at the End of the Fermentation Time with GMW Amounts, Under Mesophilic Conditions.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4713736/v1/f1f568ec061c7c523430f889.jpg"},{"id":64632851,"identity":"10d8cbee-1a6d-401d-912f-7fdf041fd5d4","added_by":"auto","created_at":"2024-09-16 21:15:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31780,"visible":true,"origin":"","legend":"\u003cp\u003eBiohydrogen Production Rate and Yield at Different GMW Amounts Under Mesophilic Conditions; ■ Rate in mL H\u003csub\u003e2\u003c/sub\u003e/ L day, ■ Yield in mLH\u003csub\u003e2\u003c/sub\u003e /g dw\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4713736/v1/a3af55e935c96f06232310b7.jpg"},{"id":64632850,"identity":"ec9f3b08-c460-493c-a865-892590a61d66","added_by":"auto","created_at":"2024-09-16 21:15:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46989,"visible":true,"origin":"","legend":"\u003cp\u003eTime-course of \u003cstrong\u003ea.\u003c/strong\u003e Cumulative Biohydrogen Formation \u003cstrong\u003eb.\u003c/strong\u003e Total Sugar Concentration under Thermophilic and Mesophilic Conditions for Different GMW Amounts.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4713736/v1/2cd2457b22b2864ce8b858ab.jpg"},{"id":64632688,"identity":"2813403a-86f3-4836-840f-3171aadfd5c5","added_by":"auto","created_at":"2024-09-16 21:07:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":40113,"visible":true,"origin":"","legend":"\u003cp\u003eBiohydrogen Production Rates and Yields at Different GMW Amounts and Temperatures; ■ Rate in mL H\u003csub\u003e2\u003c/sub\u003e/L day, ■ Yield in mL H\u003csub\u003e2\u003c/sub\u003e /g dw\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4713736/v1/5e181b382fdd58e28943c6fc.jpg"},{"id":64632853,"identity":"511420b6-c07a-4149-a2d3-b74c78e7fd48","added_by":"auto","created_at":"2024-09-16 21:15:35","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":53080,"visible":true,"origin":"","legend":"\u003cp\u003eVariations of \u003cstrong\u003e(a)\u003c/strong\u003e Cumulative Biomethane Formation \u003cstrong\u003e(b)\u003c/strong\u003e Total Sugar Concentration \u003cstrong\u003e(c)\u003c/strong\u003e Total Organic Acid Concentration with Time under Mesophilic and Thermophilic Conditions at GMW 5000 g wb\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4713736/v1/4327726ddd94a2bb760adadc.jpg"},{"id":64632689,"identity":"7c6379a7-5f2d-4d2d-a83f-318e8d633141","added_by":"auto","created_at":"2024-09-16 21:07:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":24483,"visible":true,"origin":"","legend":"\u003cp\u003eBiomethane Production Rate and Yield at Mesophilic and Thermophilic Conditions;\u003c/p\u003e\n\u003cp\u003e■Biomethane Formation Rate, ■ Biomethane Formation Yield\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4713736/v1/9dc814277d7627b618602236.jpg"},{"id":65388364,"identity":"e2c403fe-b73e-4eaf-af1f-0818bc44d7c0","added_by":"auto","created_at":"2024-09-26 20:55:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":936338,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4713736/v1/331e233c-d19c-4216-ac3e-fa7befa9efa9.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eValorization of Green Market Waste as a Renewable Energy Source\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eLarge amounts of green market wastes can be valorized to biohydrogen.\u003c/li\u003e\n \u003cli\u003eThermophilic fermentation of GMW produced more biohydrogen than mesophilic one.\u003c/li\u003e\n \u003cli\u003eHigh organic acid contents obtained from biohydrogen fermentation can be valorized to biomethane.\u003c/li\u003e\n \u003cli\u003eMesophilic conditions resulted in higher biomethane volumes than thermophilic one.\u003c/li\u003e\n \u003cli\u003eSequential biohydrogen and biomethane processes provides complete waste valorization into biofuel.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eAs a renewable energy source, biomass energy stands out with various advantages. It is suitable for energy production at all scales. It is storable, and sustainable, can be generated from organic wastes, leads to less greenhouse effect compared to fossil fuels and helps socioeconomic development, especially, in rural areas [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. On the other hand, biomass achieved from energy crops is questioned for the fuel versus food debate regarding the use of agricultural areas for this purpose instead of human nutrition. Moreover, sustainability has emerged as a concern in the production of biofuels, even when it comes to non-food plant cultivation. Therefore, the production of biofuels from non-competitive biomass has been on the energy research agenda as a solution to fuel versus food debate [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is clear that biofuel production requires a large amount of organic matter to build an efficient process. As non-competitiveness is an issue, one of the readily available organic matter rich raw material sources, without occupying agricultural land, is domestic solid waste. Studies have shown that the organic fraction of domestic solid waste is about 50%, and 70% of this fraction is disposed of in landfills [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This substantial amounts of organic matters are already in use for biogas production with low energy input through anaerobic digestion (AD) employing waste-to-energy technology for food waste treatment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe approach to biofuel production has evolved towards a more holistic approach that considers new biomass energies in addition to biogas. In order to achieve this, it is essential to include other renewable, inexpensive, and abundant non-food resources that can be diverted to on-site biofuel production. The best candidates are agricultural residues and edible plant residues such as Green Market Wastes (GMW) which are rich in organic matter and can be processed directly without the need for pre-sorting, as is the case with domestic solid wastes. With these characteristics, they have a significant economic value and enormous potential to contribute to sustainable renewable energy such as biohydrogen, bioethanol, and biogas production [\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBioethanol and biomethane are the main and well-known biofuels produced from various organic materials [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Biohydrogen production researches and applications raised in the last decades which is known as a clean energy carrier and the technology for storage and transport is constantly developing [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18 CR19\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Biohydrogen from biomass by fermentation has some limitations such as the need for pre-treatment, low production rate and yield, and a residual organic acid fraction that requires further processing either for valorization or treatment before discharge [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18 CR19\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the last decade, biohythane production gained attention for utilizing waste biomass. The main aim of these studies was biohythane (a blend of 10 to 30% biohydrogen (v/v) and 70 to 90% of biomethane (v/v)) production including sequential dark fermentation (DF, biohydrogen) and anaerobic digestion (AD, biomethane) processes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Studies on relatively new technologies point to sequential hydrogen and methane production as a respectable solution to the listed problems encountered in single-stage biohydrogen or biomethane production [\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The presence of 10\u0026ndash;30% biohydrogen in the biomethane mixture results in enhanced thermal efficiency and reduced greenhouse gas emissions to achieve carbon neutrality for global warming effects [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore; this approach helps with biohydrogen transport and storage issues. It is particularly useful for on-site processes where the hydrogen can be used directly without storage. Additionally, the organic acids released from biohydrogen fermentation are not wasted or discharged, but are used for biomethane production, providing a completely carbon neutral process.\u003c/p\u003e \u003cp\u003eIn this study; partially hydrolyzed GMW, as a lignocellulosic organic waste, was valorized in a sequential DF and AD process for biohythane production. Among the biological hydrogen production methods, DF is the most stable and efficient one and some of the important parameters are pH, temperature, gas partial pressure, Oxidation Reduction Potential (ORP), nutrients, the organic acid types produced, substrate and biomass concentration, and microorganism culture [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Many studies focused on hydrogen gas production from lignocellulosic waste biomass by DF with different operational modes and temperature [\u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Substrates such as sweetsorghum, sugar cane pulp, animal manure, waste paper towels, domastic wastes etc. were valorized giving hydrogen production yields between 0.927\u0026ndash;2.55 mol H\u003csub\u003e2\u003c/sub\u003e /mol glucose in the literature [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The hydrogen production potential from lignocellulosic wastes depend on the pretreatment applied, the type of biomass, and the microbial culture that enhance biohydrogen production yields and rates [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Physical, chemical, or enzymatic pre-treatments are needed before fermentation which unfortunately, increase the overall production cost [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Therefore, partial hydrolysis such as thermal treatment in moderate temperatures or consolidated fermentation could be key answers for reducing the operating cost [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Fermentation temperature positively affects the process such as thermophilic conditions help to eliminate hydrogen-consuming bacteria and efficient conversion of simple sugars to hydrogen [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] which may provide higher biohydrogen production yields and productivities [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnaerobic digestion (AD) as the second step in our process is a well-known process, where agricultural wastes, some industrial wastes, domestic solid wastes, etc. can be valorized for biomethane production [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The success of methane gas production in the composition of the produced biogas depends on fermentation temperature, type and amount of raw material, alkalinity (pH), substrate particle size, fermentation time, carbon to nitrogen ratio (C/N), type of bioprocess and amount of dry matter [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Different C/N ratios contained in wastes, such as wheat straw (C/N: 87/1), corn straw (C/N: 53/1), animal feces (C/N: 29/1) need careful operations by maintaining the ideal ratio for biogas production in the mixed feedstock [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Therefore; preferring hydrogen production before methane formation can alter the need of specific conditions for biomethane production. Besides, the use of the effluent of DF, with rich organic acid content, for AD will shorten the fermentation time, decrease the reactor volume. One of the questions is the fermentation and digestion temperature. Dong et al, [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] revealed that thermophilic (50-65\u003csup\u003eo\u003c/sup\u003eC) conditions gave higher biohydrogen and biomethane yields when compared with mesophilic ones. In different studies ranges of 17\u0026ndash;292 mL H\u003csub\u003e2\u003c/sub\u003e/ g VS and 150\u0026ndash;570 mL CH\u003csub\u003e4\u003c/sub\u003e/g VS gases were achieved [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGreen market wastes (GMW), which are produced in large quantities in bazaars and fruit and vegetable markets, can be utilized as raw material in energy sector, such as biohydrogen and biomethane, offering certain advantages in terms of producing high value-added products at low cost. By considering these facts, the aim of this study is to valorize the green market waste (GMW) by sequential biohydrogen (DF) and biomethane (AD) production under different temperatures using mixed culture. For this purpose, biohydrogen production potential at mesophilic conditions (37\u003csup\u003eo\u003c/sup\u003eC) at different initial substrate concentrations varying between 1000\u0026ndash;5000 g wet basis (wb) was evaluated and then, the selected substrate concentrations were fermented under thermophilic conditions to compare the effects of temperature on biohydrogen production. Finally, the organic acid-rich effluents of biohydrogen production was subjected to methanogenic organisms to evaluate the biomethane production potentials from these effluents. The novelty of the study is to modify the consolidated fermentation process by adding a simple pretreatment and then achieving an efficient biohydrogen-biomethane production from the solid-liquid mixture instead of using pretreatment supernatant in a conventional manner.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubstrate and Pre-treatment of the Substrate\u003c/h2\u003e \u003cp\u003eGreen Market Wastes (GMW) were consisted of lettuce, parsley, dill, arugula, and spring onions. The wastes were collected from the markets in the province of İzmir/ Turkey. GMW was first shredded to reduce the particle size and then, blended to get a homogeneous feedstock. A viscous liquid-like mixture was obtained from the wet GMW containing 94% moisture. Thermal pre-treatment (partial treatment) was applied to the mixture at 100\u003csup\u003eo\u003c/sup\u003eC for 1 hour. The chemical composition of 1 kg of GMW mixture is given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe organic carbon content of the raw GMW, without grinding or blending, was 439.4 g/ kg dw (dry weight). After grinding and blending processes partial liquefaction occurred. The TOC concentrations in the dried solid and liquid phases of the mixture were 491 g/ kg dw and 10.8 g/L, respectively. TOC concentration increased to 11.1 g/ L after thermal treatment in the liquid phase.\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\u003eCharacterization of Green Market Wastes after Blending (1 kg)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Nitrogen (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2940\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Phosphorus (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e298.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e-N (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e190.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e-N (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e808.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTOC in the liquid phase (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTOC in the solid phase (g/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e491\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Sugar in the liquid phase (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolid Content\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% 6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMicroorganisms\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eBiohydrogen-Producing Microorganisms\u003c/h2\u003e \u003cp\u003ePretreated anaerobic sludge (thermally treated) was used as bacterial inoculum in biohydrogen production. Anaerobic sludge was obtained from the acidogenic phase of the treatment plant of Pakmaya Industry, İzmir, Turkey. The sludge was thermally treated at pH 5 and 100\u003csup\u003eo\u003c/sup\u003eC for 1 hour under a fume hood, to eliminate methanogenic bacteria in the anaerobic sludge consortium. The pretreated anaerobic sludge was then cultivated at 37\u003csup\u003eo\u003c/sup\u003eC and 55\u003csup\u003eo\u003c/sup\u003eC to activate biohydrogen-producing \u003cem\u003eClostridium\u003c/em\u003e species. The growth media was composed of 10 g/ L peptone, 10 g/ L glucose, 3.9 g/ L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2.8 g/ L K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.25 g/ L MgSO\u003csub\u003e4\u003c/sub\u003e, 0.6 g/ L yeast extract, and 0.1 g/ L L-cysteine. Anaerobic conditions were accomplished by sparging nitrogen gas through the fermentation reactors for 10 minutes. The initial pH was adjusted to pH 7.2. Organisms that reached to the exponential growth phase in 48 hours were used as inoculum in biohydrogen production experiments.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBiomethane-Producing Microorganisms\u003c/h2\u003e \u003cp\u003eAnaerobic sludge was obtained from the methanogenic reactor of the wastewater treatment plant of Pakmaya Industry, İzmir, Turkey. The anaerobic sludge was cultivated in nutrient-rich media for 48 hours and then used as inoculum for biomethane production experiments. The growth media consisted of 0.6 g/ L yeast extract, 10 g/ L peptone, 10 g/ L glucose, 3.9 g/ L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2.8 g/ L K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 0.25 g/ L MgSO\u003csub\u003e4\u003c/sub\u003e, and 0.1 g/ L L-cysteine. Anaerobic conditions were provided by passing N\u003csub\u003e2\u003c/sub\u003e gas through the headspace of the reactors. The pH of the fermentation media was adjusted to pH 7.5 when required. The fermentation temperatures were 37\u003csup\u003eo\u003c/sup\u003eC and 55\u003csup\u003eo\u003c/sup\u003eC depending on the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Procedure\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eBiohydrogen Production\u003c/h2\u003e \u003cp\u003eThe experiments were carried out at 37 \u003csup\u003eo\u003c/sup\u003eC and 55\u003csup\u003eo\u003c/sup\u003eC in a temperature-controlled incubator over a roller shaker with 20 rpm mixing speed. The substrate concentrations were 1000, 1200, 2500, and 5000 g under mesophilic condition and 1000 and 5000 g under thermophilic one. 1 kg of wet GMW resulted in approximately 1 L of mixed solid-liquid volume after blending. Therefore, the initial reaction volume varied depending on the amount of wet GMW used (reactor volumes varied between 2\u0026ndash;10 L). The initial amount of microorganisms was 10% of the reactor volume. Silicone stoppers and caps were used to prevent gas leakage from the bottles. The initial pH was set to 7.2 at the beginning and adjusted to around 6.5 when needed with dilute NaOH solution. Anaerobic conditions were preserved by transferring nitrogen gas from the headspace of the bottles for 20\u0026ndash;40 min. Maintaining anaerobic conditions was checked with negative (\u0026lt;-250 mV) oxidation-reduction potential (ORP). The pH, total sugar, organic acid, biohydrogen percentage, and total gas volume were monitored in daily samples.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBiomethane Production\u003c/h2\u003e \u003cp\u003eFor biogas production, the 1000 g and 5000 g GMW effluents were used, in which biohydrogen was produced. The methanogenic culture was directly inoculated to reactors after biohydrogen production ceased. The fermentation was conducted at 37\u003csup\u003eo\u003c/sup\u003eC and 55\u0026deg;C. Experimental studies were terminated when the cumulative biomethane production volume reached a constant value. Experiments were carried out on rotating rollers, placed in an incubator, at 20 rpm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnalytic Methods\u003c/h2\u003e \u003cp\u003eSamples taken daily (10 mL) were centrifuged at 8000 rpm for 15 minutes and the solid phase was separated. Total sugar, acetic, butyric, propionic, and lactic acids concentrations were determined in the liquid phase. Total sugar concentration was determined by the Dubois method [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Organic acid concentrations were measured by HPLC (Agilent 1100) with a UV detector (220 nm) on Aminex HPX-87H column (9 \u0026micro;m x 300 x 7.8 mm), using 5mM sulfuric acid mobile phase with a flow rate of 0.6 mL/ min [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiohydrogen and biomethane gas samples were taken daily from the headspace of the experimental bottles with a gas-tight glass syringe. The concentrations of the gases were determined by using a gas chromatograph (Agilent 6890 N-GC) and the column was Alltech, Hayesep D 80/100 6 in. x 1/8 in. x 0.85 in. Nitrogen gas was used as a carrier gas with a flow rate of 30 mL/ min. The temperatures of the oven, injection, detector, and filament were 35\u0026deg;C, 120\u0026deg;C, 120\u0026deg;C, and 140\u0026deg;C, respectively. The volume of total gas produced was measured by the water displacement method using a solution containing 2% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and 10% NaCl. Triplicate analysis of the samples showed a standard deviation lower than 5% [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome parameters were used in characterization of the substrate. Total organic carbon concentration was carried out in Schimadzu TOC analyzer (SM 5310 B). Total Nitrogen (ASL.SCP.087.Rev.00), Total Phosphorus (SM 4500-P B SM 4500-P D), NO\u003csub\u003e3\u003c/sub\u003e-N, NH\u003csub\u003e4\u003c/sub\u003e-N concentrations were conducted according to APHA standard methods [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe yield, cumulative gas volume, and rate calculations were adapted from Ozmihci and Kargi [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and Conde and Kaparaju [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiohydrogen Production under Mesophilic Conditions\u003c/h2\u003e \u003cp\u003eDifferent GMW substrate concentrations (1000, 1200, 2500, 5000 g wb) were tested under mesophilic batch dark fermentation. Figure\u0026nbsp;1a depicts the maximum cumulative hydrogen volumes and liquid phase initial and final total sugar concentrations in fermentation media at different substrate concentrations. Cumulative biohydrogen production increased up to around 1.1 L and 1.3 L for 1000 and 1200 g wb substrate amounts. The volume substantially increased to 5 L with 2500 g wb GMW and then reached the maximum volume of 9 L at 5000 g wb substrate. The total sugar in the fermentation media was measured in the liquid phase. Its concentration was nearly 1% of the GMW at 1000 and 1200 g wb GMW, while it was only around 0.53% for 2500 and 5000 g wb experiments. That solubilized total sugar concentration was sufficiently high to support the growth of the organism and to initiate biohydrogen production. However, there was a substantial variation in total sugar concentration in the liquid phase due to the hydrolysis of the solid phase to simple sugars during fermentation. The sugar consumption was fast for the first 24\u0026ndash;48 hours of fermentation time at 1000 and 1200 g wb GMW (data not shown) resulting in a rapid biohydrogen generation phase. However, there was a delay in the production due to the required adaptation period of the microorganism at higher GMW amounts (data not shown). The final sugar concentrations in the reactors at the liquid phase were around 1\u0026ndash;3 g/ L and the sugar consumption efficiency in the liquid phase was nearly constant around 91\u0026thinsp;\u0026plusmn;\u0026thinsp;3%. The results indicated that both liquid and solid-phase sugars were used for biohydrogen production.\u003c/p\u003e \u003cp\u003eThe microbial processes can be employed to reduce costs and time. One such process is the consolidated bioprocess (CBP). In the production of biofuels, separate or simultaneous saccharification and fermentation are typically employed. However, CBP offers a distinct approach, utilizing a microbial consortium capable of producing both cellulolytic enzymes and hydrogen in the dark fermentation process. \u003cem\u003eClostridia\u003c/em\u003e species are the predominant microorganisms responsible for this function, and mixed cultures in which different species can coexist have been demonstrated to be successful in CBP [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In our study, this phenomenon also existed where biohydrogen production was accomplished through consolidated fermentation (CBP) where the microorganisms hydrolyzed GMW into simple sugars, which was then used for biohydrogen production [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe pH of the fermentation media is a good indicator of the organic acid production in dark fermentation. As seen in Fig.\u0026nbsp;1b, the variation in pH for all GMW amounts used showed that the 48 hours were critical for rich organic acid formation. pH decreased from 7.2 to around 5.1\u0026ndash;5.4 within the first 48 hours for all tested GMW amounts. Later, pH values reached a steady state for 1000 and 1200 g wb GMW indicating that organic acid production was terminated. This result was in parallel with biohydrogen production and sugar consumption which were at high rates for the first 48 h of fermentation. However, the pH fluctuation for 2500 and 5000 g wb occurred during the fermentation period. The fluctuation in pH may be attributed to the gradual release of sugar from the solid phase through biological hydrolysis, which is then converted into organic acids.\u003c/p\u003e \u003cp\u003eFigure 1c depicts the organic acid concentrations produced at the end of the fermentation period at different substrate amounts. The main organic acid types were acetic acid and butyric acids with low concentrations of propionic and lactic acid at high GMW amounts. The acetic acid concentration reached to 19.7 g/ L for 1200 g wb substrate and to around 16.55 g/L for 1000 g wb. The butyric acid concentrations were low in these substrate amounts (around 2 g/ L). On the other hand, biohydrogen-generating organic acids were almost equally produced at 2500 g wb and 5000 g wb GMW resulting in around 15 g/L total organic acid concentrations. The lower organic acid amounts in higher substrate concentrations may be due to slower simultaneous hydrolysis and fermentations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e(a) (b)\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e(c)\u003c/h2\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;1.\u003c/b\u003e The Variation of \u003cb\u003ea.\u003c/b\u003e Cumulative Biohydrogen Gas Formations, Initial and Final Total Sugar Concentrations, \u003cb\u003eb.\u003c/b\u003e pH, \u003cb\u003ec.\u003c/b\u003e Organic Acid Types and Concentrations at the End of the Fermentation Time with GMW Amounts, Under Mesophilic Conditions.\u003c/p\u003e \u003cp\u003eBiohydrogen production yields and rates with different GMW amounts are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The biohydrogen production yield and formation rate increased with increasing GMW amount up to 2500 g wb in the fermentation media. The maximum yield and rate were 35 mL/ g dw and 413 mL H\u003csub\u003e2\u003c/sub\u003e/ L day. The yield was determined based on the dry organic matter of GMW consumed during fermentation. It is clear that although maximum cumulative biohydrogen formation (CHF, Fig.\u0026nbsp;1) was obtained at 5000 g GMW, the reaction was slow mainly due to the limitations in the simultaneous microbial hydrolysis of the GMW during fermentation and the long adaptation period as mentioned above. The biohydrogen production yield at 5000 g wb GMW was the lowest compared to the other initial GMWs. In other words, the microbial hydrolysis of the GWM during fermentation was not sufficient under mesophilic fermentation temperature. Thermophilic fermentation could help enhancing the hydrolysis of the substrate, more fermentable sugars can be generated and the efficiency of the process can be improved.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eComparison of biohydrogen production under mesophilic and thermophilic conditions\u003c/h2\u003e \u003cp\u003eThe effect of thermophilic fermentation on substrate utilization, hydrolysis of GMW, and biohydrogen production was investigated. The experiments were conducted at the lowest (1000 g wb) and highest (5000 g wb) GMW tested for biohydrogen production. The results were given as comparison of mesophilic and thermophilic conditions to understand the effects of temperature. As seen in Fig.\u0026nbsp;3a, the biohydrogen production terminated after 48 h of the fermentation period at 1000 g wb GMW under mesophilic and thermophilic conditions resulting in 1.96 L and 2.2 L CHF, respectively. The biohydrogen production at 5000 g GMW was substantially higher compared to 1000 g GMW. Thermophilic conditions enhanced the biohydrogen production which continued along the fermentation period. The maximum cumulative volumes reached to 14.2 L under thermophilic condition and to 8.8 L for mesophilic one. Thermophilic fermentation resulted in almost 1.61 times more biohydrogen volume at 5000 g wb GMW.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e(a) (b)\u003c/h2\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;3.\u003c/b\u003e Time-course of \u003cb\u003ea.\u003c/b\u003e Cumulative Biohydrogen Formation \u003cb\u003eb.\u003c/b\u003e Total Sugar Concentration under Thermophilic and Mesophilic Conditions for Different GMW Amounts.\u003c/p\u003e \u003cp\u003eFigure 3b shows variation of the total sugar concentrations with time at different substrate concentrations and fermentation temperatures. The total sugar that was measured from the liquid phase of the fermentation time shows the available sugar to be converted into end products. The initial total sugar concentration in the liquid phase depended on the efficiency of partial hydrolysis. The soluble sugars increased from time to time in the fermentation media by biological hydrolysis of solid GMW particles in the media. This was observed clearly at 5000 g wb GMW tested in mesophilic conditions. Total soluble sugar in the fermentation media steadily decreased untill 192 hours and then, a sharp increase in the sugar concentration to 7.8 g/L occurred indicating that hydrolysis of the substrate took place. At the end of the fermentation period, the total sugar concentration was 2.6 g/L. The fluctuation of total sugar is due to consolidated bioprocessing where simultaneous hydrolysis and fermentation took place. The pH in the fermentation media decreased to around 5-5.5 within 24 hours due to rapid sugar consumption for all fermentation conditions, then they were nearly constant at around 6-6.5 until the end of fermentation period (data not presented). The total organic acids in mesophilic conditions consisted of acetic and butyric acid at 1000 g wb and 5000 g wb GMW were 18.53 g/ L and 15.249 g/L, respectively. In thermophilic conditions, the main organic acid was acetic acid with a concentration of 19.2 g/L in 1000 g wb GMW and 14.4 g/L in 5000 g wb GMW (data not presented). There was not a substantial increase in butyric acid production under thermophilic conditions. It is being understood from the organic acids that temperature rises also changes the end products due to dominated acid type of fermentation. The same trend, as in different substrate concentration trials at mesophilic conditions, was seen with 1000 g and 5000 g wb GMW.\u003c/p\u003e \u003cp\u003eThe biohydrogen production yields and rates for the mesophilic and thermophilic conditions with 1000 g wb and 5000 g wb GMW are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The yields increased with increasing substrates and temperatures. The highest yield was obtained as 47.5 mL H\u003csub\u003e2\u003c/sub\u003e/ g dw with 5000 g wb substrate under thermophilic conditions, but the maximum rate was 916 mL H\u003csub\u003e2\u003c/sub\u003e/ L day at mesophilic conditions. High GMW concentrations such as 5000 g wb is suitable to use under thermophilic conditions to get high volumes and yields of biohydrogen production [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBiomethane Production under Different Fermentation Temperatures\u003c/h2\u003e \u003cp\u003eConverting organic acid-rich effluent of biohydrogen-producing reactor to biomethane as a secondary product provides an economic advantage in both waste management and energy generation. Therefore, there is a need to develop methods that will enable the direct use of the biohydrogen produced. In the two-stage split system, differences in operating and production conditions, rate-limiting biological reactions, and inhibitions are more easily controlled. As previously mentioned there is no need for specific operation conditions that biogas systems need. Thus, an increase in reaction rates and productivity and a reduction in retention time can be achieved [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe effluents of 5000 g wb GMW obtained from thermophilic fermentation conditions with high organic acids content were subjected to mesophilic and thermophilic biomethane formation. Figure\u0026nbsp;5a shows the volumes of biomethane formation under mesophilic and thermophilic conditions. The cumulative biomethane volumes were 8.3 L and 5.8 L under mesophilic and thermophilic conditions, respectively. The biogas was composed of 55% of CH\u003csub\u003e4\u003c/sub\u003e, 1% of H\u003csub\u003e2\u003c/sub\u003e, and 44% CO\u003csub\u003e2\u003c/sub\u003e under mesophilic condition. The percentage of biohydrogen in total gas slightly increased to 3% for thermophilic condition, but biomethane percentage was the same as mesophilic one.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e(a)\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e(b) (c)\u003c/h2\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5.\u003c/b\u003e Variations of \u003cb\u003e(a)\u003c/b\u003e Cumulative Biomethane Formation \u003cb\u003e(b)\u003c/b\u003e Total Sugar Concentration \u003cb\u003e(c)\u003c/b\u003e Total Organic Acid Concentration with Time under Mesophilic and Thermophilic Conditions at GMW 5000 g wb\u003c/p\u003e \u003cp\u003eFigure 5b-c depicts variation of total sugar and total organic acid concentrations with time, respectively. The fermentable sugar concentration in the effluent of mesophilic biohydrogen fermentation was already limited between 750\u0026ndash;850 mg/ L. The sugar consumption profile under mesophilic methanogenesis was straight but it was varying under thermophilic one. The reason for the variation could be the progress of organic fraction hydrolysis in GMW to sugar monomers under the methanogenic phase (Fig.\u0026nbsp;5b). Similarly, total volatile fatty acid (TVFA) consumption varies in parallel to that of sugar concentrations. While TVFA was consumed for biomethane generation, the hydrolyzed sugars were converted into organic acid, which was then used for biomethane formation. This continuing hydrolysis and TVFA formation processes extend the biomethane production period with the increase in formation volume (Fig.\u0026nbsp;5c).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003e depicts the biomethane production yields and rates under mesophilic and thermophilic conditions. The biomethane formation yield (0.17 mL CH\u003csub\u003e4\u003c/sub\u003e/ g TVFA) and rate (502 mL CH\u003csub\u003e4\u003c/sub\u003e/ g TVFA day) were higher in mesophilic conditions than in thermophilic conditions (0.13 mL CH\u003csub\u003e4\u003c/sub\u003e/ g TVFA and 150 mL CH\u003csub\u003e4\u003c/sub\u003e/ g TVFA day). The main reason for the slow rate at thermophilic fermentation is due to simultaneous hydrolysis, TVFA production, and biomethane generation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e■Biomethane Formation Rate, ■ Biomethane Formation Yield\u003c/p\u003e \u003cp\u003eThe study showed that GMW is a suitable substrate for sequential biohydrogen and biomethane production. There are limited thermophilic sequential biohydrogen and biogas production studies in the literature. The one reported by Sillero et al [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] was a thermophilic, two-stage anaerobic co-digestion system (55-70\u003csup\u003eo\u003c/sup\u003eC). A mixture of sewage sludge and wine vinasse with a co-substrate of poultry manure was used. The results indicated that the addition of poultry manure to the media in thermophilic temperature (55\u0026deg;C) enhanced biohydrogen generation, yielding 27.1 mL H\u003csub\u003e2\u003c/sub\u003e/g VS and 59 mL CH\u003csub\u003e4\u003c/sub\u003e/ g VS. They concluded that hyperthermophilic temperatures also favor CH\u003csub\u003e4\u003c/sub\u003e production [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Lovato et al [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] tried mesophilic (34\u003csup\u003eo\u003c/sup\u003eC) and thermophilic (55\u003csup\u003eo\u003c/sup\u003eC) conditions with food waste. They found higher biohydrogen yields in mesophilic and higher biomethane yields in thermophilic conditions, which was the opposite what we found. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the sequential biohydrogen and biomethane production from different organic waste materials under different environmental conditions. In our study, it is clear that thermophilic conditions improved biohydrogen production. Elevated temperature increased the hydrolysis rate, which was reflected in biohydrogen production (CHF: 14.2 L). The rate when compared with mesophilic conditions was low due to the time spend in hydrolysis stage of the substrate. However, the biohydrogen was lower when compared with the literature probably due to high organic carbon content of the substrate that was valorized under consolidated fermentation conditions which needed time. The biohydrogen and biomethane production rate was comparable in both conditions with the literature except the ones using continuous fermentation mode [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The results showed that respectable amounts of biohydrogen and biomethane, which can be blended to biohythane, could be produced with GMW. Most of the sequential processes were carried out at mesophilic conditions as given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Our results are comparable with these studies. Pretreatment methods, side products such as furfurals and hydroxymethyl furfurals-5 from acid treatment of biomass and the type of sugars in the soluble form affect the biohydrogen and organic acid formation. In our study, only thermal treatment was applied for partial hydrolysis allowing organisms to reach the substrate during initial phase of consolidated fermentation. It was shown that efficient biofuel production under mesophilic conditions can be achieved with basic heat thermal treatment application and consolidated fermentation. It should be taken into consideration that consolidated fermentation processes are slower, while simultaneous hydrolysis and fermentation occurs [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. However at the end the cumulative biohydrogen and biomethane gas formations were comparable with the literature. The sequential biohydrogen and biogas production under mesophilic conditions enhanced biogas production having a high biomethane yield when compared with some studies in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Moreover, from the studies it can be concluded that sequential biohydrogen and biomethane fermentation with GMW can be adapted to continuous fermentations with high gas production potentials.\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\u003eSummary of sequential biohydrogen and biomethane production results from waste materials under different fermentation conditions.\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\u003eSubstrate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOrganism\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiohydrogen Production Rate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBiomethane Production Rate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFood Waste\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMixed culture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34\u003csup\u003eo\u003c/sup\u003eC, H\u003csub\u003e2\u003c/sub\u003e:pH 5.5, CH\u003csub\u003e4\u003c/sub\u003e: pH 7\u003c/p\u003e \u003cp\u003e55\u0026deg;C, H\u003csub\u003e2\u003c/sub\u003e:pH 5.5, CH\u003csub\u003e4\u003c/sub\u003e: pH 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.17 mL H\u003csub\u003e2\u003c/sub\u003e/ h\u003c/p\u003e \u003cp\u003e0.42 mL H\u003csub\u003e2\u003c/sub\u003e/L h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.28 mL CH\u003csub\u003e4\u003c/sub\u003e/ d\u003c/p\u003e \u003cp\u003e63.6 mL CH\u003csub\u003e4\u003c/sub\u003e/ d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSugarcane straw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMixed culture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epH 5.50, 35\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.7 mLH\u003csub\u003e2\u003c/sub\u003e/.h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.33 mL CH\u003csub\u003e4\u003c/sub\u003e/ d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVegatable waste (Onion)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMixed culture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epH 7 and 35\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.52 mL H\u003csub\u003e2\u003c/sub\u003e/h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e66.67 mL CH\u003csub\u003e4\u003c/sub\u003e/ d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMolasses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDominated by H\u003csub\u003e2\u003c/sub\u003e:\u003cem\u003eClostridium butyricum\u003c/em\u003e; CH\u003csub\u003e4\u003c/sub\u003e: \u003cem\u003eMethanobacterium beijingense, Methanothrix soehngenii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003e:pH 5.5, 35\u003csup\u003eo\u003c/sup\u003eC; HRT: 6h\u003c/p\u003e \u003cp\u003eCH\u003csub\u003e4\u003c/sub\u003e: pH 7, 35\u003csup\u003eo\u003c/sup\u003eC; HRT: 6d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e291.67 mL H\u003csub\u003e2\u003c/sub\u003e/ h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3700 mL CH\u003csub\u003e4\u003c/sub\u003e/ d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRice Straw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMixed culture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u0026deg;C, (Mixture: 1.7% H\u003csub\u003e2\u003c/sub\u003e, 58.7% CH\u003csub\u003e4\u003c/sub\u003e), continous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1062.55 mLH\u003csub\u003e2\u003c/sub\u003e/ h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1528.72 mL CH\u003csub\u003e4\u003c/sub\u003e/ d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreen market waste\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMixed culture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37\u003csup\u003eo\u003c/sup\u003eC, pH 6.5\u003c/p\u003e \u003cp\u003e55\u003csup\u003eo\u003c/sup\u003eC, pH 6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.85 mL H\u003csub\u003e2\u003c/sub\u003e/ h\u003c/p\u003e \u003cp\u003e37.6 mL H\u003csub\u003e2\u003c/sub\u003e/ h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e488.86 mL CH\u003csub\u003e4\u003c/sub\u003e/ d\u003c/p\u003e \u003cp\u003e260.1 mL CH\u003csub\u003e4\u003c/sub\u003e/ d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe sequential biohydrogen and biomethane generation of GMW by dark batch fermentation was evaluated under different conditions. High substrate concentrations were suitable for biohydrogen production under mesophilic conditions. 5000 g wb GMW produced 8.9 L CHF and 916 mL H\u003csub\u003e2\u003c/sub\u003e /L day. Thermophilic conditions favored biohydrogen production (CHF\u0026thinsp;=\u0026thinsp;14.2 L, Y\u003csub\u003eP/S\u003c/sub\u003e: 47.5 mL H\u003csub\u003e2\u003c/sub\u003e/ g dw), enhanced conversion of sugars into VFA's, and resulted in 1.6 times higher yield than that of mesophilic conditions at GMW\u0026thinsp;=\u0026thinsp;5000 g wb, but a lower rate of formation was obtained compared to mesophilic fermentation. The effluent of biohydrogen fermentation was rich in organic acid with TVFA (total volatile fatty acid) concentrations between 14\u0026ndash;20 g/L in both conditions. Thermophilic fermentation helped enhancing the hydrolysis of the substrate. Higher amounts of fermentable sugars were achieved and the efficiency of the process improved resulting with higher biohydrogen production potential.\u003c/p\u003e \u003cp\u003eMesophilic and thermophilic biomethane fermentation of some of these effluents resulted in 8.3 L and 5.7 L biomethane volume, 0.17 and 0.13 mL CH\u003csub\u003e4\u003c/sub\u003e/ g VFA yield, and biomethane production rates of 503 and 150 mL CH\u003csub\u003e4\u003c/sub\u003e/ g VFA day, respectively. Elevated temperature increased the hydrolysis yield rate, which was reflected in biohydrogen production. However, the biogas was lower when compared with the literature probably due to high organic acid concentrations causing substrate (organic acid) inhibition and causing a decrease in biomethane yield. The study showed that GMW was biologically hydrolyzed during fermentation, the sugar content of the organic waste was released to the fermentation media and they were further used for biofuel production. These results are promising and can be improved to eliminate cost-intensive pretreatment methods applied for the hydrolysis of lignocellulosic wastes. A process pathway of thermophilic biohydrogen and mesophilic biogas production can be used for sequential biohydrogen and biomethane production to achieve improved valuable end products such as biohythane.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u0026nbsp;\u003c/strong\u003eThis study was financially supported by GKE Energy in Turkey. The experiments were performed in Bioprocess Laboratory of Dokuz Eyl\u0026uuml;l University, Environmental Engineering Department. Part of this research was presented in World Hydrogen Energy Congress-26-30 June 2022, İstanbul, Turkey.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests and Funding:\u0026nbsp;\u003c/strong\u003eSerpil \u0026Ouml;zmıh\u0026ccedil;ı and İlgi Karapınarhas served on advisory board and received research funding from GKE Energy. İlknur Hacıoğlu has received scholarship support from GKE Energy. Meltem K\u0026uuml;s was an employee of GKE Energy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eSerpil \u0026Ouml;zmıh\u0026ccedil;ı and İlgi Karapınar contributed to the study conception and design. Material preparation, data collection and analysis were performed by İlknur Hacıoğlu. Meltem K\u0026uuml;s was responsible for the financial side of the study. The manuscript was written by Serpil \u0026Ouml;zmıh\u0026ccedil;ı and İlgi Karapınar.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eAll experimental data are contained within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAntonopoulou, G., Gavala, H.N., Skiadas, I.V., Angelopoulos, K., Lyberatos, G.: Biofuels Generation from Sweet Sorghum: Fermentative Hydrogen Production and Anaerobic of the Remaining Biomass. Bioresour. 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Energy. \u003cb\u003e44\u003c/b\u003e, 18785\u0026ndash;18791 (2019)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biohydrogen, Biomethane, Biohythane, Green Market Waste, Thermophilic, Mesophilic","lastPublishedDoi":"10.21203/rs.3.rs-4713736/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4713736/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHigh organic carbon wastes can be used for biofuel production for reducing raw material costs. Green market wastes (GMW) are one of the raw material candidates to generate biofuel by fermentation with low-cost production and high yield of fuel formation. Biohydrogen and biomethane are potential gaseous energy sources that can be obtained through sequential fermentation of GMW. In this study, the ground and hydrolyzed GWM (a mixture of lettuce, parsley, spring onion, and dill) solid-liquid mixture was first valorized for biohydrogen, then the organic acid-rich residue of biohydrogen fermentation was subjected to biomethane production. Biohydrogen and biomethane was produced at mesophilic (37°C) and thermophilic (55°C) temperatures. The initial GWM amount varied between 1000-5000 g wb at mesophilic biohydrogen production. The highest cumulative hydrogen formation (CHF) and the rate were obtained at 5000 g wb as CHF=8.9 L and 916 mL H\u003csub\u003e2\u003c/sub\u003e /L day, respectively. Thermophilic biohydrogen fermentation was conducted at 1000 and 5000 g wb and CHF reached to 14.2 L at 5000 g wb GMW.\u0026nbsp; The yield of hydrogen formation was 1.6 times higher at thermophilic than that of mesophilic conditions. The residues from two fermentation temperatures of 5000 g wb GMW were used to produce biomethane under both fermentation conditions. Mesophilic and thermophilic conditions resulted in cumulative biomethane formations (CMFs) of 8.3 L and 5.8 L, and biomethane production yields of 0.17 mL CH\u003csub\u003e4\u003c/sub\u003e/g VFA and 0.13 mL CH\u003csub\u003e4\u003c/sub\u003e/g VFA, respectively. The findings revealed that GMW is a suitable substrate for efficient biohydrogen and biomethane production.\u003c/p\u003e","manuscriptTitle":"Valorization of Green Market Waste as a Renewable Energy Source","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-16 21:07:30","doi":"10.21203/rs.3.rs-4713736/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"593036b9-4257-431e-9506-eb34586704a2","owner":[],"postedDate":"September 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-26T20:47:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-16 21:07:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4713736","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4713736","identity":"rs-4713736","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}