Dark Fermentation and Anaerobic Digestion for H₂ and CH₄ Production, from Food Waste Leachates

Abstract

The present study investigates a two-stage process aimed at producing biogas from food waste leachates (FWL) through an experimental approach. The first stage involves biohydrogen production via dark fermentation (DF), while the second focuses on biomethane production through anaerobic digestion (AD). The substrate consists of leachates derived from fruit and vegetable waste, which are introduced into two continuous stirred-tank reactors (CSTR1) with two different inoculum-to-substrate ratios (ISR). Dark fermentation occurs in these reactors. The effluent from the CSTRs is then fed into two additional reactors for methanogenesis. All reactors operated under mesophilic conditions. During the DF stage, hydrogen yields were relatively low, with a maximum of 8.2 NmL H₂/g VS added (ISR = 0.3) and 6.1 NmL H₂/g VS added (ISR = 0.5). These results were attributed to limited biodegradation of volatile solids (VS), which reached only 21.9% and 23.6% in each respective assay. Similarly, the removal of organic matter was modest. In contrast, the AD stage demonstrated more robust methane production, achieving yields of 275.2 NmL CH₄/g VS added (ISR = 0.3) and 277.5 NmL CH₄/g VS added (ISR = 0.5). The system exhibited significant organic matter degradation, with VS biodegradability reaching 66%, and COD removal efficiencies of 50.8% (ISR = 0.3) and 60.1% (ISR = 0.5). The primary focus of the study was to monitor and quantify the production of the two biofuels, biohydrogen and biomethane. In conclusion, this study provides an assessment of the two biochemical conversion pathways, detailing the generation of two valuable and utilizable gaseous products. This research examines the process-specific operational conditions governing gas production, with a focus on optimizing process parameters to enhance yield and overall efficiency.

1. Introduction

As modern societies develop and evolve, their needs and requirements grow, while at the same time the problems created that affect nature and the environment increase. By emphasizing developed and developing technologies and methods, it is possible to combine meeting needs with reducing or eliminating problems. Alternative fuel production from waste based on rational treatment and management could be one solution.

One of the categories of waste that is produced in large quantities and at the same time has the potential to produce alternative fuels is food waste (FW). According to the United Nations Food and Agriculture Organization (FAO), food waste is the decrease in the quantity or quality of food resulting from decisions and actions by retailers, food service providers, and consumers [1]. A more simplified definition identifies FW as biodegradable waste generated from a variety of sources, such as households, food-processing industries, restaurants, and other hospitality domains [2]. The management of FW, apart from the environmental impact, also includes a socioeconomic background. In the EU, over 58 million tonnes of food waste (131 kg/inhabitant) are generated annually [3], with an associated market value estimated at 132 billion euros [4]. On a global basis, around 1.05 billion tonnes of food waste were generated in 2022, 60% of which came from households, 28% from food services, and 12% from retail [5]. It consisted of carbohydrates, proteins, and lipids [6]. The great advantages provided by FW in terms of the production of alternative fuels are, on the one hand, its higher carbohydrate content compared to other organic wastes and, on the other hand, its biodegradability. These two properties are crucial for the high potential and rate of hydrogen production [7]. Food waste leachates (FWL) are liquid byproducts formed during the decomposition of FW. As a liquid fraction, these leachates are rich in organic matter and contain a diverse array of nutrients, carbohydrates, proteins, and lipids. This composition makes FWL, much like FW itself, a valuable resource for further processing and valorization, offering the potential for applications in bioenergy production. Particularly effective and worthy of study is the treatment of FW in two stages based on two technologies/methods: producing biohydrogen through dark fermentation in the first phase and biogas with a high percentage of methane through anaerobic digestion in the second phase.

Dark fermentation is a fermentative process in which organic and inorganic substances are converted to biohydrogen by anaerobic bacteria in the absence of oxygen and light [8]. Dark fermentation is a promising technology because it is inexpensive, has a high hydrogen production rate, and is easy to integrate with waste or wastewater treatment for large-scale biohydrogen production [9,10]. The key stage of the dark fermentation process in which biohydrogen is produced is acidogenesis. It is the second stage of the process (after the hydrolysis stage), in which the products of hydrolysis (soluble organic monomers of sugars and amino acids) are degraded by acidogenic bacteria to produce alcohols, aldehydes, volatile fatty acids (VFAs), and acetate, along with H₂ and CO₂ [11]. Numerous studies have shown that the efficiency of biohydrogen production is closely linked to the parameters of acidogenic fermentation [12].

On the other hand, anaerobic digestion (AD) is a metabolic process of a complex microorganism (facultative and stringent anaerobic) under non-toxic circumstances that converts organic matter into biogas and a stabilized organic effluent [13,14].

According to different studies, the treatment of FW using the DF process can produce biohydrogen with a yield of 10 to 100 mL H₂/gVS depending on the type of food waste (vegetables, fruit, restaurant waste, coffee waste, etc.), the type of reactor (CSTR, Batch, AnSBR, MRB) used, and the operating parameters (temperature, HRT, pH) [15]. Another study [16] reported a wide range of hydrogen production, from 1 NL H₂/L-d to 78 NL H₂/L-d, through the DF process [16]. A recent review [2] reported a biohydrogen yield obtained from FW ranging from 63 L/kg VS to 360 L/g VS in different types of experimental scales. This variability is driven by a range of factors, such as microbial dynamics, the chemical and physical properties of the substrate, reactor configuration, and key environmental and operational parameters, such as pH value and temperature [16]. According to a study [17], the DF process of FW has not been thoroughly explored. Operational pH values between 5.5 and 7.0 are considered favorable for hydrogen production through DF; however, the optimal pH is correlated with the substrate type and influenced by microbial communities. Moreover, a TS concentration higher than 15% redirects carbon flux in lactic acid accumulation [17]. Another study [18] reported that one factor influencing biogas production is the selected inoculum-to-substrate ratio (ISR). Within an ISR range of 0.05 to 0.25, an ISR of 0.14 achieved the highest biohydrogen yield, reaching up to 90 mL H₂/g VS of FW [18].

Regarding anaerobic digestion, studies present a biogas production yield of 300 to 1000 mL biogas/gVS depending on the same parameters, with the percentage of methane fluctuating from 40% to 60% [19]. In addition to pH, COD content, ammonium concentration, and VFA percentage, the inoculum-to-substrate ratio (ISR) is also a crucial factor influencing biomethane yield [20]. A study [21] investigated three ISRs, and the recommended ISR based on reference [20] achieved the highest biomethane yield in half the time of the production assays. Based on reference [2], many factors affect the acidogenesis stage during DF and AD. The most important are temperature, pH, hydraulic retention time, and organic loading rate.

The aim and novelty of the study are that it focuses on the investigation of hydrogen and biomethane production through a two-stage procedure, at two different ISRs in DF and at the same ISR in AD, under mesophilic conditions, using FWL as the substrate.

2. Results and Discussion

2.1. Bench-Scale Tests

Aiming at the appropriate execution of the two processes, the main factors were monitored in detail in all stages and all reactors of the experimental procedure.

Regarding the dark fermentation process,

Table 1. Initial and final characteristics of the bioreactors in the bench test for the DF process.

Parameter (Unit)	CSTR1 0.3 Start	CSTR1 0.3 Final	CSTR1 0.5 Start	CSTR1 0.5 Final
pH (−)	4.9	3.56	5.6	4.83
TS (g/L)	39.9	30.8	40.6	30.4
VS (g/L)	31.5	24.6	29.7	22.7
Alkalinity (g CaCO3/L)	2.1	1.8	2.0	1.7
VFAs (g HACeq/L)	- 1
COD (g/L)	36.7	32.6	31.1	28.9
NH4+ (g/L)	0.64	0.62	0.79	0.82
H2 NmL	134.8		82.2
H2 NmL/g VSadded	8.2		6.1
VSbiodegradability (%)	21.9		23.6
CODbiodegradability (%)	11.2		7.1

¹ The VFA content could not be determined by means of potentiometric titration due to the low pH values of the CSTR1.

presents the values of the main factors for the two reactors (with different ISRs) used for both their initial and final states. The pH drops significantly to more acidic values (from almost 5.5 to 3.5), while NH₄⁺ values are stable. On the other hand, the alkalinity concentration drops from 0.2 to 0.3 g CaCO₃/L in order to maintain the stability of the process. Regarding the organic load, a reduction of more than 20% is calculated for solids (VS) and approximately 10% for COD. Finally, the production of biohydrogen (H₂) is slightly higher for the experimental setup with an ISR equal to 0.3, as its production is calculated at 135 NmL or approximately 8.2 NmL/g VS_added, while for the corresponding experiment with an ISR equal to 0.5, the production amounts to 82 NmL or 6.1 NmL/g VS_added.

Accordingly, the main factors for anaerobic digestion are presented in

Table 2. Bench test biomethane results.

Parameter (Unit)	CSTR2 0.3 Start	CSTR2 0.3 Final	CSTR2 0.5 Start	CSTR2 0.5 Final
pH (−)	7.00	8.44	7.12	8.56
TS (g/L)	41.3	16.7	41.1	17.3
VS (g/L)	25.2	8.5	24.8	8.5
Alkalinity (g CaCO3/L)	2.7	7.5	2.7	7.5
VFAs (g HACeq/L)	1.6	0.9	1.4	0.8
COD (g/L)	19.1	9.4	17.3	6.9
NH4+ (g/L)	1.46	1.89	1.54	1.88
VFAs/Alkalinity	0.59	0.12	0.52	0.11
CH4 NmL	6769.1		6714.7
CH4 NmL/g VSadded	275.2		277.5
VSbiodegradability (%)	66.3		65.7
CODbiodegradability (%)	50.8		60.1

. The pH remains almost stable at optimum values [20] (between 7 and 8), as the NH₄⁺ content remains approximately constant throughout the process. Two of the most crucial factors for controlling anaerobic digestion are alkalinity and VFA concentration, while the variation of their ratio provides information about the efficiency of the process [20]. Alkalinity values increase during the anaerobic digestion process (from 2.7 g CaCO₃/L to 7.5 g CaCO₃/L), while conversely, VFA concentration decreases (from approximately 1.5 g HAC_eq/L to 0.8 g HAC_eq/L). In addition, the VFA/alkalinity ratio is also reduced (from approximately 0.55 to 0.11) in order to stabilize the AD process [20]. The efficiency of AD, both in terms of organic rate removal and biomethane production, can be reflected in the reduction in the organic load. The concentration of solids (VS) is reduced by more than 65% and COD concentration by approximately 55%. The production of biomethane is estimated at 6769 NmL or 275.2 NmL/g VS_added for an ISR equal to 0.3 and at 6715 NmL or 277.5 NmL/g VS_added for an ISR equal to 0.5.

2.2. Biogas Production

The present study focuses on the efficiency of the two processes (DF and AD) in terms of biogas production.

During the DF procedure, a total amount of 135 NmL of biohydrogen was produced from the reactor with an ISR of 0.3 and 82 NmL from the reactor with an ISR of 0.5. The limited yield of biohydrogen is due to low alkalinity levels, which further exacerbated the system’s inability to buffer against the VFA accumulation and led to a drop in pH [18,22]. The largest volume of biohydrogen was produced during the first day (first 10 h), while in the remaining two days, the production rate gradually decreased; this indicates a VFA accumulation, resulting in acidic conditions, which was confirmed by the final pH values of the CSTR1 reactors (Table 1). The yield has the same variation for both ISRs, even though the reactor with ISR 0.3 produced a higher amount of biogas.

Examining the biohydrogen production in more detail and correlating it with the volatile solid concentration (Figure 1) in the waste fed to the two reactors (CSTR1), it is observed that the degradation of the solids takes place mainly in the first hours of DF. After the end of the first day, the ratio of H₂ production to VS_added was calculated at 7.7 NmL/g VS_added for an ISR equal to 0.3 and 5.7 NmL/g VS_added for an ISR of 0.5, followed by a slight increase (until the end of the process), resulting in values of 8.2 NmL/g VS_added and 6.1 NmL/g VS_added for ISRs of 0.3 and 0.5, respectively.

In contrast to the biohydrogen production during DF, the biomethane yield during AD shows a continuously increasing trend. The biomethane production increases exponentially until approximately day 16 and then continues to increase but at a slower rate. In addition, it is noted that the two reactors, despite having different waste compositions, produce approximately the same volume of biomethane: 6769 NmL and 6715 NmL for CSTR2 0.3 and CSTR2 0.5, respectively.

Figure 2 shows the biomethane production per VS_added. As expected, the variation in values is similar to biomethane production, resulting in a final value of 275.17 NmL/g VS_added and 277.47 NmL/g VS_added for CSTR2 0.3 and CSTR2 0.5, respectively.

Produced Yields

The factors affecting the acidogenic process are pH, temperature, ISR, organic load, hydraulic retention time (HRT), reactor type, and nutrient content [2,18,23]. In our study, a significantly low biohydrogen yield was observed for ISRs of 0.3 and 0.5. Based on the literature [18,21], an optimal ISR for hydrogen production is 0.14. This ISR results in up to 90 mL H₂/g VS added [18]. Compared to our study, where the H₂ yields were 8.2 and 6.1 mL/g VS added, the yield of 90 mL/g VS added is remarkably high. However, this discrepancy can be explained by the significant variation between these outcomes. The pH of FWL was 3.25 and as more FWL was introduced into the DF reactors (CSTR1), the pH of both reactors decreased further. The optimal pH range for the DF procedure is recommended at 4.5–6 [2]. However, a low pH is an indicator of high VFA contents, as already reported in reference [16]; VFA accumulation inhibits the metabolic activity of hydrogen-producing microorganisms. pH monitoring and control were not feasible in this study due to the design of glass bottle reactors. Several studies [24] were conducted on the DF process with a controlled pH to maintain it within the suggested range to achieve a higher hydrogen yield. Both the ISR and pH were key operational factors that potentially influenced the dark fermentation process in the present study. In addition, the substrate consisted of leachates derived from FW, which means that the proportion of carbohydrates was lower compared to FW itself. As previously reported [22], the H₂ yield and the production rate are affected by the carbohydrate quantity in the substrate, pH, and the nature of the substrate.

The biomethane yield for both CSTR2 reactors, compared to the biomethane yield obtained in a previous study [25], which conducted a two-stage process, ranged from 480 to 551 CH₄ mL/g VS added. The VFA/alkalinity ratio is a crucial factor for digester stability [20,26]; as demonstrated in Table 2, the VFA/alkalinity ratio at the end of the process in both CSTR2 reactors indicates a stable and healthy digester [20], with values 0.12 for CSTR2 0.3 and 0.11 for CSTR2 0.5.

2.3. Biogas Composition

A crucial factor for the efficiency of processes in terms of biogas generation is the composition of the produced biogas, mainly the content of biohydrogen (for the DF phase) and biomethane (for the AD phase).

The following table (

Table 3. Biogas composition of DF and AD processes.

Test	Time (Hrs)	CH4 (%)	H2 (%)	CO2 (%)	H2S (%)	Other Gases (%)
	Dark Fermentation
CSTR1 0.3		0.0	40.3	57.2	-	2.5
CSTR1 0.5		0.0	38.3	56.4	-	5.3
	Anaerobic Digestion
CSTR2 0.3	24	6.3	0.1	64.9	n.d. 1	28.7
	48	10.9	0.2	80.9	0.117	8.0
	144	36.9	0.0	61.6	0.375	1.1
	192	49.5	0.0	48.9	0.243	1.4
	240	65.9	0.0	32.2	0.103	1.8
	312	74.3	0.0	23.7	0.057	1.9
	360	73.7	0.0	24.6	0.041	1.7
	408	73.5	0.0	25.3	0.027	1.1
	480	81.2	0.0	17.6	0.019	1.2
CSTR2 0.5	24	7.3	0.0	53.5	n.d. 1	39.2
	48	14.6	0.4	77.7	0.108	7.1
	144	45.7	0.0	52.3	0.246	1.7
	192	64.4	0.0	33.8	0.140	1.7
	240	72.5	0.0	25.8	0.087	1.7
	312	72.4	0.0	26.2	0.066	1.4
	360	77.6	0.0	21.0	0.049	1.3
	408	79.0	0.0	19.6	0.041	1.4
	480	79.8	0.0	18.6	0.033	1.6

¹ n.d.: not detected.

) presents the composition of the produced biogas for both experimental configurations (different ISRs) at different times. Obviously, as will be presented in detail below, the percentage of the desired products (biohydrogen for DF and biomethane for AD) in the produced biogas increased through the implementation of processes. The percentage of biohydrogen is estimated at the end of DF at approximately 40%, while the percentage of biomethane rises to 80%.

Figure 3 illustrates the composition of the biogas produced during the dark fermentation process for the two different values of ISR. The biohydrogen content is at the same level for both ISRs: 40.3% for 0.3 and 38.3% for 0.5, respectively. The highest percentage of biogas content is carbon dioxide: 57.2% and 56.4% for ISRs of 0.3 and 0.5, respectively. Finally, other gases such as monoxide, hydrogen sulfide, nitrogen, etc. are present in lower proportions.

Figure 4 and Figure 5 present the composition of the biogas produced during anaerobic digestion.

More specifically, Figure 4 illustrates the composition of the biogas produced in the CSTR2 reactor, which was fed by CSTR1 with an ISR equal to 0.3 at different times. As initially observed, the biogas produced is mainly composed of CO₂ (approximately 65%), while as the process progresses, the digestion of the organic waste leads to a higher yield of the desired gas (biomethane), stabilizing the percentage above 80%. The end of the experimental process leads to the production of biogas with a content of 81.2% in biomethane, 17.6% in CO₂, and only 1.2% in other gases.

Accordingly, in CSTR2, which was fed by CSTR1 with an ISR of 0.5 (Figure 5), at the beginning of the process, the required gas was found in a low percentage; however, throughout the test duration, the content of biomethane increased, ultimately constituting 80% of the biogas produced. The percentage of CO₂ was determined at 18.6%, and the content of other gasses was calculated at 1.6%.

2.4. H₂S Percentage

According to [6], the sulfur content (%) in FW ranges from 0.09% to 0.7%. Due to its presence, it is crucial to quantify the H₂S percentage generated during anaerobic processes. Hydrogen sulfide is toxic, and its presence in a combined heat and power (CHP) combustion engine results in the formation of SO₂ and SO₃ oxides, which are highly corrosive to pipelines, instruments, equipment, and metal surfaces [27]. Figure 6 illustrates the H₂S composition (%) for the two anaerobic processes. As observed, the H₂S content decreased during the DF procedure. In the AD process, the H₂S on the 1st day was low; an increase was observed during the digestion until the 6th day, reaching 0.375% for CSTR2 0.3 and 0.246% for CSTR2 0.5. After the 6th day, the H₂S content increased until the end of the process, with values of 0.02% and 0.03% for CSTR2 0.3 and CSTR2 0.5, respectively. The highest H₂S content was produced during the first days of the AD process. The H₂S peak (6th day) does not coincide with the methane production peak (10th day, as shown in Figure 4 and Figure 5), as the low pH and high CO₂ generation at the starting phase of the digestion led to significant H₂S release [28].

3. Materials and Methods

3.1. Materials

3.1.1. Food Waste Leachates

The used FWL consisted of leftovers composed of 50% fruits and 50% vegetables. The leftovers were chopped into pieces ranging from 2 to 3 cm and then stored in a 10 L container at 4 °C, allowing them to ripen until an adequate liquid fraction was observed, which could then be utilized for the objectives of this study. The fruits and vegetables were collected from the canteen of the research center. The collected leachates were used as the substrate in the bench-scale test. The composition of the FWL was determined by means of pH, chemical oxygen demand (COD), total solids (TS), volatile solids (VS), volatile fatty acids (VFAs), ammonium (NH₄⁺), and alkalinity. The chemical characterization of FWL is presented in

Table 4. Main characteristics of anaerobic sludge and food waste leachates.

Parameter (Unit)	AS	FWL
pH (−)	8.18	3.25
TS (g/L)	47.0	39.6
VS (g/L)	25.9	30.0
Alkalinity (g CaCO3/L)	2.7	1.8
VFAs (g HACeq/L)	0.37	- 1
COD (g/L)	8.2	41.9
NH4+ (g/L)	1.3	0.53

¹ The VFA content could not be determined by means of potentiometric titration due to low pH values of the FWL.

.

3.1.2. Anaerobic Sludge

As inoculum, anaerobic sludge (AS) obtained from a commercial mesophilic anaerobic digester plant located in the area of Eordaia (Western Macedonia, Greece), was used. The AS was preheated at 100 °C for 1 h to suppress methanogen activity [21] for the dark fermentation process. A study [29] highlighted that heat treatment (>90 °C) suppresses the activity of hydrogen-consuming methanogenic archaea, thereby selecting for spore-forming bacteria like Clostridium, Bacillus, and Thermoanaerobacterium, which are efficient hydrogen producers. Regarding the anaerobic digestion procedure, the AS was used as received from the anaerobic digester unit. In Table 4, the chemical characterization of AS is presented.

3.1.3. Experimental Procedure

Both processes were based on the inoculum-to-substrate ratio (ISR). The ISR was determined based on the VS content of the test materials. The experimental procedures for DF and AD were carried out in the laboratories of CERTH in Ptolemaida, Greece. The assessment of the BHP and BMP assays was conducted based on the ISR. According to reference [20], the ISR can be expressed as the ratio of the VS concentration of the inoculum to the COD content of the substrate to (g VS_inoc/g COD_sub) or as the ratio of inoculum VS to substrate VS (gVS_inoc/gVS_sub). The BMP batch tests in this study were designed following the recommended VS_inoc-to-VS_sub ratio value [20], taking into account the VS content of both materials.

Regarding the DF procedure, two CSTR bench-scale bioreactors (CSTR1) with a nominal volume of 1 L and a working volume of 0.75 L were used. ISRs of 0.3 and 0.5 were studied to determine the biohydrogen production yield. Similarly, two CSTRs (CSTR2) with nominal volume and working volumes of 2.5 L and 1.7 L, respectively, were used for the AD process. Τhe CSTR2 bioreactors were inoculated with non-pretreated AS and the contents of the two CSTR1 bioreactors after the completion of the DF procedure. The ISR for biomethane production was 0.5 g VS AS/g VS effluent of the DF process [20,30]. Figure 7 illustrates the experimental pattern, while Figure 8 represents the flow diagram of the process.

3.1.4. HRT

HRT refers to the average time feedstock remains in a digester [26]. In a two-stage fermentation system, the first stage of HRT typically ranges from 1 to 3 days [2,23]. During these days, VFAs and hydrogen are produced. In the second stage, the methanogenic stage, 10 to 30 days are required for biomethane generation [2]. Since acidogenic bacteria grow faster than methanogens, the HRT is shorter in the first stage [26].

3.2. Methods

3.2.1. Analytical Methods

The measurements of TS, VS, COD, NH₄⁺, and alkalinity were carried out according to APHA Standard Methods [31]. The pH was estimated using a digital pH meter (Hanna, HI2260, Woonsocket, RI, USA). The VFA content was calculated as described by Mota et al. (2015) [32] and expressed as acetic acid equivalents (HAC_eq). COD content and NH₄⁺ concentration were quantified using a HACH DR2800 spectrometer (Hach Lange GmbH Headquarter, D-40549 Düsseldorf, Germany).

The produced gas composition was identified and quantified by means of a microGC biogas analyzer (microGC 490, Agilent Technologies, Agilent, Santa Clara, CA, USA). The biohydrogen and biomethane potential yield were carried out using the flow meter unit from the Automated Methane Potential Test System II (AMPTS II, BPC Instruments AB Mobilvägen 10 SE-223 62 Lund, Sweden).

3.2.2. Potential Yield

The generated biogas from each reactor passed through a 3 M NaOH aqueous solution to absorb CO₂ and impurities. The tests were conducted under mesophilic conditions (35 +/− 2 °C). The bench scale bioreactors were flushed with nitrogen gas to achieve anaerobic conditions.

The purified biogas was passed through a flow cell unit (each bottle was equipped with an individual flow cell), and gas productivity was evaluated by water displacement. The digital signal was recorded using software. The results of the BioHydrogen Potential (BHP) and BioMethane Potential (BMP) assays are expressed as normalized mL (NmL) H₂ and CH₄ per gram VS added, respectively. The BHP process lasted 3 days [2,18], and the duration of BΜP was 21 days.

3.2.3. MicroGC

The composition of the produced biogases was determined by means of a microGC biogas analyzer. The microGC system was equipped with two independent column channels. Each channel was coupled with a μTCD detector. The first channel was equipped with a molsieve 5A column. Argon gas was used as a carrier gas for the chromatographic separation of H₂, O₂, N₂, CH₄, and CO. A PoraPlot U column was installed on the second channel. Helium was utilized as a carrier gas for the determination of CO₂ and H₂S. The length of each column was 10 m. The column channels were heated at 110 °C and held for all analysis time. The analysis time was 3 min. The produced gas composition was carried out in triplicate. The biogas samples were taken before the NaOH trap.

4. Conclusions

This study investigated the performance of a two-stage anaerobic digestion process, where the first stage, dark fermentation (DF), was conducted in two continuous stirred-tank reactors (CSTRs) with different inoculum-to-substrate ratios (ISR = 0.3 and ISR = 0.5). The second stage, anaerobic digestion (AD), utilized the effluent from the DF reactors after 3 days as the substrate. Both ISR and pH monitoring were identified as critical factors for maximizing hydrogen production during the DF process.

The use of a series of continuously stirred tank reactors (CSTRs) offers a practical and efficient approach for the simultaneous treatment of complex substrates, such as food waste, while producing both biohydrogen and biomethane. This configuration allows for optimized conditions that enhance the production of both gases, making it a promising solution for processes involving multi-component feedstocks.

The evaluation of two different ISRs during the DF stage demonstrated that a lower ISR led to a greater volume of biohydrogen production, likely due to the higher carbohydrate content in the substrate. Biohydrogen production in the DF phase was almost complete within the first 24 h. However, after the first day, the biohydrogen production rate significantly declined, resulting in a lower cumulative yield.

A key factor influencing biogas yield is the composition of the waste streams used. FWL produced lower amounts of biogas compared to fresh FW.

Both stages of the process produced high-quality biogas, with the DF stage achieving up to 40% biohydrogen and the AD stage exceeding 80% biomethane.

Upon reviewing the article and comparing the corresponding values with those found in the literature, it is clear that the ISR values (0.3 and 0.5) were not the most efficient for hydrogen production. This highlights the need for further research into ISR and the determination of optimal values, which should be a priority for future studies and strategies. Additionally, it is particularly valuable to investigate the effectiveness of the two-stage process on various substrates, as this will allow for a comprehensive evaluation, not only in terms of hydrogen and methane production but also regarding the reduction in organic load. Given the complexity of the two processes and their sensitivity to a variety of factors, it is crucial—and an important area for future research—to explore and optimize variables such as pH, temperature, acid concentration, and the balance between substrate and biomass. Finally, scaling up the two-stage process and evaluating its performance on a larger scale is essential to determine its feasibility for application in real-world settings.