Effect of Exogenous Inoculation on Dark Fermentation of Food Waste Priorly Stored in Lactic Acid Fermentation

Abstract

Lactic acid fermentation has recently been shown to be a robust storage strategy for food waste prior to conversion to biohydrogen through dark fermentation. However, the importance of initial microbial communities and, more particularly, exogenous microorganisms on the conversion of lactic acid-rich stored substrate is not yet fully elucidated. This study investigates the impact of introducing exogenous inoculum to lactic acid-rich stored food waste prior to biohydrogen production in dark fermentation. Results showed exogenous inoculation produced a statistically significant increase in biohydrogen production rate (R_m) by 199%, 250%, 137%, 130%, 19%, and 10% compared to non-inoculated stored food waste after food waste storage at 4 °C, 10 °C, 23 °C, 35 °C, 45 °C, and 55 °C, respectively. Interestingly, no impact on the maximum production yield (P_m) was observed, but exogenous inoculation increased the accumulation of acetate, up to 160% more compared to endogenous inoculum. The main hydrogen-producing bacteria (HPB) were affiliated with Clostridium sp., while Prevotella_9 sp., another known HPB, was found after the fermentation of the food waste stored at 23 °C. In this study, the interest of exogenous inoculation to convert food waste stored by lactic acid fermentation was demonstrated through an increase in production rate along with higher accumulation of co-products, e.g., acetate. Such findings are promising for further development of process coupling, combining storage and conversion by fermentation of complex food waste.

1. Introduction

For ameliorating the negative impacts of fossil fuel utilization on the environment, hydrogen is expected to play a key role in decarbonization efforts, with a wide range of applications including the industrial, transportation, and domestic sectors [1]. Nowadays, the production of hydrogen is dominated by fossil fuel-based processes, with less than 1% produced from electrolysis worldwide [2]. If the sources of electricity, not always renewable, and the overall lifecycle of electrolysis-based technologies are considered, the net impact on global warming potential is still not negligible [3]. To overcome this issue, renewable-based hydrogen is gaining more and more interest, particularly biomass-based renewable sources, as they have the lowest impact when considering the overall lifecycle of the technology [4]. In particular, dark fermentation is one of the promising technologies for the biological production of hydrogen, credited with a high production rate or productivity and the ability to convert a wide range of substrates [5,6]. Dark fermentation was previously investigated as a means to valorize various types of substrates, such as end-of-life dairy products [7], tequila vinasse [8,9], cheese whey [10], and lignocellulosic biomass such as grass silage [11].

Food waste is one of the most promising substrates for biological conversion, and more particularly by dark fermentation, in regard to their high biodegradability and high abundance [12,13]. Utilizing food waste as a substrate for dark fermentation can solve both issues of reducing the pollution of biowaste and, at the same time, producing bioenergy from renewable resources through the generation of biohydrogen-rich gas [5]. The suitability of using food waste as a substrate for dark fermentation also entails an inherent issue of organic carbon losses either during transport or storage due to their high biodegradability [12]. In the management of food waste conversion for valorization, particularly bioenergy production, storage is rarely an avoidable step [14] but seldom considered. The focus of study on dark fermentation is mostly on improving the performance of conversion of substrate to biohydrogen, but not on the storage part, hence a similar emerging topic of lactate-driven dark fermentation [6,9,15,16,17,18,19,20]. Research investigating storage of substrate prior to conversion to bioenergy mostly considered conversion to biomethane subsequent to storage [14,21,22,23,24]. In the context of dark fermentation for biohydrogen production, lactic acid fermentation has been discarded as a storage method prior to dark fermentation, as lactic acid bacteria were considered detrimental to dark fermentation [20]. However, it has recently been shown to also have positive effects, such as initiating granule formation in high-rate fermentative hydrogen production systems and hydrogen production from lactose fermentation [25].

For solving this storage prior to dark fermentation issue, lactic acid fermentation has recently been proposed as a robust storage strategy that can be utilized at a wide range of temperatures and reduce the needs in energetically costly refrigeration [26]. In this previous study, focus was given on the storage condition (temperature), and the subsequent step of conversion to biohydrogen by dark fermentation was using endogenous microbial communities without using any exogenous inoculum [26].

At the end of food waste storage by lactic acid fermentation, various metabolic end products can accumulate, such as lactate, acetate, and ethanol [26]. The metabolite composition can potentially alter the microbial communities, as different concentrations of various organic acids were previously shown to affect the microbial populations and associated metabolic pathways in dark fermentation [27]. Furthermore, endogenous and exogenous/added microbial communities can either show positive or negative interactions, having an impact on the performance in dark fermentation [28]. In the previous article published by authors on coupling lactic acid fermentation as a storage method and dark fermentation for biohydrogen production [26], attention was given to the storage parameter (storage temperatures), whereas in the second part of conversion to biohydrogen through dark fermentation, only endogenous inoculum was utilized for standardization, hence did not include different inoculation strategies.

Considering these factors, this study aims to evaluate the impact of introducing exogenous inoculation to food waste previously stored by lactic acid fermentation. The effect was estimated by measuring the microbial performances and pathways in the dark fermentation for conversion of the stored food waste to biohydrogen.

2. Results and Discussion

2.1. Exogenous Inoculation of Food Waste Stored in LAF Increases Hydrogen Maximum Production Rate in Dark Fermentation

Biohydrogen potential (BHP) tests were carried out with exogenous inoculation to evaluate the impact of adding exogenous microorganisms on metabolic pathways in dark fermentation. These results were compared to BHP tests carried out with only endogenous microorganisms as the sole microbial resources, as reported by Roslan et al. (2023) summarized in

Table 1. Modified Gompertz Fitting comparisons between BHP tests performed using endogenous inoculum and exogenous inoculum (this study) from food waste stored in LAF at various temperatures ¹. Endogenous inoculum experimental data from [26].

Storage Temperature (°C)	Pm, Maximum Production (mL/gVS)		Rm, Maximum Production Rate (mL/gVS·d)		λ, Lag Phase (d)
	Endogenous	Exogenous	Endogenous	Exogenous	Endogenous	Exogenous
4	80 ± 6 a	57 ± 3 a	46 ± 6 a	135 ± 15 abc	0.4 ± 0.1 a	0.4 ± 0.0 a
10	89 ± 0 a	85 ± 3 a	53 ± 10 ab	183 ± 14 c	0.7 ± 0.0 ab	0.4 ± 0.0 a
23	79 ± 5 a	74 ± 10 a	72 ± 18 abc	151 ± 92 abc	1.1 ± 0.0 c	0.8 ± 0.4 bc
35	94 ± 20 a	62 ± 29 a	69 ± 14 abc	167 ± 97 bc	0.5 ± 0.1 ab	0.4 ± 0.0 a
45	84 ± 2 a	64 ± 8 a	58 ± 12 ab	66 ± 7 abc	0.6 ± 0.0 ab	0.4 ± 0.0 a
55	141 ± 35 b	94 ± 10 a	76 ± 21 abc	82 ± 17 abc	0.9 ± 0.1 bc	0.4 ± 0.0 a

¹ Statistical analysis was performed for each parameter (P_m, R_m, and λ) with 12 conditions (endogenous, exogenous, and all temperatures) for each parameter analysis. Groups sharing the same superscript letters are statistically the same or have no statistical difference.

[26]. Exogenous inoculation did not impact the maximum biohydrogen production yield (P_m), as statistical analysis showed no group differentiation, except at food waste stored at 55 °C. The higher BHP for food waste stored at 55 °C can be attributed to thermal degradation causing a heat pre-treatment effect. Here, BHP using endogenous microbes was statistically different and higher (141 ± 35 mL/gVS), while exogenous inoculation stored at the same temperature shared the same statistical group with all other conditions, at 94 ± 10 mL/gVS. A similar observation was shown by Dauptain et al. (2020), where the food waste inoculated with exogenous inoculum had lower cumulative biohydrogen production at 79 ± 13 mL/gVS as compared to food waste without exogenous inoculation at 156 ± 23 mL/gVS, indicating a negative interaction between endogenous and exogenous microbial communities [28]. In a similar study by Villanueva-Galindo, Pérez-Rangel et al. (2023), food waste pre-fermented for lactic acid accumulation at 37 °C, effluent was centrifuged and submitted to different dilution concentrations ranging from 5 to 25 g/L, much higher than the lactic acid concentrations accumulated in the storage reactors in this study, and subsequently converted to biohydrogen through dark fermentation using either thermally pretreated sludge and native microbial communities [29]. Exogenous inoculation did not produce any statistically different maximum biohydrogen yield [29]. This observation of no changes in biohydrogen yield might be due to the fact that the available biodegradable materials from the substrate are the same, despite the difference in the microbial communities consuming the substrate in dark fermentation.

A strong difference was shown for the maximum production rate of biohydrogen (R_m), where a clear effect of exogenous inoculation was observed. The lowest R_m of 46 ± 6 mL/gVS·d was found for food waste stored at 4 °C and using only endogenous microbes. In counterpart, the highest R_m of 183 ± 14 mL/gVS·d was reached for food waste stored at 10 °C and with exogenous inoculation. In all cases, exogenous inoculation resulted in higher R_m, with the highest difference found for food waste stored at 10 °C with an increase of 250% from its endogenous control of 53 ± 10 mL/gVS·d, followed by 199% for food waste stored at 4 °C, 137% at 23 °C, 130% at 35 °C, 19% at 45 °C, and 10% at 55 °C. The increase in maximum production rate might be attributable to the acclimatization of the inoculum, where the endogenous microorganisms were acclimatized to low pH during storage in lactic acid fermentation for 15 days, whereas the exogenous inoculum did not undergo the same environmental conditions.

The lag phase (λ) was lowered for all cases by exogenous inoculation, except for food waste stored at 4 °C. The difference in lag phase might be due to the adaptation of endogenous microbial communities to the low pH during storage in lactic acid fermentation (pH 3.6 ± 0.3), which the exogenous inoculum was not adapted to, as it was added at the beginning of the BHP tests, after storage of food waste in lactic acid fermentation.

No methane gas was observed throughout dark fermentation for all experimental conditions, despite the inoculum used not being heat-pretreated to deactivate the methanogens. This can be attributed to the pH of dark fermentation in this study, where it was adjusted to pH 6 at the beginning of dark fermentation and subsequently dropped due to the accumulation of metabolites and buffered with MES, whereas the growth of methanogen typically requires pH 6.8 to pH 7 [30].

2.2. Acetate Accumulation Increases with Exogenous Inoculation and Affects Butyrate Accumulation at Food Waste Stored at Lower Temperature of 4 °C

Table 2. Final metabolite concentration accumulated by the end of BHP tests, comparing between the outcomes of endogenous inoculum [26] and exogenous inoculation (this study) ¹.

Storage Temperature (°C)	Final Metabolite Concentration After BHP Test (gCOD/L)								Hydrogen Production Efficiency (HPE)
	Lactate		Acetate		Butyrate		Caproate
	Endogenous	Exogenous	Endogenous	Exogenous	Endogenous	Exogenous	Endogenous	Exogenous	Endogenous	Exogenous
4	-	-	1.2 ± 0.2 cde	1.8 ± 0.1 ef	2.8 ± 0.4 a	4.3 ± 0.4 b	-	-	0.5 ± 0.1 ab	0.2 ± 0.0 a
10	-	-	0.9 ± 0.1 bc	2.2 ± 0.0 f	3.9 ± 0.1 ab	3.8 ± 0.1 ab	-	-	0.5 ± 0.0 ab	0.3 ± 0.0 ab
23	-	-	0.5 ± 0.5 ab	1.9 ± 0.2 f	3.2 ± 0.1 ab	3.9 ± 0.2 ab	-	-	0.6 ± 0.2 b	0.3 ± 0.1 a
35	-	-	0.7 ± 0.0 bc	1.8 ± 0.3 def	3.2 ± 0.8 ab	4.0 ± 0.3 ab	0.7 ± 1.2 a	-	0.6 ± 0.0 b	0.2 ± 0.1 a
45	0.1 ± 0.2 a	-	0.0 ± 0.0 a	1.1 ± 0.2 bcd	3.4 ± 0.8 ab	3.0 ± 1.0 ab	-	-	0.9 ± 0.2 c	0.4 ± 0.1 ab
55	-	-	0.0 ± 0.0 a	1.3 ± 0.3 cde	3.6 ± 0.1 ab	3.2 ± 0.5 ab	-	0.4 ± 0.8 a	1.2 ± 0.1 d	0.5 ± 0.1 ab

¹ Statistical analysis was performed based on the types of metabolites, with 12 conditions (endogenous, exogenous, and all temperatures) for each metabolite analyzed. Groups sharing the same superscript letters are statistically the same or have no statistically significant difference.

reports the metabolite distribution at the end of the BHP tests operated with food waste stored at various temperatures, by comparing the metabolism of endogenous microbial communities and the effect of adding exogenous microorganisms. The main metabolites that accumulated after fermentation were acetate and butyrate, with traces of lactate and caproate. The absence of lactate at the end of the BHP tests, especially after adding exogenous inoculum, reflected a full conversion of lactate, which had accumulated during storage, to hydrogen and other co-metabolites alongside a full conversion of ethanol. Statistical analysis clearly showed that the introduction of exogenous inoculum positively impacted the accumulation of acetate. Regardless of the food waste storage temperature, exogenous inoculation increased the amount of acetate accumulated, the highest of 2.2 ± 0.0 gCOD/L for the food waste stored at 10 °C and the lowest at 1.1 ± 0.2 gCOD/L and 1.3 ± 0.3 gCOD/L for the food waste stored at 45 °C and 55 °C, respectively. Interestingly, at these temperatures (45 °C and 55 °C), no acetate accumulation was observed in BHP operated with only the endogenous microorganisms. For the food waste stored at 4 °C, exogenous inoculation increased acetate accumulation by 58% at 1.8 ± 0.1 gCOD/L as compared to 1.2 ± 0.2 gCOD/L with no inoculum. The percentage increase was the highest for the food waste stored at 35 °C, with a percentage increase of 160%, accumulating 1.8 ± 0.3 gCOD/L of acetate. This is followed by a 159% increase for food waste stored at 10 °C and a 146% increase for food waste stored at 23 °C.

Further analysis on HPE indicated that for all cases except food waste stored at 45 °C and 55 °C, both endogenous and exogenous inoculation, homoacetogenesis was indicated with HPE values less than 1. For food waste stored at 45 °C and 55 °C, no acetate accumulated with the endogenous inoculum, while exogenous inoculation showed accumulation of 1.1 ± 0.2 gCOD/L and 1.3 ± 0.3 gCOD/L of acetate, suggesting the role of heat during storage in disabling the acetate-producing pathways in the BHP tests and the exogenous inoculation’s role in enabling them.

As for butyrate accumulation, exogenous inoculation did not lead to any statistically important difference, except for food waste stored at 4 °C, where exogenous inoculation increased the accumulation of butyrate by 54% at 4.3 ± 0.4 gCOD/L as compared to 2.8 ± 0.4 for endogenous inoculum.

2.3. New Genera Emerge with Exogenous Inoculation, with Clostridium sp. Maintained as the Main HPB for Both Endogenous and Exogenous Inoculum

Figure 1 shows the relative abundance of bacterial genera found at the end of BHP tests with exogenous inoculation for food waste previously stored in LAF at various temperatures. Clostridium sp. was the main genus found in all reactors, with 58 ± 3%, 63 ± 3%, 56 ± 5%, 69 ± 1%, 38 ± 2%, and 27 ± 6% for food waste stored at 4 °C, 10 °C, 23 °C, 35 °C, 45 °C, and 55 °C, respectively. Clostridium sp. is considered one of the most important genera of hydrogen-producing bacteria (HPB) due to its high biohydrogen yield, fast growth, ability to convert a wide range of substrates, and high resistance to harsh conditions [31]. The resistance to harsh conditions was observed in the previous study by Roslan et al. (2023), where Clostridium sp. emerged in dark fermentation, despite previously being stored at low pH for 15 days in LAF [26]. Furthermore, several Clostridium species were reported to be able to consume lactate together with acetate as carbon and energy sources for biohydrogen production through the pyruvate-ferredoxin oxidoreductase pathways [31]. Exogenous inoculation caused the emergence of several genera not found in the tests operated with only endogenous microorganisms. In particular, the emergence of Prevotella_9 sp., Prevotella_7 sp., and Paraclostridium sp. was observed. This can be attributed to the source of exogenous inoculum used in this study, which was from urban wastewater treatment plants. For the food waste stored at 23 °C, Prevotella_9 sp. was found with a high relative abundance of 18% ± 7%. Prevotella is a Gram-negative, non-sporing bacterium that can be found in human feces and rumen [32]. Some species are found to be strictly anaerobes [32], consistent with this study considering the source of the exogenous inoculum and the anaerobic conditions of the BHP tests. Similarly, Prevotella_9 sp. was observed in a study by Zhao et al. (2024), where it emerged as the dominant HPB [33].

Figure 2 shows the relationships between process parameters, such as the storage temperatures, the type of inoculation, and the process outcomes. Interestingly, and despite being known as an efficient HPB, Clostridium sp. showed an inverse correlation with P_m and instead exhibited a closer relationship with the accumulation of acetate. Indeed, further correlation study showed a Pearson coefficient of R = 0.7 between the genus and acetate. This is in line with the review that most homoacetogenesis in dark fermentation was associated with Clostridium sp., making it difficult to be controlled, as members of the same genus are responsible for biohydrogen production [25]. The accumulation of acetate is also highly correlated with exogenous inoculation (Pearson coefficient, R = 0.8). Similar acetate accumulation increase after exogenous inoculation was reported by Dauptain et al. (2020), where dark fermentation of food waste with endogenous inoculum and exogenous inoculum accumulated 13% of acetate (gCOD/gCOD total) and 29% of acetate (gCOD/gCOD total), respectively [28]. Despite both conditions (endogenous and exogenous inoculum) having Clostridium sp. as the main genus with the maximum relative abundance, the difference in acetate accumulation indicated that either different species were involved or a change in metabolic pathways occurred. In this study, butyrate showed no strong correlation with either biohydrogen production or acetate accumulation, despite butyrate being one of the main pathways of biohydrogen production, alongside the acetate pathway [34]. Compared to a previous study operated with endogenous inoculum, where butyrate accumulation positively correlated with biohydrogen production [26], the low correlation in this study using exogenous inoculation might be due to a shift in metabolites, particularly acetate. Exogenous inoculation also closely correlated with the relative abundance of Caproiciproducens sp. Despite Caproiciproducens sp. being normally associated with the accumulation of caproate in dark fermentation, this genus was also found to be closer related to acetate production in a study performed on thermophilic fermentation of sugarcane vinasse by Fuess et al. (2024) crediting the versatility of this genus with acetate as one of the fermentation products [35].

3. Materials and Methods

3.1. Substrate

The substrate used in this study was freshly reconstituted food waste. The composition of the food waste was based on the food waste composition usually found in France as described by Noguer et al. (2022), consisting of a mixture of minced beef (15%), yogurt (10%), mixed berries (15%), breaded fish (10%), French fries (20%), mixed vegetables (7.5%) including broccoli, long beans, carrots, and potatoes, mixed carrots (7.5%), and bread (15%), representing 62% carbohydrates, 22% proteins, and 16% lipids by total mass [36]. The food waste was priorly stored by operating lactic acid fermentation at 10% TS (total solids) [37] for 15 days and under six different temperatures (4 °C, 10 °C, 23 °C, 35 °C, 45 °C, 55 °C) as previously reported by Roslan et al. (2023) [26].

Prior to storage in lactic acid fermentation (LAF), the food waste had a biodegradable COD value of 1.28 ± 0.2 gCOD/VS [36], a 215.6 ± 273.2 μm particle size (measured at a 0–2000 µm range), a total COD of 122.8 g/L, and a biohydrogen potential of 76.8 ± 11.9 mL/gVS, directly converted to biohydrogen through dark fermentation without storage, as a control [26]. Lactate, ethanol, and acetate accumulated throughout the storage of the food waste depending on the storage temperatures [26]. Total solids (TS) and volatile solids (VS) were determined using the APHA standard methods [38]. The TS and vs. of the food waste composition were 34.8 ± 0.1% TS and 33.7 ± 0.1% vs. prior to dilution [36], whereas after dilution, it was 9.8 ± 0.2% TS and 9.5 ± 0.2% vs. Due to the high amount of organic acids accumulated after storage, the TS and vs. values were corrected to take into account the volatilization of organic acids during drying (Information S1 in Supplementary Material) [39]. The correction was carried out to prevent underestimation of TS and VS. values and consequently overestimating the yield of gas. The metabolite concentrations in stored food waste are as described in

Table 3. Stored food waste total solids (TS) and volatile solids (VS) in percentage (% w/w basis) and concentrations of metabolites in stored food waste [26].

Storage Temperature (°C)	%TS and %VS	Metabolite Concentrations in Stored Food Waste (gCOD/L)
		Lactate	Acetate	Ethanol	Propionate
4	9.6 ± 0.1, 9.3 ± 0.1	3.8 ± 0.2	0.9 ± 0.1	1.1 ± 0.2	-
10	10.1 ± 0.1, 9.7 ± 0.1	5.5 ± 0.1	0.9 ± 0.0	8.5 ± 3.4	-
23	9.5 ± 0.2, 9.1 ± 0.2	13.1 ± 0.2	1.3 ± 0.0	22.5 ± 3.7	0.6 ± 0.5
35	9.1 ± 0.4, 8.7 ± 0.4	15.3 ± 2.0	1.1 ± 0.1	6.3 ± 0.6	-
45	9.5 ± 0.1, 9.2 ± 0.1	6.4 ± 1.0	0.6 ± 0.1	3.2 ± 0.4	-
55	9.5 ± 0.2, 9.2 ± 0.2	2.2 ± 0.7	-	-	-

.

3.2. Inoculum

The microbial inoculum corresponded to activated sludge sampled from the activated sludge process of the urban wastewater treatment plant in Narbonne, as previously used by Noguer, Escudié et al. (2022) [36]. After collection, the sludge was dewatered and centrifuged at 9000 rpm for 5 min while maintaining the temperature at 4 °C (Avanti JXN-26 Centrifuge, Beckman Coulter, Indianapolis, IN, USA). The supernatant was removed, and the biomass was freeze-dried and stored at −80 °C for long-term usage of the inoculum [40]. The inoculum had a volatile solid content of 0.75 ± 0.4 gVS/g. The freeze-dried inoculum was directly introduced into the dark fermentation reactor bottles without any heat pretreatment, as heat shock was shown to potentially deactivate lactate-utilizing hydrogen-producing bacteria [41].

3.3. Biohydrogen Potential (BHP) Test

BHP batch tests were performed according to a standardized protocol for the biohydrogen potential test [42]. The BHP tests using exogenous inoculum in this study were conducted at the same time as the BHP tests using endogenous microbial communities as reported by Roslan et al. (2023). For that, 20 g of stored food waste were introduced into a 600 mL glass reactor bottle, in triplicates for each storage temperature, for a total of 18 replicates. To buffer the pH in the reactor, 20 mL of 1 M MES (2-(N-Morpholino) ethanesulfonic acid) was added. Thereafter, 250 ± 7 mg of inoculum was added to each bottle to have a substrate-to-inoculum ratio of 10 on a vs. basis. The pH was adjusted to pH 6 using 8 M of NaOH, and Milli-Q water was added for a final working volume of 200 mL, diluting the stored food waste and its accumulated metabolites (Table 3). The bottle was sealed using chlorobutyl rubber caps to allow for headspace gas sampling with needles. The headspace of the bottle was purged with nitrogen gas to ensure anaerobic conditions and was subsequently placed in a water bath with a temperature maintained at 37 °C. The headspace was connected to a micro-gas chromatograph using a needle. The BHP test was stopped when no additional hydrogen production was observed after 3–5 days.

3.4. Gas Composition Analysis

The headspace pressure and gas composition in the BHP bottles were automatically sampled by a micro-gas chromatograph (SRA l-GC R3000, SRA Instruments, Marcy-l’Etoile, France), and gas composition was analyzed every two hours by thermal conductivity detection and a GC equipped with a PoraPlot U (PPU) 8 m column at 70 °C and 20 psi with helium carrier gas for CO₂ gas analysis and a Molsieve 5A 10 m column at 80 °C and 30 psi with argon as carrier gas for H₂, N₂, CH₄, and O₂ gas analysis [27].

3.5. Metabolites Analysis

At the end of the BHP tests, two mL of liquid sample was sampled from each BHP bottle and centrifuged at 13,400 rpm for 15 min. The supernatant was analyzed for organic acids quantification, and the centrifugation pellet was frozen at −20 °C for microbial analysis. The supernatant samples were acidified with 0.1 M H₂SO₄ and filtered with a 0.2 μm nylon filter (Fisherbrand, Illkirch, France). Organic acids were analyzed using high-performance liquid chromatography (HPLC) (Thermo Scientific Dionex Ultimate 3000, Illkirch, France), coupled with a refractive index detector (ERC RefractoMax 520, Data Apex, prague Czech Republic), performed at a flow rate of 600 mL/min using a column (Aminex HPX-87H Ion Exclusion, Biorad, Marnes-la-Coquette, France) at 50 °C with a protective pre-column (Bio-Rad Micro-Guard Cation H⁺, Biorad, Marnes-la-Coquette, France). The metabolite concentrations are expressed in gCOD/L with the conversion of each organic acid’s concentration based on its COD equivalence [42].

3.6. Microbial Analysis

To observe the relative abundance of bacteria in the BHP tests, microbial analysis was performed as previously reported by Noguer et al. (2022) [36]. The biomass stored after centrifugation (13,400 rpm for 15 min) underwent DNA extraction, utilizing the FastDNA SPIN kit for soil according to the manufacturer’s guidelines (MP Biomedicals, LCC, Irvine, CA, USA). Universal primers were used to target the V3–V4 region of the 16S rRNA. The PCR mix comprised MTP Taq DNA Polymerase (Sigma-Aldrich, Saint Louis, MS, USA) (0.05 u/μL concentration) together with enzyme buffer, with forward primer (344F: ACGGRAGGCAGCAG) and reverse primer (802R: TACCAGGGTATCTAATCCT), both at a concentration of 0.5 mM, dNTP at a concentration of 0.2 mM, and sample DNA at 5–10 ng/μL, added with water reaching a final volume of 50 μL. A thermal cycler (Mastercycler, Eppendorf, Germany) was used for 35 cycles of denaturation for 1 min at 95 °C, annealed for 1 min at 65 °C, and elongation for 1 min at 72 °C. Following 35 amplification cycles, 10 min of final elongation was performed at 72 °C. PCR amplifications were verified using a bioanalyzer (2100 Bioanalyzer, Agilent, Santa Clara, CA, USA). The GenoToul platform was used for sequencing of the reaction (Toulouse, France https://www.genotoul.fr (accessed on 1 July 2022)) utilizing the Illumina Miseq sequencer (2 × 300 pb paired-end run). Mothur version 1.48.0 was used to analyze raw sequence reads, cleaning, assembly, and quality checking, and for alignment and taxonomic outlines, SILVA release 132 was used. For data processing and figure development, Microsoft Excel with Power Query was used, with genera less than 1% relative abundance grouped as “others”.

3.7. Data Analysis

3.7.1. Gas Production

The volume of gas production was determined by the periodic pressure increase measurement in the headspace of the bottles [26]. The pressure of the headspace was measured every two hours by the micro-gas chromatograph, and the total volume of hydrogen gas production was determined using the following equations:

$$\Delta N\left(n\right)=\left[y\left(n\right)P\left(n\right)V_h/RT]-[y\left(n-1\right)P\left(n-1\right)V_h/RT\right],$$

(1)

$$\Delta V\left(n\right)=\Delta N\left(n\right) \left(R{T}_0\right)/P_0 ,$$

(2)

where ∆N(n) is the change in number of moles of gas at sampling time n, y(n) is the percentage of gas composition analyzed from the gas chromatograph at sampling time n, P(n) is the measured pressure in the headspace by the micro-gas chromatograph at sampling time n, V_h is the headspace volume of the bottle, R is the gas constant (8.314 J/mol·K), T is the temperature (K) of sampled gas, ∆V(n) is the change in volume of gas at sampling time n, T₀ is the reference temperature at 273.15 K, and P₀ is the reference pressure at 10⁵ Pa. The cumulative gas production volume corresponds to the sum of the change in volume of gas of all sampling times.

3.7.2. Model Fitting for Gas Production

To obtain the maximum production (P_m), the maximum production rate (R_m), and the time of lag phase (λ), data of cumulative gas production were fitted to a modified Gompertz Equation, as first suggested by Lay et al. (1996) [43],

$$H_t=P_m·exp\left\{-exp\left[\left(R_m·e\right)/P_m \left(\lambda -t\right)+1\right]\right\},$$

(3)

where H_t is the cumulative hydrogen production, and t is the fermentation time. P_m, R_m, and λ were determined using the Microsoft Excel Solver function.

3.7.3. Hydrogen Production Efficiency for Determination of Homoacetogenesis

To assess the extent of the association of acetate production to either hydrogen production or homoacetogenesis, the following equation describing hydrogen production efficiency (HPE) is utilized as suggested by Perat et al. (2024) [34]:

$$HPE=H_{2}experimental/\left(2\left(Acetate+Butyrate\right)\right),$$

(4)

where the values of H₂, acetate, and butyrate are on a molar basis. A ratio of equal to one indicates no occurrence of homoacetogenesis, whereas a ratio of less than one indicates the occurrence of homoacetogenesis, based on the estimation of theoretical acetate and butyrate production for biohydrogen production [34].

3.7.4. Statistical Analysis

To verify any statistical difference between the groups of results, particularly between endogenous and exogenous inoculation at various temperatures, a one-way ANOVA test was carried out using the built-in Microsoft Excel Data Analysis tool. If a statistical difference was found (p < 0.05), Tukey’s test was used to differentiate statistically different groups with Rstudio’s emmeans, multicomp, and multicompView libraries. To observe the relationships between process parameters (storage temperatures and inoculation), process outcomes (metabolite accumulation, hydrogen production, and relative abundance of genera), principal component analysis (PCA) was performed using RStudio utilizing the factoextra library. For the same relationships, the Pearson correlation coefficient was determined using the Microsoft Excel Data Analysis tool for correlation.

4. Conclusions

In this study, exogenous inoculation increased the rate at which biohydrogen was produced from food waste stored by lactic acid fermentation by up to 250% (53 ± 10 mL/gVS·d vs. 183 ± 14 mL/gVS·d for food waste stored at 10 °C). No effect on the maximum biohydrogen yield was, however, observed. Acetogenesis increased with exogenous inoculation by up to 160% in dark fermentation (0.7 ± 0.0 gCOD/L vs. 1.8 ± 0.3 gCOD/L of acetate for food waste stored at 35 °C). Butyrate increased in dark fermentation for the food waste stored at the low temperature of 4 °C (2.8 ± 0.4 gCOD/L vs. 4.3 ± 0.4 gCOD/L of butyrate). Clostridium sp. remained the genus with the highest relative abundance, with the emergence of other genera after exogenous microbial addition, and more particularly Prevotella_9 sp. Caproiciproducens sp. relative abundance positively correlated with exogenous inoculation and with acetate accumulation. Overall, LAF is a robust strategy to store FW prior to biohydrogen production through DF, and this study demonstrated that exogenous inoculation is a potential strategy for increasing the production rate and productivity of biohydrogen and acetate–producing fermentation reactors.