International Journal of Hydrogen Energy 108 (2025) 113–120

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International Journal of Hydrogen Energy

journal homepage: www.elsevier.com/locate/he

Biohydrogen production by co-digestion of food waste and corn

industry wastewater
Monserrat Vázquez-López, Iván Moreno-Andrade *
Laboratory for Research on Advanced Processes for Water Treatment, Unidad Académica Juriquilla, Instituto de Ingeniería, Universidad Autónoma de México, Blvd.
Juriquilla 3001, 76230, Querétaro, Mexico

A R T I C L E I N F O A B S T R A C T

Handling Editor: Ibrahim Dincer Food waste (FW) and corn industry wastewater (cornWW) are residues with a high potential for hydrogen
production in the dark fermentation process. The study aimed to identify the co-digestion of FW and cornWW
Keywords: ratios for improving hydrogen production and evaluating the synergistic effect on biohydrogen production po­
Biohydrogen tential (BHP) in batch test. The inoculum consisted of a mixed culture plus the native microbiota of each sub­
Co-digestion
strate. The adjustment of the initial pH to 7.5 was compared to the co-digestion without control of the initial pH
Dark fermentation
of the experiments. The highest hydrogen and metabolites production were obtained with a co-digestion FW:
Food waste
Corn industry wastewater cornWW ratio. When no pH adjustment is applied, pH values above 10 completely inhibit the metabolic activity
of bacteria in the process. The cornWW not only provided alkalinity to the system but also can be applied to
adjust the organic matter content of FW, adjusting the initial concentration of organic matter. The growth of
microbial communities was formed mainly by Clostridium sp. The co-digestion of FW:cornWW can be technically
feasible to improve the production of H2 through the dark fermentation process.

1. Introduction added value, such as H2 and organic acids [6,7]. Biohydrogen produc­
tion from industrial organic waste with high chemical organic demand
Rapid global population growth has led to an imbalance in global (COD) as acid cheese whey [8,9], tequila vinasse [10] and municipal
environmental resources (farmland, fresh water, energy, and biological organic solid waste [11], has been reported as successful study cases.
resources), generating social and environmental problems and contrib­ One of the strategies that has been implemented to improve the DF
uting to a decline in human well-being [1]. Respecting fossil energy process is co-digestion, where two or more organic wastes are used to
sources, Salamen et al. [2] reported that reserves would last about 40 achieve a balance of nutrients and reduce the adverse effects of substrate
years longer. Hence, as they run out, all renewable energy sources need inhibitors to increase the biogas production, biochemical products
to be researched and prioritized to avoid the generation of gases that (organic acids) and improve process stability [12].
contribute to air pollution and climate change in the processing and use Among the organic waste potential substrates for H2 production, the
of coal, oil, and gas [3,4]. A viable alternative to substituting fossil fuels food waste (FW) and wastewater from the corn industry (cornWW) are
is hydrogen (H2) for its high energy content (122 kJ/g). In addition to considered highly biodegradable substrates, low or zero cost, and highly
this, in H2 combustion, only water vapor is produced. Leveraging a available [13]. Regarding FW, it is estimated that about a third of the
biological process offers the dual advantages of renewability and uti­ world’s food produced is wasted [14]. FW is organic waste generated by
lizing of organic waste as a feedstock, thereby achieving a twofold restaurants, food processing plants, homes, businesses, and institutions
benefit: clean energy generation and waste reduction [5,6]. [15]. The FW composition varies significantly from one source to
A renewable alternative to generate H2 is dark fermentation (DF), another; this variation depends mainly on lifestyle, economic situation,
which is defined as an intermediate stage of the anaerobic digestion waste management standards, and industrial structure [16]. Generally,
process. In this process, the metabolic activity of methanogenic micro­ the moisture content is around 70–80% (wet base) and ST (total solids)
organisms is inhibited. The fermentative microorganisms involved from 20% to 30%, of which 90% corresponds to SV (volatile solids). The
transform substrates rich in carbohydrates into by-products of high- FW is composed mainly of carbohydrates, proteins, and lipids [17],

* Corresponding author.
E-mail address: imorenoa@ii.unam.mx (I. Moreno-Andrade).

https://doi.org/10.1016/j.ijhydene.2024.03.315
Received 31 August 2023; Received in revised form 15 February 2024; Accepted 25 March 2024
Available online 1 April 2024
0360-3199/© 2024 The Authors. Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
M. Vázquez-López and I. Moreno-Andrade International Journal of Hydrogen Energy 108 (2025) 113–120

characterized by having an acid pH and a high content of biodegradable gSV/kginoculum.

organic matter [18].
CornWW includes corn for food processing, corn starch and flour 2.2. Analytic methods
production, including the nixtamalization process to produce soft tor­
tillas and tortilla chips. In the nixtamalization process, the starch is The physicochemical parameters: moisture, total solids (TS), volatile
partially or entirely gelatinized, lipids are saponified, and corn proteins solids (VS), fixed solids (FS), alkalinity, and total Kjeldahl nitrogen
are solubilized, transforming it into a product known as nixtamalized (TKN) were determined according to standard methods [38]. pH mea­
dough, which is used for the production mainly of tortillas and other by- surements were done using a digital pH meter (Hanna, Hi5522). Density
products derived from corn [19,20]. The wastewater from the nixtam­ was obtained by dividing the feedstock’s weight by the residue’s volume
alization process is called “nejayote”, and in this paper will be named in [39]. The Chemical Oxygen Demand (COD) was determined by the
general as cornWW. For example, in Mexico, approximately 14.4 million HACH Reactor Digestion Method.
m3 of nejayote are generated per year, which has a final disposition in The carbohydrates were determined by the phenol-sulfuric acid
municipal sewage systems or water bodies without any treatment, rep­ method described by Dubois et al. [40], proteins were performed using
resenting a great concern to reduce and pollute water resources [21]. the method proposed by Lowry et al. [41], and lipids were analyzed
CornWW is characterized for having an extremely alkaline pH between using the reaction with sulfuric acid in the presence of
10 and 14, a high concentration of chemical oxygen demand (COD) in a sulpho-phosphovainillin described by Mishra et al. [42]. The analysis of
range of 1670–40000 mgO2/L and alkalinity around 180–3260 mgCa­ metals was performed by atomic absorption by the flame method with
CO3/L [21–24]. the equipment AVANTA PM (GBC Scientific) (APHA, 2005). For the
Over the past few years, interest has grown regarding the treatment determination of total organic carbon (TOC) and total inorganic carbon
and valorization of cornWW, including agricultural (28%), environ­ (TIC), a total organic carbon analyzer equipped with a solid sample
mental (11%) and chemical engineering (10%), and only 4.7% are module was used (TOC-LCSH, Shimadzu Corporation) (APHA, 2005).
focused on the possible use for energy production. Corn WW has been Volatile fatty acids (VFA) such as acetic, butyric, and propionic acids
proposed as a growing medium, reducing the chemical oxygen demand were quantified by HPLC in a chromatography Agilent (model 1260,
(COD) and the production of biomass with high protein content [25]. Agilent Technologies) equipped with a diode matrix detector (DAD),
The hemicellulose in nejayote has also been used to create added-value with a refractive index detector (RID) and a coupled column AMINEX
chemicals such as furfural using photocatalytic hydrolysis [26]. Cas­ HPX-87 H. A 5 mM solution of H2SO4 was used as an eluent at a flow rate
tro-Muñoz and Yánez-Fernández [27] employed a microfiltration pro­ of 0.6 mL/min with a column temperature of 50 ◦ C.
cess, followed by two ultrafiltration processes, to valorize different
fractions of cornWW that can be applied in various industrial sectors, 2.3. Experimental design
such as animal feed, carbon supply for biotechnological processes, food
additives, cosmetics industry, among others. For example, cornWW can To determine the Biochemical Hydrogen Potential (HBP), mono and
be used as a substrate to obtain specific phenolic compounds by applying co-digestion of FW and cornWW experiments were carried out. The ra­
a membrane process [28]. Application of novel technologies such as the tios used were 100:0, 10:90, 20:80, 30:70, 40:60, and 0:100 (v:v; FW:
membrane application for sub-products and H2 separation or CO2 direct cornWW), which corresponded to a concentration of 28, 23.2, 28.4,
capture [29,30] has been demonstrated to improve the process 33.6, 38.8, and 18 gSV/L, respectively. The substrate/inoculum ratio
applicability. was 2.7 according to Ref. [37], the working volume was 360 mL, and all
CornWW has been applied as co-substrate with vinasse [31], brewery reactors were shaken intermittently (60 s of work every 3 min). Exper­
wastewater [32], and abattoir wastewater [33] for producing H2 iments were carried out comparing the effect of initial pH in the test,
through dark fermentation. Co-digestion of FW and cornWW for H2 running a set of experiments with the resulting pH from different sub­
production has not been reported. This co-digestion could overcome strates (pH was variable according to ratios of FW:cornWW) and
some technical limitations of FW, such as rapid acidification, reducing comparing to experiments with the initial pH adjusted to 7.5 at the
the growth of lactic acid bacteria, which is the main bottleneck of beginning of the process. Both sets of experiments were carried out at 37
mono-digestion with FW [11,34]. This approach also eliminates the ± 1 ◦ C.
need for water to dilute FW for H2 production. This paper aims to Finally, a purge was performed to achieve an anaerobic atmosphere
evaluate the co-digestion of different FW:cornWW ratios, identifying the once the bottles were sealed, displacing the oxygen with nitrogen gas for
best conditions for the highest H2 production. 30 s. The final time of the test was when the H2 production curve was
asymptotic or when the last three records of accumulated H2 were less
2. Material and methods than 5% [37]. Throughout the process, the volume of gas generated was
normalized to a pressure of 1 atm and a temperature of 0 ◦ C, which was
2.1. Feedstocks and inoculum expressed as NmL. At the end of the process, samples were taken from
each Schott bottle for corresponding analysis.
CornWW was obtained from a local tortilla-producing factory (called Tests of the BHP were performed in duplicate with the Automatic
tortilleria) in Querétaro, Mexico, sampling a volume of 40L, which was Biogas Production Monitoring System (Automatic Methane Potential
stored in plastic containers at − 4 ◦ C to ensure the preservation of its Test System II, AMPTS II, Bioprocess Control, Sweden AB). This system
properties until its use. On the other hand, the FW was collected from the consists of three units: Unit A is the sample incubation unit, which has
municipal market of Querétaro, Mexico. To obtain a representative 15 glass Schott bottles of 500 mL; Unit B is the carbon dioxide absorption
sample, the characterization was carried out considering the sampling unit with 15 glass Schott bottles of 100 mL filled with 80 mL NaOH 3 M
Mexican municipal solid waste guidelines (NMX-AA-015-1995) [35]. and 0.4% timolphthalein pH indicator (w/v), and unit C is the device
The FW was crushed in an industrial mill to obtain a particle size < 5 mm with 15 injection mold flow cells in a water tank for measuring the
and later stored at − 20 ◦ C until use. The inoculum was obtained from a volume of gas generated using the principle of liquid displacement.
mesophilic anaerobic digester of a flour industry. A thermal shock pre­
treatment was performed at 105 ◦ C ± 5 ◦ C for 24h to eliminate mainly 2.4. Microbial community analysis
methanogenic archaea and hydrogen-consuming bacteria (homoaceto­
genic) [36]. After heat treatment, the material was disintegrated with a At the end of the dark fermentation process, samples were taken to
mortar and sieved through a mesh sieve #20 (850 μm) [37]. The volatile analyze the microbial community. The samples were from the mono-
solids (VS) content in the dry inoculum was 759.52 ± 0.60 digestion of FW, cornWW, and co-digestion of FW:cornWW 40:60

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ratio. The samples of biomass were preserved at − 4 ◦ C until its analysis. Table 1
Genomic DNA was extracted from biomass samples using the Power­ Physicochemical composition of food waste and corn industry wastewater.
Soil® DNA isolation kit (MOBIO, USA) according to the manufacturer’s Parameters FW cornWW
instructions. The DNA concentration was quantified by spectropho­
pH 4.6 ± 0.07 11.7 ± 0.01
tometry using a NANODrop 2000c (Thermo Scientific, USA). The DNA Moisture 91.2 ± 0.1 % 97.6 ± 0.1 %
was submitted to the Research and Testing Laboratory (RTL, Lubbock, Alkalinity 0.05 ± 0.01 g CaCO3/L 3.03 ± 0.02 g CaCO3/L
USA) for Illumina MiSeq sequencing using the bacteria primers 357wF- COD 181.0 ± 2.8 gO2/kg 27.9 ± 0.4 gO2/L
785R of the 16S rDNA. To determine the identity of each sequence, the TS 87.9 ± 0.4 g/kg 23.4 ± 0.2 g/L
VS 72.8 ± 0.458 g/kg 17.75 ± 0.193 g/L
procedure described in RTL Genomics (http://www.rtlgenomics.com/d FS 15.1 ± 0.075 g/kg 5.69 ± 0.052 g/L
ocs/Data_Analysis_Methodology.pdf). Finally, a Pearson’s correlation Carbohydrates 18.1 ± 0.832 g/kg 12.93 ± 0.095 g/L
matrix was performed to determine the possible relationships between Proteins 6.2 ± 0.585 g/kg 3.82 ± 0.052 g/L
the microbial community, fermentation yield, and metabolites gener­ Lipids 0.1 ± 0.004 g/kg 0.01 ± 0.007 g/L
Density 1080.0 ± 0.1 g/kg 1012.8 ± 0.1 g/L
ated. The analysis was made using Past 4 software version 4.15 [43].
TNK 2.15 ± 0.17 g/kg 0.85 ± 0.37 g/L
TOC 620.2 ± 3.2 g/kg 8.2 ± 0.1 g/L
2.5. Data analysis TIC 0.00 ± 0.00 g/kg 0.07 ± 0.02 g/L
Ca+2 0.0 mg/kg 70.9 mg/L
Co+2 0.0 mg/kg 0.1 mg/L
2.5.1. Gompertz model Cu+2 2.1 mg/kg 1.2 mg/L
The experimental data of cumulative hydrogen production showed a Fe+2 2456.0 mg/kg 590.6 mg/L
diauxic behavior. For this reason, data were fitted to the modified Mg+2 1728.0 mg/kg 19.6 mg/L
Mn+2 65.3 mg/kg 0.4 mg/L
Gompertz model according to Kim et al. [44]. The model considers two
Na+1 1230.5 mg/kg 14.4 mg/L
terms. The first term represents the early H2 production from readily Ni+2 2.0 mg/kg 0.4 mg/L
degradable materials with a particular rate R1, and the second term Zn+2 35.0 mg/kg 11.7 mg/L
expresses the later H2 production from degradable materials with a
particular rate R2 [45].
( ( ) ) ( arabinoxylans, sugars, hydroxycinnamic acids, vitamins and proteins
BHP = H1 ∗ exp − exp
R1 ∗ 2.71
(λ1 − t) + 1 + H2 ∗ exp that generate a high COD around 1.7–40.1 g O2/L [51]. In this case, the
P1 COD of the cornWW was 27.9 ± 0.4 g O2/L.
( ) )
R2 ∗ 2.71 Both residues have the presence of micronutrients that are essential
− exp (λ1 − t) + 1
P2 for microbial metabolism and H2 production. In this case, metal ions
such as Na+, Mg 2+, Zn 2+ y Fe 2+ can stimulate enzyme activity and thus
Where: BHP is the cumulative hydrogen production (mL), H1 and H2 facilitate H2 synthesis. These are of great importance given that each
hydrogen production (mL), R1 and R2 maximum hydrogen production metal ion has a specific effect on the bacterial cell during fermentation
rate (mL/h), λ1 and λ2 are the lag phase time (h), and t is the fermen­ [52].
tation time (h). Note: subscripts 1 and 2 refer to the early and later H2 Due to their physicochemical composition and high carbohydrate
production. The office package (Excel) was used using the solver tool to content compared to proteins and lipids, FW and cornWW are potential
calculate the kinetic parameters of the Gompertz model. substrates for producing H2. Numerous studies have demonstrated a
positive correlation between H2 production and the carbohydrate con­
2.5.2. Co-digestion performance index (CPI) tent of the substrates [11]. Conversely, it has been shown that protein
The CPI is defined as the specific hydrogen yield of co-digestion and lipid content have no significant contribution to the production of
divided by the weighted average specific hydrogen yield of mono- H2 [53]. On the other hand, the alkalinity of cornWW increases the
digestion of each feedstock [46]. This indicator will be used to iden­ buffer potential of the process. It has been reported that maintaining a
tify the ideal mixing ratio of the feedstocks. A CPI >1 means a synergistic pH of 5.5 throughout the process can enhance the production of H2 [54,
effect of co-digestion, and a CPI <1 indicates an antagonistic impact 55]. Similarly, the cornWW alkalinity (>3 g CaCO3/L) provided suffi­
[47]. cient buffer capacity to adapt to an increase in VFA in the DF process
[56]. The use of cornWW for FW treatment avoids the addition of
2.5.3. Statistical analysis chemical agents to control alkalinity and pH, resulting in an initial pH
Data were reported as mean, and standard deviations were calcu­ close to neutral, ensuring values higher than 4 during the DF process,
lated. Results were subjected to analysis of variance (ANOVA), and avoiding inhibition of microorganisms [57].
mean differences were compared by Tukey test (P < 0.05) using Minitab
17 (https://www.minitab.com). 3.2. Biochemical hydrogen potential (BHP)

3. Results and discussion Tests of the BHP showed that the tests with variable pH showed the
highest H2 production in a co-digestion with a FW:cornWW ratio of
3.1. Physicochemical composition of FW and cornWW 40:60 (initial pH 8.2) with an accumulated volume of hydrogen was
1110 ± 44 NmL H2. For the ratios 0:100 (pH 12.4) and 10:90 (pH 11.7),
Characterization parameters of FW and cornWW are shown in the H2 accumulated volume of H2 was zero (Fig. 1A). Moreover, in ex­
Table 1. The FW macromolecules conformation is mainly composed of periments where the initial pH was adjusted to 7.5, H2 was generated in
carbohydrates, followed by proteins and low lipids content. The pH of all evaluated ratios (Fig. 1B). A high H2 yield can be achieved by
these residues was 4.6 ± 0.07. The composition regarding humidity and working in a pH range of 5.5–7.5 in mesophilic conditions [58]. The
solids are within the ranges reported by Li [17] and Pramanik et al. [48]. ratio with the highest accumulated H2 volume was 40:60 (1618 ± 122
High carbohydrate content in FW can positively influence the H2 gen­ mLH2), followed by 30:70 (1256 ± 5). The co-digestion of substrates
eration [49]. The cornWW showed an alkaline pH (>11) due to the increased the H2 production in all the cases except in 10:90 without
remaining lime used during the nixtamalization process (>80%) [50]. adjustment of pH, where the process was completely inhibited. The pH
CornWW is considered highly polluting because it is composed of sus­ adjustment showed a significant difference in the H2 production (p <
pended solids such as corn pericarp, endosperm residues, non-starch 0.05). Not adjusting initial pH in values higher than 8 affected the H2
polysaccharides, and granules also contains dissolved solids such as production because bacteria could not initiate the fermentation process

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M. Vázquez-López and I. Moreno-Andrade International Journal of Hydrogen Energy 108 (2025) 113–120

only in mono-digestions and a diauxic behavior in co-digestions (Fig. 1).

The highest H2 production was obtained at 40:60, adjusting initial pH to
7.5. Table 2 shows the kinetic parameters of data adjusted to the
Gompertz model. Regarding lag time (λ), the time was lower in tests
where the initial pH was adjusted to 7.5, indicating that the microor­
ganisms present were better adapted at the start of the process. In the
case of co-digestion, the diauxic H2 generation results in a second lag
time (λ2) from 13 to 34 h, depending on the FW:cornWW ratios.
The diauxic behavior resulted in two maximum volumetric hydrogen
production rates (VHPRmax) and maximum H2 production for each
curve, where the first data is higher due to the presence of fermentation
of soluble COD in substrates (Table 2). It has been reported that diauxic
behavior is associated with carbon catabolite repression [60]. Therefore,
this phenomenon is likely due to two completely different substrates in
the mixture, which provide different carbon sources. Due to the
complexity of these carbon sources, their metabolism may occur at
different intervals. Nevertheless, it is not exhorting that a slightly
extended duration in mono-digestions would have the same effect since
García-Depraect & León-Becerril [61] reported a diauxic behavior in the
production of H2 from fruit and vegetable waste, and it was attributed to
a temporary availability in different carbon sources with specific
metabolization rates.
Only some studies are related to evaluating the PHB of cornWW with
other complex substrates. García-Depract et al. [31] investigated the
co-digestion of vinasse and cornWW with the best ratio of 80:20 (v:v),
obtaining 8.1 NmLH2/L/h and longer lag time phases. Differences with
our study are related to the substrate composition since FW has been
demonstrated to have higher VHPR than vinasses due to the initial
content of easy-to-degrade compounds in FW and the presence of in­
hibitors in tequila vinasses. In another study, De Angel-Acosta et al. [32]
tested the co-digestion of cornWW and wastewater from the brewery,
Fig. 1. Kinetics of the biochemical hydrogen potential: (A) Initial pH variable showing that the 40:60 and 60:40 ratios (pH of 5.8 and 6, respectively)
resulting from the FW:cornWW mixture, and (B) Experiments with the initial
resulting in VHPRmax of 33.7 and 46.2 mLH2/L h, respectively. For this
adjusted to pH 7.5 independently of the FW:cornWW ratios.
case study, the lag time (7 h) agrees with the data obtained in our study.
The comparison with other studies suggests that the complexity of the
as their metabolic activity was inhibited, resulting in zero H2 production different substrates leads to the differences in process beyond the
[59]. co-digestion with cornWW. For all the cases, co-digestion of acid sub­
strates with cornWW, offers the opportunity to save chemical agents to
3.2.1. Kinetic parameters adjusted to the Gompertz model regulate the pH due to its alkalinity.
In general, the experimental results of the H2 production tests were
well adjusted to the Gompertz model, in all cases the values of R2 were
higher than 0.99. The adjustment showed a behavior in sigmoid curves

Table 2
Parameters of adjustment of data to the modified Gompertz model.
Condition FW/cornWW H1 (mL) H2 (mL) R1 (NmL/h) R2 (NmL/ λ1 (h) λ2 (h) VHPRmax1 (NmL/L VHPRmax2 (NmL/L
ratio h) h) h)

Variable pH 100:0 65.0 ± 2.8 – 28.1 ± 1.9 – 19.6 ± – 86.1 ± 16.7 –

0.3
40:60 646.7 ± 783.5 ± 220.8 105.5 ± 32.8 ± 7.1 ± 0.2 15.9 ± 292.9 ± 34.7 91.1 ± 34.9
145.7 12.5 12.6 6.7
30:70 564.9 ± 38.8 677.9 ± 105.6 70.6 ± 13.8 27.3 ± 7.7 ± 0.1 18.0 ± 196.0 ± 38.4 76.0 ± 32.3
11.6 3.9
20:80 484.1 ± 68.0 703.6 ± 336.9 51.7 ± 37.1 34.1 ± 1.9 14.4 ± 33.7 ± 157.2 ± 83.9 113.8 ± 32.0
3.5 7.7
10:90 – – – – – – – –
0:100 – – – – – – – –
Initial pH 100:0 324.6 ± 10.2 – 15.4 ± 0.8 – 5.9 ± 0.1 – 42.9 ± 2.3 –
7.5 40:60 805.2 ± 48.5 1242.5 ± 93.1 ± 8.1 64.4 ± 9.0 6.7 ± 0.2 29.5 ± 258.7 ± 22.5 178.9 ± 25.0
488.6 6.7
30:70 656 ± 17.0 639 ± 40.0 93.2 ± 8.7 42.0 ± 6.8 7.4 ± 0.1 22.8 ± 258.8 ± 24.2 116.7 ± 18.8
2.6
20:80 453.6 ± 21.2 871.1 ± 84.4 85.1 ± 0.9 15.3 ± 0.1 8.2 ± 0.2 15.1 ± 236.3 ± 2.5 42.5 ± 0.4
1.8
10:90 368.1 ± 38.6 640.7 ± 1.9 60.8 ± 5.2 7.9 ± 0.3 8.7 ± 0.9 18.6 ± 168.9 ± 14.5 23.3 ± 2.9
0.7
0:100 267.3 ± 17.4 – 20.7 ± 4.2 – 15.3 ± – 57.4 ± 11.8 –
0.5

Note: VHPRmax was calculated according to García-Depraect et al [31].

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3.2.2. Specific hydrogen production were acetate and butyric, followed by propionate. The lactic acid was
Regarding the volume of H2 generated by the grams of volatile solid formed when high-content cornWW was present in the co-digestion
removed (VSremoved), a significant difference was found in all the pro­ (Fig. 3B). This is likely due to Bifidobacterium sp. (Fig. 4A), a genus
portions tested (p < 0.05) (Fig. 2). It is noted that the highest specific H2 known for lactate production from starch [34]. Fig. 3B shows that the
production was obtained in the 40:60 ratio independently of the initial highest total VFA concentration was obtained in the 40:60 ratio, with
pH (241.8 and 265 mLH2/VSremoved, for adjusted and non-adjusted pH, the concentrations of acetate, butyric, and propionic were 4.96, 5.04,
respectively), achieving a carbohydrate removal percentage higher than and 1.84 g/L, respectively. The highest H2 production is the highest total
70% in both cases. With the initial pH adjusted, the 60:40 ratio showed a VFA generation. Regarding tests with variable pH, it showed that the
higher yield than with a variable pH (8.2) since this condition facilitated 40:60 ratio generated a lower VFA concentration than experiments with
the substrate hydrolysis, enhanced hydrogenation activity and meta­ initial pH adjusted to 7.5, achieving a concentration of 2.2, 4.0, and 1.2
bolic pathways [9,62]. When the initial pH was not controlled, the g/L of acetate, butyric and propionic, respectively (Table 2. Supple­
highest yields were achieved in the ratios of 30:70 and 20:80, implying mentary material).
that hydrogen-producing bacteria can adapt temporarily to alkaline pH The metabolites obtained exhibited a similar composition to the co-
levels of 9.4 and 10.5. digestion of abattoir wastewater with CornWW. However, completely
On the other hand, in the experiments where the initial pH was different metabolites were observed in vinasse and CornWW co-
adjusted to 7.5, the carbohydrates removal of cornWW was 60.3%, digestion, where substrate was primarily converted into lactate, fol­
while for FW only 23.9% carbohydrates were removed (Table S1 sup­ lowed by lactate metabolism with other VFAs for H2 production [65]. A
plementary material) because the substrates are completely different significant finding of the co-digestion of FW: CornWW is its potential
matrices, one from solid waste and the other from wastewater. Due to its implementation to mitigate issues associated with the excessive growth
nature, wastewater can favor mass transfer in the system because of its of lactic acid bacteria and subsequent accumulation of lactic acid, which
low solids content [63]. is a common challenge in dark fermentation using FW. However, it is
The better mass transfer promoted by the cornWW increased in the imperative to ascertain whether this behavior persists in a continuous
removal rate and H2 production. However, in the proportions 10:90 and system concerning the obtained VFAs.
0:100, the H2 and VFA were negligible; this can be associated with an Furthermore, all co-digestions obtained a final pH of around 5 in­
excessive alkalinity affecting the metabolic activity of bacteria for per­ dependent of the initial pH, except for 10:90 and 0:100 without pH
formance, reflected in a final pH higher than 10, inhibiting the process. adjustment, where the final pH was higher than 10, resulting in a
A slight removal of solids and carbohydrates is evident in the ratios complete inhibition of the dark fermentation process. The initial pH and
10:90 and 0:100 (Table S1 supplementary material). However, this concentration of each substrate used directly affected the production of
consumption of organic matter does not appear to be directly related to metabolites. Carbohydrate consumption decreases the pH (VFA pro­
H2 production. Instead, it is conceivable that carbohydrates, amino duction), and the final pH within the range (5.5 and 6.0) has been
acids, and lipids, may contribute to the synthesis of phenolic com­ associated with high yields [34]. Considering that pH is one of the
pounds, as secondary metabolites [64]. crucial operational parameters due to its relationship with enzymatic
Although all samples indicated a synergistic effect due to the co- activity for substrate metabolism and H2 production [9,34], alkalinity in
digestion of both substrates, the 40:60 ratio showed the highest co- cornWW avoid possible pH inhibition. The final alkalinity in
digestion performance index of 3.44, correlated to the highest specific
H2 production and carbohydrates removal. The cornWW in the co-
digestion not only has the role as substrate contributing to the organic
matter content and regulating the alkalinity of the system but also can be
used as a diluent, increasing the moisture of the mixing, improving the
contact area between the inoculum and the substrate and regulating the
pH (e.g., decreasing pH from 12.4 to 8.2 in 40:60 ratio, Fig. 2) increasing
VFA and H2 production.

3.2.3. Metabolites related to the production of hydrogen

In general, the predominant metabolites in all ratios of FW:cornWW

Fig. 3. Metabolites generated in dark fermentation: A) initial pH variable, and

Fig. 2. Specific hydrogen production of the different FW:cornWW ratios. B) initial pH adjusted to 7.5.

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Fig. 4. A) Microbial community analysis in the substrates and in the experiment at the highest H2 production, and B) Pearson’s correlation matrix for the bio­
hydrogen production, metabolites, and microbial community.

co-digestion experiments (>2.2 gCaCO3/L) indicates that an initial pH volume of 1618 ± 122 NmL H2 and a carbohydrate removal of 78 ±
of 7.5 improves the H2 production [58]. 1.8%, significantly higher than the rest of the ratios. Clostridium sp. was
demonstrated to be the dominant microorganisms in co-digestion at the
3.2.4. Microbial community analysis highest H2 production, displacing the growth of bacteria such as Exi­
Microbial communities present at the end of fermentations from FW, guobacterium sp. present as native microbiota from cornWW. The
cornWW, and co-digestion of FW:cornWW with the highest H2 produc­ cornWW not only provided alkalinity to the system but also can be
tion were markedly different (Fig. 4A). During the fermentation of FW as applied to adjust the organic matter content of FW, adjusting the initial
a sole feedstock, it was mainly dominated by Clostridium sp. (75%), concentration of organic matter and increasing the H2 production.
followed by C. butyricum (13.3%). According to Pearson’s correlation Further studies to make the process economically feasible are related to
matrix, Clostridium sp. showed a positive correlation with the production the evaluation and optimization of the long-term operation of contin­
of H2 and a generation of metabolites such as acetate, butyrate, and uous or discontinuous processes, research on pathways for generating
propionic (Fig. 4B). Concerning C. butyricum, it only had a positive added-value DF sub-products (VFA and metabolites), and application of
correlation with the presence of lactate. The presence of C. butyricum novel technologies as the membrane application for sub-products and H2
could contribute to lactate production, while Clostridium sp. contributes separation or CO2 direct capture, promoting sustainable development
to the co-digestion of lactate and acetate for butyrate and H2 production and the circular economy of DF processes.
[66].
Regarding the fermentation of cornWW the bacteria involved were Declaration of competing interest
completely different. The most abundant bacteria were Exiguobacterium
sp. (37.6%), followed by C. butyricum (35.7%), C. sartogoforme (14.0%), The authors declare that they have no known competing financial
and Clostridium sp. (1.9%). Exiguobacterium sp. and C. sartogoforme interests or personal relationships that could have appeared to influence
positively correlated with lactate generation [67,68]. This is probably the work reported in this paper.
because Exiguobacterium sp. favored starch hydrolysis present in
cornWW in addition to lactate generation [67,69]. For this reason, the Acknowledgments
highest concentration of lactate (>2 g/L) was generated in the cornWW
fermentation (Fig. 3B). On the other hand, Exiguobacterium sp. was The financial support granted by DGAPA-UNAM through the PAPIIT
probably found in the native microbiota of cornWW as it has been re­ project IN102722 is gratefully acknowledged. The project Grupos
ported to be alkaliphilic and non-spore-forming [67]. However, in this Interdisciplinarios de Investigación of the Institute of Engineering
substrate type, lactate-producing bacteria growth was favored. The UNAM, is also acknowledged. Monserrat Vázquez-López would like to
presence of Clostridium sp. and the presence of metabolites such as thank CONAHCYT for the scholarship (702623). Gloria Moreno and
lactate and propionic were key to producing H2. Jaime Perez are acknowledged for their technical assistance.
In the case of the mixture composed of both substrates, although it
was mostly formed by cornWW, the microbial composition had a similar Appendix A. Supplementary data
tendency to the microbial composition of FW fermentation. The FW
microbial community was dominant during the FW and cornWW co- Supplementary data to this article can be found online at https://doi.
digestion, displacing the cornWW microbiota. In the co-digestion, an org/10.1016/j.ijhydene.2024.03.315.
abundance of Clostridium sp. and C. butyricum reached 91% and 5.3%,
respectively, confirming that the presence of Clostridium sp. in this co- References
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