Dark fermentative biohydrogen production using Opuntia spp.

K. Carrillo-Verástegui1; C. Escamilla-Alvarado1,*; C. Escárcega-González1; J. Cano-Gómez1; D.

Paniagua-Vega2; J. Hernández-Duque3
1Grupo de Ingeniería y Bioprocesos Sustentables, Centro de Investigación en Biotecnología y Nanotecnología, Facultad de

Ciencias Químicas, Universidad Autónoma de Nuevo León, Parque de Investigación e Innovación Tecnológica km 10 Autopista al
Aeropuerto Mariano Escobedo, 66629, Apodaca, Nuevo León, México
2Departamento de Química Analítica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Dr. José Eleuterio

González 235, Mitras Centro, 64460, Monterrey, México

3Departamento de Química, Universidad Autónoma de Aguascalientes, Avenida Universidad 940, 20131, Aguascalientes

Aguascalientes, México
* Corresponding author: cea_escamilla@yahoo.com.mx
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ABSTRACT

In a future with problems such as global warming, water scarcity and decreasing fertile land for
cultivation, crassulacean acid metabolism plants such as the cactaceae Opuntia spp. (nopal) could perform
as feedstock for biofuels. Although the use of nopal is widely regarded as a functional food, it can be used
for the production of biomethane and biohydrogen. Since there are few studies focused on the production
of biohydrogen from nopal, the main objective of this work was to evaluate the biohydrogen potential of
three varieties of Opuntia spp.

Opuntia biomass from three different sources were used: edible Opuntia ficus-indica (eOfi) acquired
from the local market, Opuntia engelmannii (Oe) sampled from the region, and Opuntia ficus-indica var.
Copena (Ofic) that was retrieved from field plantation. Two consecutive experiments were followed. Firstly,
biohydrogen potential was assessed using eOfi and Ofic at three inoculum to substrate ratios (ISR): 1, 0.6
and 0.2; at 35 °C, with manual stirring twice a day and 50 mL fermentation volume. Secondly, after
identifying the best ISR, biohydrogen potential from Ofic was compared against that of Oe. The
biohydrogen production kinetics were adjusted to the modified Gompertz equation.

The main effects analysis results from the first experiment showed that ISR had a profound negative
effect on maximum biohydrogen production, and on hydrogen production rate as well. The main effects
of Opuntia biomass source revealed Ofic was a better biomass for biohydrogen production than eOfi. The
assay with Ofic at ISR 0.2 gave the best results for maximum biohydrogen production (435.6 mL H2) and
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hydrogen production rate (10.34 mL H2/h); lag phase was 8.31 h. Furthermore, the biohydrogen
performance from Ofic was also better than that of Oe.

In conclusion, the best type of Opuntia (investigated in this work) for biohydrogen production, in terms
of maximum production and minimum lag phase, was Ofic at ISR 0.2.

Keywords: biohydrogen; dark fermentarion; nopal; Opuntia

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1. Introduction

Problems such as water scarcity, global warming, decrease of fertile land and droughts have
affected crops in recent years, so it has been said arid and semi-arid regions will increase around
the world, covering more than a third of the current global land area. In this situation, crassulacean
acid metabolism plants such as Opuntia spp. could serve as feedstock for several processes,
including biofuels and bioproducts production [1]. Although biofuels production such as
biomethane and bioethanol have been studied, biohydrogen production experiments with Opuntia
are still scarce.

Moreno-Dávila et al. [2] studied the fermentative production of hydrogen in packed bed reactors
to evaluate pH (4 and 11.32) and incubation temperature (16, 35, 45 and 55 ºC) in the performance
of hydrogen production from dairy processing wastewater. Purges from a brewery wastewater
treatment plant were used as inoculum. O. imbricata and the latter anaerobic inoculum were used
to form a biofilm, while the substrate used was dairy processing wastewater. The maximum yield
was 12.73 mmol H2/g COD when the initial conditions were COD of 21.1 g, pH 11.32, and 16 ° C.
After such work, Cepeda-Rodríguez et al. [3] pretreated O. imbricata and used it as a support for
the production of hydrogen at anaerobic conditions. Maximum hydrogen production was 13.42
mmol H2 and 1.86 mmol H2/(L*h) when O. imbricata was treated with KIO3 0.02 M at 90 ° C.

Although these works highlight Opuntia use in hydrogen production, it was mainly used as a
packed bed for biofilm formation. So the fermentation of Opuntia spp. as the main substrate is a
topic that has not yet been studied and is therefore of interest. In consequence, this study focuses
on the use of the Opuntia spp. at three ISR impacts on biohydrogen production.

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 2. Materials and Methods

2.1 Inoculum preparation

Sludge from a lab-scale anaerobic digester mounted following the Poggi-Varaldo method
[4] and operated according to Rodríguez-Valderrama et al. [5], was used as inoculum. Anaerobic
digester was fed weekly with vegetable and fruit residues at a mass retention time of 40 days.
Inoculum sampled from the digester was subjected to a heat treatment (96 ºC, 2 h) in order to
inhibit methanogenic archaea [6].

2.2 Substrate preparation

Three Opuntia types were used: edible Opuntia ficus-indica (eOfi) acquired from a local market
in Monterrey, Nuevo León, Opuntia engelmannii (Oe) sampled from the region and Opuntia ficus-
indica var Copena retrieved from field plantation in General Zepeda, Coahuila. The solid residues
of Opuntia from an extraction process (methanol 80 % at 23 °C for 1 h), were retrieved by
centrifugation and dried in a convection oven at 90 ° C for 24 h. Dried biomass was ground in an
electric coffee grinder (RJ44-OPP-BLACK, Chefman) and sieved to a particle size of 210 µm.

2.3 Hydrogen production experimental setup

The hydrogen production assay was carried out in duplicates in 124 mL serological bottles. The
operating volume was kept at 50 mL in all experiments. The substrate and pretreated inoculum
were placed in the bottles depending on inoculum to substrate ratios (ISR) proposed (1, 0.6 and
0.2). A duplicated control was performed with pretreated inoculum. The bottles were capped with
butyl and sealed with an aluminum cap. Experiments were carried out in a fermentation chamber
at 35 ° C. Reactors were stirred daily and production was measured with the displacement of a 1
M NaOH solution in inverted measured cylinders.

2.4 Experimental design

Three sets of experimental designs were carried out to evaluate and quantify hydrogen
production by dark fermentation of three types of Opuntia biomass. In the first design, a general
factorial design 3x2, the hydrogen production potential was evaluated using two substrates from
Opuntia cladodes (eOfi and Ofic) at three ISR and 35 °C. In the second experimental design, an
unifactorial experiment, the effect of ISR on hydrogen production was evaluated using the residual
biomass from the best extraction method obtained in a previous assay [7]. In the last experimental
design, the maximum hydrogen production of the Opuntia types was compared against that of the
extraction residues for the three species at the best ISR of the previous experiments. When daily
hydrogen production was unsignificant, assays were concluded and the digested sludge was
characterized.

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 2.5 Biomass characterization

Total solids (TS) and volatile solids (VS) were determined as described in APHA/AWWA/WEF
[8]. Volatile fatty acids (VFA) and alkalinity were measured as reported in Anderson et al. [9]. The
pH was measured according to NMX-AA-25-1984 [10].

3. Results and Discussion

Biohydrogen potential by dark fermentation for Opuntia spp. as substrate, it is not reported in
the literature, so it is very important to know the production and the maximum yield this biomass
can achieve. Parameters to modified Gompertz, Boltzmann and first-order models are enlisted in
Table 1. In Fig. 1, Ofic biomass was better than eOfi by 14% in terms of maximum cumulated
production, and by 40% in terms of the lag phase.

Table 1. Parameters of the modified Gompertz, Boltzmann and first-order models for eOfi and Ofic at
three ISR.
eOfi Ofic
Parameters 1 0.6 0.2 1 0.6 0.2
Modified Gompertz
Hmax (mL H2) 64.0 78.6 368.9 68.7 78.5 435.6
rmax (mL H2/h) 2.8 6.3 12.8 2.2 3.8 10.3
 (h) 14.6 11.6 14.1 9.2 4.5 8.3
R2 0.999 0.984 0.979 0.994 0.990 0.993
Boltzmann
Hmax (mL H2) 63.0 78.0 360.9 66.4 77.5 424.1
rmax (mL H2/h) 2.8 6.2 13.1 2.2 3.9 10.5
t50 (h) 26.7 18.0 29.1 25.0 15.2 30.1
R2 0.997 0.974 0.973 0.988 0.979 0.986
First-order
Hmax (mL H2) 115.9 100.1 509.0 121.9 86.2 545.8
k (1/h) 0.013 0.031 0.016 0.013 0.046 0.018
R2 0.886 0.891 0.908 0.945 0.965 0.957

The model that statistically gives the best fit to the curves is the modified Gompertz equation.
An important piece of information offered by the sigmoidal Boltzmann equation is the t 50, the time
in which 50% of the maximum accumulated production is reached; the lag phase and the t 50 is
lower for ISR 1 and 0.6, however, the production of both was lower compared to those of ISR 0.2.

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 Fig. 1. Cumulative hydrogen production using (A) eOfi and (B) Ofic at three ISR.

Table 2 shows the final characterization of the digestate. The high concentration of VFAs for
both species at ISR 0.2 can be highlighted; these can be incorporated into a photofermentation
process for the production of hydrogen (Rodríguez-Valderrama et al., 2020).

Table 2. Properties of the residual digestate for eOfi and Ofic at three ISR.
eOfi Ofic
Property 1 0.6 0.2 1 0.6 0.2
TS (%bh) 12.29 14.18 15.94 12.95 14.72 16.74
VS (%bs) 42.84 47.09 57.76 40.59 44.38 49.13
pH 7.35 6.95 6.04 6.95 6.46 6.20
Alkalinity (mg CaCO3/L) 8890 10900 14375 8855 9825 11325
VFA (mg CH3COOH/L) 3276 4200 6510 2952 4050 5070
Notes: TS, total solids; VS, volatile solids; VFA, volatile free acids

Regarding the second experiment in which the solid extraction residues were used, the best
maximum production for residual Ofic (R-Ofic) was at ISR 0.2 (Fig. 2). The kinetic parameters for
the modified Gompertz model of this series of experiments are found in Table 3.

Table 3. Biohydrogen production from R-Ofic modified Gompertz parameters.

ISR Hmax (mL H2) rmax (mL H2/h)  (h) R2
1 61.63 1.73 34.45 0.9935
0.6 80.93 3.16 30.87 0.9946
0.2 234.63 12.44 34.93 0.9703
Notes: Hmax, Maximum hydrogen production; rmax: maximum hydrogen production rate;  lag phase.

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 Fig 2. Cumulative hydrogen production using an extract residue R-Ofic at three ISR.

The solid residues resulted in 50% lower maximum production than the biomass prior to
extraction; this is 435 mL H2 for Ofic compared to 234 mL H2 for R-Ofic; this comparative can be
seen in Fig. 3. Among the investigated species, the extraction residue that had the best hydrogen
production was R-Oe with 318 mL H2; nevertheless within the raw biomass, Ofic was still the best
Opuntia type as its maximum hydrogen production was 431 mL H2.

Table 4. Biohydrogen production from Opuntia spp. at ISR 0.2 modified Gompertz parameters.
Biomass Hmax (mL H2) rmax (mL H2/h)  (h) R2
R-eOfi 180.89 4.91 29.61 0.997
R-Oe 318.47 9.60 36.55 0.984
R-Ofic 234.63 12.44 34.93 0.99
eOfi 269.53 10.89 26.71 0.995
Oe 317.08 8.43 32.60 0.986
Ofic 431.6 11.99 28.46 0.99

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 Fig. 3. Comparative of the cumulative hydrogen production between three Opuntia types as (A) raw
biomass and (B) an extract residual biomass.

4. Conclusion

The maximum hydrogen production with Opuntia spp. by dark fermentation of commercial and
cultivated species using ISR 0.2 is reported for the first time being 369 and 435 mL H2 respectively.
Also, incorporation of extraction prior to dark fermentation resulted in a 25% decrease in hydrogen
production.

Acknowledgments

The authors would like to thank Dr. Dulce Yaahid Flores Rentería and Dr. Alberto Sandoval
Rangel for their contribution in collecting and identificating Opuntia ficus-indica var. Copena used
in this work. Kenia Carrillo-Verástegui would like to thank CONACyT for its financial support on
scholarship No. 751366.

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XXI International Congress of the Mexican Hydrogen Society