Biocatalysis and Agricultural Biotechnology 23 (2020) 101507

Contents lists available at ScienceDirect

Biocatalysis and Agricultural Biotechnology

Addition of electron shuttling compounds and different pH conditions for

hydrogen production by a heat-treated sludge
Yair A. Del Angel-Acosta a, Luis H. Alvarez a, b, *, Refugio Bernardo Garcia-Reyes a,
María Teresa Garza-González a, Julián Carrillo-Reyes c
a Universidad Autónoma de Nuevo León (UANL), Facultad de Ciencias Químicas, Av. Universidad S/N, Cd. Universitaria, San Nicolás de los Garza, C.P. 66455, Nuevo
León, Mexico
b Instituto Tecnológico de Sonora (ITSON), Departamento de Ciencias Agronómicas y Veterinarias, 5 de Febrero 818 Sur, C.P. 85000, Cuidad Obregón, Sonora, Mexico
c Laboratory of Research on Advanced Processes for Water Treatment, Instituto de Ingeniería, Unidad Académica Juriquilla, Universidad Nacional Autónoma de

México, Blvd. Juriquilla 3001, Querétaro, C.P. 76230, Mexico

ARTICLE INFO ABSTRACT

Keywords: In the present work, the effect of anthraquinone-2-sulfonate (AQS) and its reduced form anthrahydrox-
Dark fermentation yquinone-2-sulfonate (AH2QS), and different pH values were evaluated during a dark fermentation process us-
Biohydrogen
ing heat-treated anaerobic granular sludge for hydrogen production. The highest maximum hydrogen produc-
Electron shuttling compounds
tion (1.99 mmol) was obtained by adding the reduced electron shuttling compound (AH2QS) at pH 6. This ex-
Gompertz model
Heat-treated anaerobic sludge
perimental condition also reflected the highest substrate consumption efficiency and the lowest lag phase in
relation to the AQS and the control (only sludge). In contrast, the lag phase and maximum hydrogen produc-
tion were not impacted by AH2QS addition at initial pH of 6.4 and 7.8. Regardless of the initial pH value, the
hydrogen production rate was increased after the addition of the electron shuttling compounds, which could
be attributable to the performance of either the oxidized and reduced form of the electron shuttling com-
pound. These findings demonstrate the feasibility of AH2QS addition during dark fermentation at pH 6.4 for
improving the hydrogen production rate. Exoelectrogenic microorganisms identified in the inoculum could be
responsible to improve the dark fermentation process in assays with electron shuttling compounds.

1. Introduction
During the last decades, the continuous population growth, as well
as the accelerated industrial development, have increased the global
energy demand. It is reported that approximately 80% of the global
energy requirement depends on fossil fuels (Das, 2001). Unfortu-
nately, the combustion of these fuels generates gaseous byproducts
that could contribute to global warming and the greenhouse effect on
earth (Das, 2001). Hydrogen has been proposed as a new energy car-
rier since water is the only combustion byproduct (Zhang et al.,
2006). Hydrogen can be obtained from a wide variety of raw materi-
als under different processes such as steam reforming, coal gasifica-
tion, electrolysis of water, among others. However, these conventional
physicochemical processes require high electrical-energy levels that
turn them poorly attractive from a sustainable point of view (Das,
2001). Dark fermentation has been reported as a suitable method for
hydrogen production since biological processes require less energy.
Also, they are usually operated at room temperatures and are able to
produce hydrogen all day long without any source of light.
Dark fermentation is an interesting approach for energy recovery
by the feasibility to couple it to wastewater treatment. Nonetheless,
low production yields have been observed in this process, achieving
values under the theoretical value of four mole of hydrogen per mole
of glucose, in the case of ideal acetate fermentation (Nath and Das,
2004). To improve the hydrogen production rate, extracellular elec-
tron shuttling compounds were added to pure cultures of Clostridium
and Enterobacter (Hatch and Finneran, 2008; Zhang et al., 2009). It
has been suggested that the reduced electron shuttles can directly pro-
vide additional electrons during fermentative hydrogen production in-
creasing production rates or yields. The possible mechanisms to eluci-

* Corresponding author. Instituto Tecnológico de Sonora (ITSON), Departamento de Ciencias Agronómicas y Veterinarias, 5 de febrero 818 Sur, C.P. 85000,

Cuidad Obregón, Sonora, Mexico.

E-mail address: luis.alvarez@itson.edu.mx (L.H. Alvarez).

https://doi.org/10.1016/j.bcab.2020.101507
Received 11 December 2019; Received in revised form 15 January 2020; Accepted 17 January 2020
Available online 21 January 2020
1878-8181/© 2020 Elsevier Ltd. All rights reserved.
Y.A. Del Angel-Acosta et al.
date the role of the reduced electron shuttling compounds during hy-
drogen production are still unclear. However, some authors had as-
sumed that these reduced molecules interact with fermentative elec-
tron transport systems as a secondary electron donor molecule and
hydroquinones molecules directly transfer electrons to ferredoxin or
nicotinamide adenine dinucleotide (NAD+) (Hatch and Finneran,
2008). The presence of extracellular electron donors alters the intra-
cellular NADH/NAD+ ratio by replacing functions of menaquinone
and modify membrane-linked enzymatic reaction, favoring hydrogen
production (Ye et al., 2012).
The studies for hydrogen production in the presence of electron
shuttling compounds were conducted each one at a different initial
pH condition. Nonetheless, considering that quinone compounds are
influenced by pH to be oxidized or reduced (Uchimiya and Stone,
2009), it is hypothesized that different pH conditions may have an ef-
fect on hydrogen production in presence of electron shuttling com-
pounds. On the other hand, it was also documented that phosphate
addition to the medium increases both the rate and maximum hydro-
gen production using a microbial consortium (Davila-Vazquez et al.,
2011). Then, in the present work, the role of anthraquinone-2-
sulfonate (AQS), its reduced form (anthrahydroxyquinone-2-sulfonate,
AH2QS), and three pH conditions (one buffered with phosphates)
were evaluated during the hydrogen production by dark fermentation
using a heat-treated anaerobic sludge. The use of heat-treated sludge
for this type of process instead of pure cultures could represent a bet-
ter approximation for applications on a real scale.
2. Materials and methods
2.1. Inoculum, chemicals, and basal medium
Anaerobic granular sludge used was collected from an up-flow
anaerobic sludge blanket reactor (UASB reactor) installed in a brew-
ery industry (Sonora, Mexico). The sludge was stored at 4 °C until it
was used and contained 9.6% of volatile suspended solids (VSS). To
suppress the methanogenic activity, the sludge was disintegrated with
a sieve of 0.7 mm followed by a heating treatment at 85 °C for 1.5 h
in a hot water bath (Mu et al., 2006). In order to maintain the homo-
geneous heating, the biomass was stirred by recurring 1-min air injec-
tions (every 15 min) during the thermal treatment. AQS and palla-
dium-coated aluminum catalyst were purchased from Sigma Aldrich.
The basal medium for all bioassays was prepared as follows (mg L−1):
NH4H2PO4 (4500), K2HPO4 (125), MgCl2·6H2O (100), ZnCl2 (75),
FeSO4·6H2O (25), MnSO4·7H2O (15), Na2MoO4·2H2O (12.5),
CuSO4·5H2O (5), CoCl2·6H2O (3) (Davila-Vazquez et al., 2011).
2.2. Chemical reduction of AQS
A stock solution of AQS (40 mM) was prepared in a serum bottle
using the basal medium and palladium as a catalyst for the reduction
as previously was described by Hernández et al. (2012). The bottle
was sealed with a rubber stopper and aluminum cap and was flushed
with hydrogen gas for 5 min. The solution was stored in a shaker until
the reduction was accomplished, which was evidenced by a change in
the color to orange. AH2QS is the reduced form of AQS, this reduced
electron shuttling compound solution was supplied to microbial incu-
bations for hydrogen production experiments.
2.3. Hydrogen production with AQS and AH2QS
Hydrogen production by dark fermentation was evaluated in batch
systems and all experiments were performed in triplicate. The kinetics
tests were carried out in 120 mL serum glass bottles inoculated with
1 g VSS L−1 of heat-treated anaerobic sludge and provided with 80 mL
of basal medium. The basal medium was adjusted to evaluate the ef-
2
Biocatalysis and Agricultural Biotechnology 23 (2020) 101507
fect of pH as follows: 1) pH 6.0, adjusted by adding concentrated HCl;
2) pH 6.4, adjusted and buffered by adding Na2HPO4 at 2000 mg L−1;
and 3) pH 7.8, corresponding to the pH value obtained after to pre-
pare the basal medium solution described in section 2.1. Different ex-
perimental assays were prepared to evaluate the effect of AH2QS and
AQS, including controls without electron shuttling compounds (only
sludge) and endogenous with AH2QS. After to add the basal medium
to the bottles, they were sealed with rubber stoppers and aluminum
caps. The anaerobic conditions were established by flushing in the
headspace a gas mixture composed of N2/CO2 (80%/20%). A first
pulse of glucose (1.1 mmol L−1 or 0.2 g L−1) was added to activate the
inoculum during 9 h with incubation at 150 rpm and 35 °C; then, a
second pulse of glucose to obtain a concentration of 11.1 mmol L−1
(2 g L−1). Electron shuttling compounds were added from a stock solu-
tion (1 mL) of AH2QS or AQS for a concentration of 500 μM, which
was in accordance of previous studies (Ye et al., 2013, 2012). The ex-
periments were finally incubated at 35 °C and 150 rpm. Initial and fi-
nal liquid samples were withdrawn for carbohydrates determination
for substrate consumption efficiency. Gas samples were taken every
three hours to measure hydrogen production and to confirm that
methane was not produced.
Experimental data of hydrogen production were used to determine
the kinetic parameters using the modified Gompertz model (Lin and
Lay, 2004a) as follows:
(1)
where H(t) is the accumulative hydrogen production (mmol), t is the
fermentation time (h), λ is the time of lag-phase (h), Hmax is the max-
imum hydrogen production (mmol), and Rmax is the hydrogen pro-
duction rate (mmol h−1). These kinetic parameters were estimated us-
ing a nonlinear estimation and least squares, both implemented in the
STATISTICA 8.0 software.
2.4. Analytical methods
Glucose concentration in liquid samples was determined according
to the total carbohydrates method (Dubois et al., 1956). Gas samples
(10 μL) were injected in a gas chromatograph (Thermo Scientific,
Trace 1310) for the hydrogen determination. The chromatograph was
equipped with a thermal conductivity detector (TCD) and a 30 m TG-
BOND (Thermo Scientific) column packed with 5 Å molecular sieve
(0.32 mm × 30 μm). The injector, oven and detector temperatures
were set at 150 °C, 40 °C and 200 °C, respectively. Nitrogen was used
as the carrier gas with a flow rate of 1.5 mL/min.
2.5. DNA extraction and sequencing
To evaluate the potential exoelectrogenic activity in the inoculum,
the community was characterized based on the 16s rDNA gene. Ge-
nomic DNA was extracted from the previously heat-treated anaerobic
sludge. For DNA extraction, a PowerSoil® DNA isolation kit (MOBIO,
USA) was used according to its manufacturer's instructions. The in-
tegrity and concentration of the DNA samples were evaluated by run-
ning samples on an agarose gel stained with SYBR Green (1%) and
quantified by spectrophotometry using a NANODrop 2000c (Thermo
Scientific, USA). Then, DNA was submitted to Research and Testing
Laboratory (RTL, Texas, USA) for Illumina MiSeq sequencing, using
the universal primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R
(GGACTACHVGGGTWTCTAAT) of 16s rDNA gen. Procedure for the
sequence analysis and further analysis of the identified OTUs (Opera-
tional Taxonomic Units) was conducted as described by Barragán-
Trinidad et al. (2017).
Y.A. Del Angel-Acosta et al. Biocatalysis and Agricultural Biotechnology 23 (2020) 101507

3. Results and discussion
3.1. Effect of redox compounds and pH
Fig. 1 shows the results of the accumulative hydrogen production
in batch systems under all experimental conditions tested. The highest
Fig. 1. Accumulative hydrogen production in batch systems with the oxidized
(AQS) and reduced (AH2QS) electron shuttling compounds at 35 °C and differ-
ent initial pH conditions. Symbols represent experimental data and the solid
lines represent the data from the Gompertz model. In all cases the standard
deviation was ≤10%.
hydrogen production was obtained in AH2QS amended cultures at pH
6 with a value of 1.99 mmol, followed by 1.81 mmol with only sludge
treatment, and 1.54 mmol by incubation with AQS. The accumulative
hydrogen production with AH2QS at pH 6 was higher than the cul-
tures with AQS at pH 6 after the incubation period, achieving an in-
crement of 1.26-fold. Nonetheless, similar values were obtained at pH
6.4 and 7.8, with an increment of 1.06- and 1.03-fold, respectively, in
AH2QS incubations respect to AQS. Nevertheless, compared to incuba-
tions containing only sludge, the hydrogen production with AH2QS
was higher only at pH 6, but similar responses were observed in the
treatments at pH 7.8. Hydrogen production at pH 6 with AH2QS
started 10–12 h early the other treatments (Fig. 1). The endogenous
control did not show hydrogen production at any tested condition.
Substrate consumption in AH2QS incubations was higher than the
consumption achieved in other treatments and under the three pH
values tested (Table 1). For instance, at pH 6, the substrate consump-
tion was 62% with AH2QS, 51% with only sludge, and 45% with AQS.
For the pH values of 6.4 and 7.8, the substrate consumptions for all
conditions were in the range of 90–94%, achieving the highest values
for incubations with AH2QS. Final pH values for all treatments were
in the range of 4.77–4.96 (Table 1), which indicates the production of
organic acids due to the substrate fermentation. The hydrogen molar
yield associated with substrate consumption decreased according to
pH increments (Fig. 2A). The hydrogen molar yields obtained at pH 6
were the highest observed compared to pH 6.4 and 7. The values ob-
tained were 3.9 mmol hydrogen for assays with AQS and only sludge
and 3.6 mmol hydrogen with AH2QS, in all cases per mmol of glucose
consumed. These results are closed to the theoretical value of 4 mmol
per mmol of glucose consumed when acetate is the main organic
product. At pH 6.4 and 7.8, a similar response was observed between
the three experimental conditions evaluated. Even though the sub-
strate consumptions were higher at pH 6.4 and 7.8 compared to pH 6
(Table 1), it was not reflected in increments in the hydrogen molar
yield. Previous studies documented that under acidic conditions (pH
4–6) the hydrogen yield was higher compared to pH values of 6.5 and
7 (Fang and Liu, 2002; Lin and Chang, 1999). The pH also affects the
metabolism pathways during hydrogen production. Butyrate and ac-
etate are the most abundant products between pH conductions of 4–7,
but the production of propionate is drastically promoted at pH 6.5
and 7 (Fang and Liu, 2002), which limits the hydrogen molar yield as
observed in this study.
During the last years, there has been an increased interest in re-
newable energy technologies like anaerobic digestion of organic bio-
mass or wastes to produce gaseous biofuels as hydrogen. Between the

Table 1
Kinetic parameters of the modified Gompertz equation for hydrogen production estimated from experimental data in batch systems at 35 °C and different initial
pH of the medium.

Treatment Substrate consumption (%) Final pH Hmax λ Rmax Time for Hmax R2

(mmol) (h) (mmol h−1) (h)

Initial pH 6.0
Only sludge 51.4 ± 3.1 4.84 ± 0.1 1.81 ± 0.10 16.9 ± 1.4 0.170 ± 0.01 36 0.998
AQS 45.7 ± 5.5 4.86 ± 0.2 1.59 ± 0.07 18.0 ± 1.2 0.234 ± 0.01 30 0.995
AH2QS 62.2 ± 2.5 4.77 ± 0.2 1.99 ± 0.04 5.9 ± 0.5 0.203 ± 0.01 18 0.998
Initial pH 6.4
Only sludge 91.9 ± 4.5 4.78 ± 0.2 1.86 ± 0.11 3.8 ± 0.28 0.356 ± 0.02 21 0.998
AQS 90.3 ± 4.0 4.77 ± 0.1 1.68 ± 0.05 4.9 ± 0.32 0.324 ± 0.02 21 0.999
AH2QS 94.1 ± 1.2 4.83 ± 0.3 1.78 ± 0.07 5.5 ± 0.50 1.405 ± 0.08 9 0.999
Initial pH 7.8
Only sludge 92.0 ± 6.5 4.80 ± 0.2 1.17 ± 0.10 6.5 ± 0.53 0.162 ± 0.01 24 0.995
AQS 91.8 ± 4.6 4.96 ± 0.1 1.17 ± 0.08 7.1 ± 0.67 0.174 ± 0.01 21 0.998
AH2QS 94.1 ± 4.2 4.89 ± 0.3 1.21 ± 0.06 6.2 ± 0.55 0.208 ± 0.01 21 0.959

AQS: anthraquinone-2-sulfonate; AH2QS: anthrahydroxyquinone-2-sulfonate; λ: time of lag-phase; Hmax: maximum hydrogen production; Rmax: hydrogen pro-
duction rate; R2: Determination coefficient.

3
Y.A. Del Angel-Acosta et al. Biocatalysis and Agricultural Biotechnology 23 (2020) 101507

Table 2
Different pretreatment methods to enrich hydrogen producing inoculum and
their effect in hydrogen yield.

Pretreatment method Inoculum Substrate Hydrogen Reference

yield

(mmol
H2/mmol
substrate)

Heat at 100 °C for 15 min Digested Glucose 1.78 Wang and Wan
sludge (2008)
Heated in boiling water Anaerobic Glucose 1.10 Hu and Chen
bath for 30 min sludge (2007)
Heat at 65 °C for 30 min Waste Glucose 2.30 Baghchehsaraee
activated et al. (2008)
sludge
Heat at 100 °C for 45 min Waste Glucose 1.84 (Lin and Lay
activated (2004b))
sludge
Heat at 80 °C for 30 min Anaerobic Glucose 3.84 Wang et al.
sludge (2011)
Heat at 85 °C for 90 min Anaerobic Glucose 3.9 - AQS This study
with 1 min of air sludge 3.9 - only
injection every 15 min sludge
3.6 -
AH2QS
Acid, pH 3 for 24 h Waste Glucose 1.81 Chang et al.
Fig. 2. Hydrogen molar yield (A) and specific hydrogen production (B) by activated (2011)
heat-treated sludge under all experimental conditions tested. sludge
Acid, pH 3–4 for 24 h Anaerobic Sucrose 1.30 Mu et al.
strategies to improve hydrogen production by anaerobic bacteria sludge (2007)
Basic, pH 10 with 2 N Anaerobic Sucrose 3.06 Zhu and Béland
through dark fermentation are the control of bioreactor parameters NaOH for 30 min digested (2006)
(pH, temperature, biomass and hydraulic retention times, etc.) and sludge
the use of appropriated substrates and microorganisms (Ghimire et Aeration for one week Waste Glucose 1.96 Ren et al.
al., 2015). Moreover, the addition of electron shuttling compounds, as activated (2008)
sludge
reduced quinones, has also been used to evaluate their impact on hy-
drogen production by using a pure culture of Clostridium beijerinckii
(Hatch and Finneran, 2008; Ye et al., 2013, 2011). In the present while control at pH below 5.5 resulted in the partial substrate degra-
study was evaluated the role of AH2QS, AQS, and pH on hydrogen dation (Fang and Liu, 2002).
production, but using a heat-treated anaerobic granular sludge, which
may suppose a more realistic scenario for engineering applications 3.2. Kinetic parameters from the Gompertz model
compared to pure cultures. Nonetheless, to suppress the hydrogen
consumers such as methanogens and homoacetogens commonly found One of the most used mathematical expressions is the three para-
in anaerobic cultures (Wang and Wan, 2009). There are different pre- meters (lag-phase time, maximum production potential and hydrogen
treatment methods to enrich the inoculum for hydrogen production production rate) Gompertz model, which represents the experimental
(Table 2). Although until now it has not been clarified the specific data in this study in a proper manner, evidenced by the good fit ob-
role of reduced electron shuttles on hydrogen production, but it is served (R2 > 0.95, Table 1). However, this model cannot be used as a
suggested that AH2QS interact with fermentative electron transport predictive model due that the biochemical nature of the fermentation
systems as a secondary electron donor compound, possibly by trans- process is not considered (Valentín-Reyes et al., 2018). Table 1 shows
ferring electrons to ferredoxin (Hatch and Finneran, 2008). the kinetic parameters estimated from the experimental data with the
The results suggest that AH2QS and pH influenced the kinetic pa- modified Gompertz model. Depending on the experimental conditions
rameters and substrate consumption. Some studies conducted without tested, the addition of AH2QS impacted positively some kinetics para-
the addition of electron shuttling compounds reports that hydrogen meters of the Gompertz model. The maximum hydrogen production
production is highly influenced by pH, with a better performance be- (Hmax) in incubations with AH2QS was higher than the other treat-
tween pH values of 5.5–7 (Ghimire et al., 2015) and a notable de- ments at pH 6 (1.99 mmol) and pH 7.8 (1.21 mmol). For the hydro-
crease of the hydrogen activity above pH 7 (Tang et al., 2008; gen production rate (Rmax), the best response in incubations with
Wongthanate et al., 2014), which is in concordance with the results of AH2QS was observed at pH 6.4, followed by pH 7.8, but at pH 6, the
the present study. The low substrate consumption for all treatments at highest value was obtained with AQS. In the incubations with AH2QS
pH 6, adjust with HCl, could be attributable to the decrement in the at pH 6.4, which was buffered with phosphate, Rmax was 3.9- and
pH. The production and accumulation of organic acids caused a de- 4.3-fold higher than the treatments with only biomass and AQS, re-
crease below pH 5 resulting in low final pH and affecting substrate spectively; this was reflected in a substantial decrease in the time to
consumption and hydrogen production (Zhang et al., 2013). In addi- achieve Hmax. The specific hydrogen production rates in the incuba-
tion, it was reported that the gradual decrease in pH (up to pH 4–5) tion at pH 6.4 with AH2QS had a value of 17.5 mmol L−1 h−1, which
can affect hydrogen production due to inhibition of iron-containing was substantially higher than the other treatments (Fig. 2B). The lag
hydrogenase, which is responsible to oxidize ferredoxin (Kapdan and phase (λ) at pH 6 was also decreased in AH2QS incubations compared
Kargi, 2006), and by the inactivation of the microorganisms to the values with AQS and with only biomass. At pH 6.4, the lag
(Penniston et al., 2016). In contrast, sensitive control of pH within the phase with the electron shuttling compounds were higher than the in-
range of pH 5.5 to 7 resulted in complete substrate consumption, cubations without them (Table 1).

4
Y.A. Del Angel-Acosta et al.
The addition of electron shuttling compounds seems to be not able
to favor the accumulative hydrogen production at pH 6.4 and 7.8 as
compared to incubations with only sludge. With exception of the re-
sults at pH 6, the accumulative hydrogen production, Hmax, and lag
phase values were similar between the treatments with AH2QS, AQS,
and only sludge. The acidic condition (pH 6) could be responsible to
favor decrements in the lag phases and increment in Hmax in AH2QS
amended cultures. Under that condition (pH 6) it is less favorable to
suffer an autoxidation on the electron shuttling compound (AH2QS)
compared to pH 7.8 (Uchimiya and Stone, 2009), which would limit
their capacity to act as secondary electron donor for the metabolism
of fermentative bacteria, limiting also their capacity to improve the
hydrogen production. In addition, notable differences in the hydrogen
production rates were achieved with AH2QS at pH 6.4 and 7.8. In a
similar manner to this study, no differences in the total hydrogen pro-
duction were found using C. beijerinckii, reduced AQDS, and glucose,
compared with their respective controls, under the pH conditions of
6.0 (Hatch and Finneran, 2008) and 6.8 (Ye et al., 2012). Nonethe-
less, an increase on specific hydrogen production rate was observed
by using the reduced form of AQDS (Ye et al., 2012).
In spite of that substrate consumption efficiencies in incubations at
pH 6.4 (buffered with phosphates) were higher (90–94%) than pH 6,
this was not reflected in increasing the Hmax, nonetheless, the hydro-
gen production rates (Rmax) were substantially improved, especially
in AH2QS incubations (Table 1). The increase in Rmax in incubations
at pH 6.4 could be associated with a possible synergy between phos-
phate and AH2QS. Phosphate addition in pH 6.4 assays improved the
Rmax 2.09- and 1.38-fold respect to their corresponding values with
only sludge and AQS, respectively at pH 6. But, in the presence of
AH2QS at pH 6.4, the Rmax was 6.9-higher than their respective val-
ues observed at pH 6. The effect of phosphate on hydrogen production
was previously evaluated, documenting an improvement on Hmax
and Rmax using heat-treated sludge (Davila-Vazquez et al., 2011).
Phosphate addition (pH 6.4) was reported also responsible in decreas-
ing the microflora lag phase and functions as alkalinity and phospho-
rous donors (Lin and Lay, 2004a).
3.3. Microbial diversity in heat-treated anaerobic granular sludge
The 16s rDNA bacterial sequencing recovered 30 096 reads in the
inoculum sample, classified in 198 different OTUs, with a Shannon-
Weiner diversity index of 2.9. The two main phyla were Proteobacte-
ria and Firmicutes, grouped in 47 different genera; where
Pseudomonas, and other 5 genera were subdominant (Fig. 3). The ap-
Fig. 3. Microbial diversity at genus level in the heat-treated anaerobic granu-
lar sludge used as inoculum for hydrogen production.
5
Biocatalysis and Agricultural Biotechnology 23 (2020) 101507
plied heat treatment was able to suppress the methanogenic activity,
without methane detection in the biogas produced in every evaluated
condition. In addition, the selection of exoelectrogenic microorgan-
isms in the inoculum, such as genera Pseudomonas, Clostridium and
Bacillus assured the hydrogen production; and also, the possibility of
evaluating the effect of AQS and AH2QS as electron shuttling com-
pounds.
The dominant genera Pseudomonas and Clostririum has been widely
reported to power microbial fuel cells (Jiang et al., 2016; Logan,
2009; Nimje et al., 2009). Their exoelectrogenic activity depends on
different mechanism, for instance Pseudomonas sp. synthetize
phenazine, a redox mediator (Pham et al., 2008), the metabolism of
this microorganisms could responsible to reduce the electron shuttles
as AQS. Clostridium butyricum can make a direct electron transfer by
membrane-bound cytochromes (Park et al., 2001) and Bacillus also
produce redox metabolites (Nimje et al., 2009).
4. Conclusion
The addition of anthrahydroxyquinone-2-sulfonate (AH2QS) during
dark fermentation improves the maximum hydrogen production and
substrate consumption when fermentation started at pH 6. The lag
phase was also shortened compared to the control experiments in the
absence of the reduced electron shuttling compound (AH2QS). When
the fermentation process was carried out at an initial pH of 7.8 and
6.4, the lag phase was not affected by AH2QS addition, whereas the
maximum hydrogen production decreases 46% and 10%, respectively.
However, specific hydrogen production rate values and substrate con-
sumption increase with the addition of the reduced electron shuttling
compound. Additionally, it is important to mention that the reduced
electron shuttling compounds during dark fermentation using heat-
treated anaerobic sludge is a promising strategy for improved hydro-
gen production, but a rigorous pH monitoring and adjustment is es-
sential to ensure the optimal electrochemical performance of quinones
and guarantee an improved hydrogen production process. Finally, the
existence and variety of the correct exoelectrogenic microorganisms in
the inoculum, play a major role in the dark fermentation process to
promote the direct interspecies electron transfer, a mechanism that re-
sults in an improvement in the overall metabolism of the microbial
community.
Author contribution statement
Luis H. Alvarez and Refugio B. Garcia defined the experimental as-
says and advised the PhD student. Yair Del Angel, PhD student, con-
ducted all laboratory tasks related to biohydrogen production and
prepared the manuscript. Julian Carrillo conducted the microbial
analysis. Maria T. Garza supported the administration of the resource.
Acknowledgment
Yair A. Del Angel-Acosta would like to thank CONACYT for the
scholarship received during his doctoral studies. All authors also
thank the Facultad de Ciencias Químicas of the Universidad
Autónoma de Nuevo León for the infrastructure and to the CONACYT
(project 236129) for funding this research.
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