Research Article

Received: 25 April 2021 Revised: 19 June 2021 Accepted article published: 15 July 2021 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.6853

Transient shifts in hydraulic retention times

improve the methane production from
ruminal hydrolysates of agave bagasse
Miguel Vital-Jácome, Martín Barragán-Trinidad, Germán Buitrón* and
Guillermo Quijano

Abstract
BACKGROUND: Valorization of lignocellulosic wastes such as agave bagasse from tequila industries can be enhanced using effi-
cient biomass pretreatments. However, the next challenge requires finding the best process conditions to produce methane
and remove chemical oxygen demand (COD) from the pretreatment hydrolysates. In this work, an innovative feeding strategy
based on alternating hydraulic retention times (HRT) was investigated to improve methane production from ruminal hydroly-
sates of Agave tequilana bagasse.
RESULTS: The ruminal hydrolysate provided a substrate rich in volatile fatty acids (VFA) for direct use in the methanogenic pro-
cess. The alternating feeding strategy increased the volumetric methane production rate by up to 25% (from 0.76 ± 0.05 to
0.95 ± 0.03 L CH4 L−1 d−1) compared to operating at a constant feeding rate, equivalent HRT of 15 h, and organic loading rate
(OLR) of 8 g COD L−1 d−1. The alternating HRT operation improved VFA conversion without modifying the methane yield, and
the COD removal was increased by 14%. The system stability, measured through the alkalinity index, was maintained during
the proposed feeding strategy and after the increase in OLR.
CONCLUSION: The proposed feeding strategy is a promising tool for improving methane production from agave bagasse
hydrolysates. The strategy was robust and easy to apply, which could expand its use for a wider variety of substrates.
© 2021 Society of Chemical Industry (SCI).

Keywords: agave bagasse; alternating feeding; lignocellulosic biomass; methane production; ruminal pretreatment

INTRODUCTION challenge for exploiting the bagasse energy potential to produce

The demand for energy due to the rising human population has
led to the search for renewable energy, including biofuels, which
by 2018 provided around 9% of the global energy supply.1 Biogas
and biomethane produced from municipal organic wastes, waste-
water treatment or agro-industrial residues are now feasible alter-
natives to fossil fuels worldwide.2-4 Using these residues for
renewable energy production has a lower environmental impact
and improves waste management without compromising food
security.5 Many of these wastes are composed of lignocellulosic
biomass, whose complex and recalcitrant nature can complicate
its degradation during anaerobic digestion processes.6 In this
regard, the biodegradability of lignocellulosic biomass can be
improved using physical, chemical, physicochemical and biologi-
cal pretreatment methods.7 In Mexico, great interest is devoted to
agave bagasse as one of the lignocellulosic biomasses whose
revalorization needs to be addressed for reducing the environ-
mental impacts of the mezcal and tequila industries.8,9 The hydro-
lysates obtained from the pretreatment methods applied to
agave bagasse are suitable for producing methane (CH4) by
anaerobic digestion.10 However, after pretreatment, a significant
CH4 is related to the digester operating conditions.
During the anaerobic process, the main objectives are maintain-
ing a stable operation with an adequate biogas flow rate while
optimizing CH4 production and removing chemical oxygen
demand (COD).11,12 To achieve these objectives, several control
strategies have been developed over the years for anaerobic
digestion systems; the vast majority focused on controlling the
substrate feeding rate as the principal variable to manipulate.12
The most straightforward control strategies, such as on/off and
feedback control algorithms (proportional integral derivative con-
trol, PID), are the simplest and easiest to implement. However,
their performance in anaerobic systems is often limited due to
* Correspondence to: G Buitrón, Laboratory for Research on Advanced Processes
for Water Treatment, Unidad Académica Juriquilla, Instituto de Ingeniería,
Universidad Nacional Autónoma de México, Blvd. Juriquilla 3001, 76230
Querétaro, Mexico. E-mail: gbuitronm@iingen.unam.mx
Laboratory for Research on Advanced Processes for Water Treatment, Unidad
Académica Juriquilla, Instituto de Ingeniería, Universidad Nacional Autónoma
de México, Querétaro, Mexico
1

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 www.soci.org
the nonlinear nature of the process.12 In contrast, advanced strat-
egies such as fuzzy logic controls and artificial neural networks are
powerful for modeling and controlling anaerobic systems, but
they have a high complexity level and usually require a large
amount of information regarding the process variables.12,13 More-
over, most control strategies are only tested in simulation models;
laboratory-scale or pilot-scale reactors are rarely applied in full-
scale digesters, mainly because of the lack of online monitoring,
the lack of adequate instrumentation and the high cost associated
with sophisticated control methods.12,14
Process instability has been reported when extreme distur-
bances occur in anaerobic bioreactors.15 However, it was evi-
denced that the application of temporary organic shock loads in
a fermentative process associated with shifts in the hydraulic
retention time (HRT) increase up to 40% the productivity of biohy-
drogen.16 Inspired by such temporary shifts in the hydraulic reten-
tion time, a control strategy was developed for the anaerobic
digestion of agave bagasse hydrolysates.17 The strategy is based
on a fast extremum-seeking approach and uses the anaerobic
digestion model number 2 (AM2) model.18 Such strategy alter-
nates between the HRT values below and above the optimal
model HRT value. Under this operation, the digester is expected
to achieve a CH4 production that will oscillate close to the theoret-
ical maximum. The operation under alternating HRT was tested
with numerical simulations, predicting a potential increase in
CH4 production.17 Nevertheless, this strategy's hypothesis still
needs to be confirmed experimentally for biogas production,
which can be done in a simple way and before implementing
complex control algorithms.
This investigation presents a proof of concept of an innovative
feeding strategy to increase CH4 production from ruminal hydro-
lysates of agave bagasse in an upflow anaerobic sludge blanket
(UASB) reactor. The feeding strategy was based on alternating
HRT values, leading to an alternating organic loading rate (OLR).
The UASB performance under alternating and constant HRT was
compared at average OLR values of 6, 8 and 13 g COD L−1 d−1.
The performance of both feeding strategies was evaluated in
terms of CH4 production rate, COD removal and the profile of vol-
atile fatty acids (VFA) in the effluent.
MATERIALS AND METHODS
Lignocellulosic biomass and ruminal fluid
Agave (Agave tequilana Weber; var. Azul) bagasse was obtained
from a distillery located in Valle de Amatitán, Jalisco, México.
The average composition of the agave bagasse was: 56 ± 2%
Table 1. Composition of ruminal hydrolysate obtained from
pretreatment
Parameter Value
−1
Soluble COD (g L ) 11.44 ± 46
Soluble carbohydrates (g L−1) 1.31 ± 0.08
CODEq. acetate (g L−1) 1.90 ± 0.10
CODEq. propionate (g L−1) 1.88 ± 0.06
CODEq. butyrate (g L−1) 2.60 ± 0.16
Other VFA (g L−1) 1.55 ± 0.08
COD, chemical oxygen demand; CODEq, equivalent COD; VFA, volatile
fatty acids.
2
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M Vital-Jácome et al.
cellulose, 11 ± 1% hemicellulose, 15 ± 1% lignin and 11 ± 1%
extractive compounds; 93 ± 3% total solids and 90 ± 1% volatile
solids (VS). The ruminal fluid came from beef cattle and was
harvested at the municipal slaughterhouse TIF-412, located in
Santiago de Querétaro, Querétaro, México. The sample of ruminal
fluid was rapidly transferred to the laboratory and used straight
away. The ruminal fluid's solids content was 0.13 ± 0.01 g TS g−1
and 0.11 ± 0.01 g VS g−1, respectively.
Agave hydrolysate
Agave bagasse hydrolysis with ruminal fluid was carried out in a
100 L reactor with 80 L working volume at room temperature.
The fermentation was carried out with a bagasse/ruminal fluid
ratio of 9 (g TS g−1 TS), an initial solids content of 10% (w/v) and
McDougall medium as the mineral salt medium.19 The reactor
was operated at room temperature and manually homogenized
(once a day) for 7 days; the pH was maintained at a value of
7 by NaOH addition. At the end of the fermentation, the reactor
content was filtered, and the supernatant (ruminal hydrolysate)
was acidified at pH 2 with phosphoric acid and preserved at 4 °
C. Table 1 presents the composition of the hydrolysate obtained
after ruminal fermentation. Before use, the ruminal hydrolysate
was diluted with tap water to achieve COD concentrations of 5.0
and 8.1 g L−1. The hydrolysate was also supplemented with min-
eral medium reported by Angelidaki et al.,20 based on the follow-
ing stock solutions (g L−1): solution A – NH4Cl, 100; NaCl, 10;
MgCl2.6H2O, 10; CaCl2.2H2O, 5; solution B – K2HPO4.3H2O, 200;
solution D, trace metal solution (g L−1) – FeCl2.4H2O, 2; H3BO3,
0.05; ZnCl2, 0.05; CuCl2 2H2O, 0.038; MnCl2.4H2O, 0.05;
(NH4)6Mo7O24.4H2O, 0.05; AlCl3, 0.05; CoCl2.6H2O, 0.05; NiCl2.6H2O,
0.092; EDTA, 0.5; concentrated HCl, 1 mL; Na2SeO3.5H2O, 0.1;
solution E, vitamin mixture (mg L−1) – biotin, 2; folic acid, 2; pyri-
doxine acid, 10; riboflavin, 5; thiamine hydrochloride, 5; cyanoco-
balamine, 0.1; nicotinic acid, 5; p-aminobenzoic acid, 5; lipoic acid,
5; DL-pantothenic acid, 5. The stock solutions – A, 10 mL; B, 2 mL;
D, 1 mL; E, 1 mL – were added per liter of diluted hydrolysate
and the mixture was neutralized to pH 7.5 with 10 mol L−1 NaOH
addition. Sodium bicarbonate was added to the hydrolysates for
an initial NaHCO3/COD ratio of 0.6 g g−1.21
Inoculum
The methanogenic inoculum was anaerobic granular sludge from
a full-scale UASB reactor treating brewery effluents at Guadala-
jara, Jalisco, México, containing 0.101 g TS g−1 and 0.086 g VS
g−1. The sludge's specific methanogenic activity of 0.71 g CODCH4
g−1 VS d−1 was previously determined using glucose as the sub-
strate in a batch experiment.
Experimental set-up
The experimental set-up consisted of a glass UASB reactor with a
working volume of 2 L and headspace of 0.2 L (71 cm height,
6 cm internal diameter) (Fig. 1). The reactor was equipped with
a coil heat exchanger coupled to a thermo-bath to maintain the
working temperature at 35 °C. A peristaltic pump was used for liq-
uid recirculation, maintaining the liquid ascending speed at
3 m h−1. During reactor operation, biogas flow rates were mea-
sured online with a continuous flow sensor (μFlow, Bioprocess
Control, Lund, Sweden) coupled to a programmable logic control-
ler (PLC) for data acquisition. The volumen is reported as Normal
Liters (NL) considering Standard temperature and pressure condi-
tions of 273.15 K and 1 bar.
J Chem Technol Biotechnol 2021
Methane production from ruminal hydrolysates of agave bagasse
Figure 1. Schematic diagram of the UASB methanogenic reactor.
Bioreactor operation
The reactor was inoculated with 10 g VS L−1 anaerobic granular
sludge. The reactor operation was divided into four different
phases. During phase I or start-up, the reactor was fed with rumi-
nal hydrolysate at a concentration of 5 g COD L−1 and HRT of 20 h,
corresponding to an OLR of 6 g COD L−1 d−1. During phase II, the
HRT was decreased to 15 h, corresponding to an OLR of 8 g COD
L−1 d−1. The biogas flow rate was continuously monitored, and
the CH4 and CO2 content were determined periodically to calcu-
late the volumetric CH4 production rate (VMPR), expressed at
standard conditions for temperature (273 K) and pressure (1 atm).
Effluent samples were taken daily to evaluate the alkalinity, COD
removal and CH4 yield. The concentration of VFA in the effluent
was determined at the end of each phase. Steady state was
reached at the end of each phase, defined by a stability criterion
derived by obtaining for at least three consecutive days a coeffi-
cient of variation of <10% in the VMPR.
Alternating feeding strategy
Phase III began after reaching the steady state in phase II, and the
alternating feeding strategy programmed in the PLC was started.
This strategy consisted of alternating between two HRT values – a
long HRT of 20 h and a short HRT of 10 h – each maintained for
4 h. According to previous experiments (data not shown), apply-
ing the short HRT for more than 4 h led to granular biomass disin-
tegration and potential biomass washout. By maintaining the
long HRT for 4 h, the resulting average HRT was 15 h (OLR of 8 g
COD L−1 d−1), matching the HRT and OLR used in phase II. The
alternating feeding strategy was maintained in phase IV, but the
ruminal hydrolysate concentration was increased to 8.1 g COD
L−1, corresponding to an OLR of 13 g COD L−1 d−1. Effluent sam-
ples were taken daily to evaluate the alkalinity, COD removal
and CH4 yield. The concentration of VFA in the effluent was deter-
mined at the end of phases III and IV. The steady state was consid-
ered when a VMPR coefficient of variation of <10% was recorded
on at least three consecutive days.
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Alkalinity
The alkalinity index (⊍) corresponded to the ratio between alkalin-
ity due to VFA to the total alkalinity (Eqn (1)) and was used as a
control parameter to determine the stability of the anaerobic pro-
cess. An ⊍ value between 0.2 and 0.4 is recommended for a stable
digester operation.22 Alkalinity was determined from a 10 mL
effluent sample taken daily from the methanogenic reactor. Each
sample was titrated with 0.05 mol L−1 H2SO4 from its initial pH to
a pH of 5.75 and the consumed volume was noted (related to the
bicarbonate alkalinity, volpH 5.75); then, the titration continued
until a pH of 4.3 was obtained, measuring the consumed volume
(related to the total alkalinity, volpH 4.3):23
VFA alk volpH 4:3 −volpH 5:75
⊍= = ð1Þ
Total alk volpH 4:3
Analytical methods
Soluble COD concentration was determined using the colorimet-
ric closed reflux method (Hach 435, range from 0 to 1500 mg COD
L−1). The contents of solids (TS and VS) of the agave bagasse,
ruminal fluid, anaerobic sludge and effluent samples were quanti-
fied based on standard gravimetric methods.24 Soluble carbohy-
drates were measured using the colorimetric phenol–sulfuric
acid method.25 Biogas composition was analyzed with a gas chro-
matograph (SRI Instruments Model 8610C, Champaign, IL,USA)
equipped with a thermal conductivity detector (TCD) and two
steel columns (2 m in length; 0.79 mm in diameter). The injector,
column and detector temperatures were 90, 110 and 150 °C,
respectively. N2 was used as the carrier gas at a flow rate of
20 mL min−1. The concentration of VFA in the effluent (acetate,
propionate, butyrate, isobutyrate, valerate, isovalerate and capro-
ate) was analyzed with a gas chromatograph (Agilent Technolo-
gies 7890B, USA) equipped with an HP-FFAP column and a
flame ionization detector. Injector and detector temperatures
were maintained at 190 and 210 °C, respectively. The initial col-
umn temperature was 60 °C (1.5 min), which was then increased
to 90 °C (15 °C min−1), then to 170 °C (25 °C min−1) and main-
tained for 4 min. N2 was used as the carrier gas at a flow rate of
2.5 mL min−1.
Statistical analysis
Experimental results are expressed as mean ± standard deviation.
The differences among measures at the different phases were
determined by analysis of variance tests (one-way ANOVA), fol-
lowed by the post hoc Tukey's multiple comparisons test
(⊍ < 0.05) to identify specific means that are significantly different
from each other.
RESULTS AND DISCUSSION
Ruminal hydrolysate
VFA represented 70% of the soluble COD present in the hydroly-
sate (Table 1), with acetate, propionate and butyrate as the most
abundant VFA. Similar hydrolysate composition has been previ-
ously reported for this type of pretreatment.26 According to the
literature, the microorganisms and enzymes in the rumen hydro-
lyzed the cellulose and hemicellulose into hexoses and pentoses,
which can be further converted into pyruvate and then into VFA
made up of six or fewer carbon atoms.26 The solubilization of
agave bagasse in terms of COD was 0.13 g COD g−1 TS, equivalent
to a hydrolysis efficiency of 15% under the present working
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conditions (considering a ratio of 1.10 g COD g−1 TS for agave
bagasse), consistent with the efficiency reported by Valdez-
Vazquez et al.10 of 14.5% for agave bagasse pretreated with rumi-
nal fluid. The relatively low hydrolysis efficiency is partly related to
the high content of lignin in agave biomass (14–47%) compared
to other lignocellulosic residues such as corn cob (2.9–9%), wheat
straw (20–25%) or sugarcane bagasse (14–22%).27 Lignin has an
inhibitory effect because it reduces the access of cellulolytic
enzymes to the cellulose matrix.7
Hydrolysis efficiencies for agave bagasse in the range of 36–48%
have been reported using chemical pretreatments (diluted
acid),10,28 and up to 80% using combinations of chemical/hydro-
thermal with enzymatic pretreatments.29,30 However, the use of
corrosive chemicals, generation of inhibitory compounds, high
energy demand and requirement of commercial non-reusable
enzymes strongly limit these pretreatments at full scale.26 Bagasse
pretreatment based on ruminal fluid avoids the use and storage of
toxic chemicals and the requirement of high-temperature and
-pressure conditions, making this process affordable and environ-
mentally friendly compared with their physical–chemical counter-
parts.31 Another advantage of using ruminal microorganisms for
pretreatment relies on the direct conversion of structural
4
Figure 2. Time course of (a) VMPR during reactor operation, and (b) box and whisker plot of VMPR at steady state.
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M Vital-Jácome et al.
polysaccharides into VFA instead of sugars (hexoses and pentoses)
obtained by other pretreatment methods.26 The latter simplifies
the anaerobic digestion process since VFA enters directly into the
methanization stage, allowing shorter HRT values using the ruminal
hydrolysates rather than the chemical, hydrothermal or enzymatic
ones. Besides CH4 production, other applications of ruminal hydro-
lysates are related to obtaining and purifying valuable products
such as ethanol, VFA and medium-chain fatty acids (MCFA).26
Regarding the main limitations of agave bagasse pretreatment
with ruminal biota, we must consider the low conversion efficiency
and the long process time of the pretreatment; however, these
drawbacks can be overcome by performing more exhaustive stud-
ies to establish process parameters that allow maximizing the solu-
bilization of particulate material of agave bagasse, including
(i) long-term experiments where acclimation issues to the substrate
can be ruled out, and (ii) discontinuous systems in which the hydro-
lytic activity of ruminal microorganisms can be increased by uncou-
pling HRT from solid retention time (SRT).32,33
Effect of feeding strategy on CH4 production
Reactor start-up was carried out in experimental phase I at an HRT
of 20 h. Figure 2(a) shows that the steady state was reached
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between day 11 and 17, achieving a stable VMPR of 0.53 ± 0.03
NL CH4 L−1 d−1. In phase II, the HRT was reduced from 20 to
15 h, increasing the OLR from 6 to 8 g COD L−1 d−1. During this
phase, the steady state was reached between days 24 and
29 (Fig. 2(a)), achieving a VMPR of 0.76 ± 0.05 NL CH4 L−1 d−1,
which was significantly higher by 43% concerning phase I (Fig. 2
(b)). Therefore, the increase in CH4 production from phase I to
phase II was attributed to the increase in OLR. During phase III,
the proposed feeding strategy was started, alternating the HRT
between 20 and 10 h, with an average HRT of 15 h. The steady-
state operation in phase III was reached between days 32 and
37, achieving a VMPR of 0.95 ± 0.03 NL CH4 L−1 d−1 (Fig. 2(a)).
Despite operating at the same average HRT and with the same
OLR as in phase II, the VMPR increased significantly by up to
25% during phase III (Fig. 2(b)). The behavior observed in phase
III agrees with that expected by the model predictions of Vargas
et al.,17 who estimated a VMPR enhancement by using the alter-
nating feeding strategy. In phase IV, the COD in the influent was
increased from 5 to 8 g COD L−1, increasing the OLR from 8 to
13 g COD L−1 d−1 but maintaining the same alternating feeding
strategy and the same average HRT of 15 h as in phase III. The
steady-state operation was reached between days 39 and 42, with
a VMPR of 1.49 ± 0.02 NL CH4 L−1 d−1 being recorded (Fig. 2(a)).
This VMPR value represented an increase of 57% compared with
that obtained in phase III. The high and stable production of CH4
during phase IV showed that the feeding strategy was robust at
higher OLR values. Compared to phase II (OLR of 8 g COD L−1
d−1), the VMPR recorded in phase IV was up to 96% higher, which
confirmed the superior VMPR under the proposed alternating
feeding strategy compared to the same bioreactor operated at a
constant feeding rate and equivalent HRT and OLR. Principal com-
ponents of biogas were CH4 and CO2. H2S was not measured since
it was not expected from lignocellulosic biomass. The average
CH4 content in the biogas during the experiment was 70 ± 1%,
the other 30 ± 1% being CO2; no H2 or CO was detected
(Table 2). Due to this CH4 content, such biogas can be used for
generating electric and thermal energy in combined heat and
power (CHP) engines.4
Table 2 compares the results herein obtained with studies using
lignocellulosic hydrolysates obtained with other pretreatment
methods at bench scale. The VMPR values herein obtained were
in agreement with values reported for acid, alkali and hydrother-
mal hydrolysates of lignocellulosic substrates such as sugarcane
bagasse, pelletized grass, oat straw and wheat straw,34-38 as well
as with acid hydrolysates of agave bagasse.28,39 The VMPR in
phase IV was comparable with that previously reported for other
pretreatments based on commercial enzymes, acid/base and
hydrothermal processes.36,40 Furthermore, using ruminal hydroly-
sates allowed the use of shorter HRT values while increasing OLR
compared with previous studies. Hence compact and efficient reac-
tors can be implemented with ruminal hydrolysates. It is important
to stress that direct comparisons among the hydrolysates pre-
sented in Table 2 cannot be made due to their different composi-
tions. For instance, up to 64% of the COD present in hydrolysates
obtained with commercial enzymes may come from the enzymatic
cocktail itself,40 leading to overestimated VMPR values. However, it

Table 2. Results from the reactor's steady-state operation

Reactor HRT OLR (g COD CH4 COD removal VMPR CH4 yield (L CH4
Substrate configuration Hydrolysate (h) L−1 d−1) (%) (%) (NL CH4 L−1 d−1) kg−1 COD) Reference

Sugarcane UASB Hydrothermal 18.4 2.4 — 85.7 — 270 34

bagasse
Pelletized CSTR Alkali 480 3.3 — 59 0.73 223 35
grass
Oat straw UASB Enzymatic 24 11.5 — 83 3.5 360 36
(commercial
enzymes)
Oat straw UASB Acid 24 12 — 88 1.5 300 36
Wheat straw UASB Hydrothermal 24 8.2a — — 2.1 256b 37
Wheat straw UASBc Hydrothermal 48 2.8 66.9 71.2 — 267 38
Agave AnSBR Acid 90 1.3 — 72.8 0.3 260 28
bagasse
Agave AnSBR Acid 90 2.7 — 80 0.86 280 39
bagasse
Agave UASB Enzymatic 14 20 70 90 6.4 319 40
bagasse (commercial
enzymes)
Agave UASB Ruminal Phase I 20 6 69.5 55.8 ± 5.0 0.53 ± 0.03 158.1 ± 21.1 Present
bagasse Phase II 15 8 69.0 53.9 ± 1.2 0.76 ± 0.05 184.1 ± 12.7 study
Phase III 15d 8 71.3 61.3 ± 1.7 0.95 ± 0.03 201.6 ± 4.1
Phase IV 15d 13 68.0 53.8 ± 5.3 1.49 ± 0.02 213.5 ± 18.9
a
OLR in g VS L−1 d−1.
b
Methane yield in mL CH4 g−1 VS.
c
Thermophilic conditions (55 °C).
d
Operation under the alternating feeding strategy proposed.
AnSBR, anaerobic sequencing batch reactor; COD, chemical oxygen demand; CSTR, continuous stirred tank reactor; HRT, hydraulic retention time;
OLR, organic loading rate; UASB, upflow anaerobic sludge blanket; VMPR, volumetric methane production rate.
5

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must be highlighted that a short HRT can be set with ruminal
hydrolysates while supporting a VMPR comparable with hydroly-
sates obtained with other pretreatments. In this regard, a detailed
techno-economic analysis is required to establish the most efficient
and cost-effective pretreatment for agave bagasse.
CH4 yield, COD removal and effluent VFA
The biochemical methane potential for agave bagasse hydrother-
mally treated was reported to be as high as 252 L CH4 kg−1 VS
added (178 L CH4 kg−1 COD)41 – very close to the CH4 yield
obtained in the present study and representing about 59% of
the theoretical value (426 L CH4 kg−1 VS) calculated by Kaparaju
et al.38 The CH4 yield and COD removal were key performance
parameters evaluated in the present study (Fig. 3a). A gradual
increase in the CH4 yield value was observed after each phase
(Table 2). The statistical analysis revealed that the CH4 yield was
not significantly affected by individual factors such as the HRT
reduction (phase I vs. phase II), the implementation of the alter-
nating feeding strategy (phase II vs. phase III) or the increase of
the OLR (phase III vs. phase IV). However, the CH4 yield was signif-
icantly affected by the combination of two or more factors, such
Figure 3. Time course of (a) CH4 yield and COD removal during reactor operation, and (b) VFA effluent concentration at steady state.
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M Vital-Jácome et al.
as the HRT reduction plus the feeding strategy (phase I vs. phase
III) or the HRT reduction plus the feeding strategy plus the
increase of the OLR (phase I vs. phase IV). The CH4 yield values
obtained under the feeding strategy in phase III and phase IV were
201.6 ± 4.1 and 213.5 ± 18.9 L CH4 kg−1 COD, respectively, which
agree with the average biochemical methane potential of 219 L
CH4 kg−1 COD reported by Valdez-Vazquez et al.10 for agave
bagasse hydrolysates from several pretreatment methods. These
results indicate that it is possible to achieve CH4 yield values close
to the hydrolysates' maximum anaerobic biodegradability when
operating under the alternating feeding strategy here proposed.
No significant differences were observed between COD removal
values at the steady state of phase I and phase II after HRT reduc-
tion. However, significant differences were observed between
phase II and phase III mediated by implementing the alternating
feeding strategy, increasing COD removal by 14%. Afterwards,
no significant changes were observed in COD removal between
phase III and phase IV due to increased OLR (Table 2). These
results showed that the feeding strategy improves the COD
removal, which explains the 25% increase observed in VMPR with-
out significant changes in CH4 yield. Figure 3(b) shows the
J Chem Technol Biotechnol 2021
Methane production from ruminal hydrolysates of agave bagasse
Figure 4. Time course of pH and alkalinity index during reactor operation.
concentration of VFA in the reactor effluent at the end of the four
experimental phases investigated. Under operating conditions at
constant HRT (phases I and II), up to seven VFA were detected,
including acetate, propionate, butyrate, isobutyrate, valerate, iso-
valerate and caproate. Under the alternating feeding strategy
(phases III and IV), four to five VFA were detected, including propi-
onate, butyrate, isobutyrate and isovalerate, all of which have
been reported to have slower degradation rates among
VFA.42-44 Comparing the effluent of phase II and phase III, the
absence of acetate in phase III should be noted. This result indi-
cates that the feeding strategy favors acetate degradation, which
is the direct substrate for methanogens to methanogenesis. The
presence of acetate in phase IV effluent was attributed to a slight
accumulation related to the increase in OLR. Differences in the
type and concentration of the VFAs identified in the effluent
strongly suggested that the alternating feeding strategy medi-
ated changes in the activity of the microbial community. In this
context, improvements in CH4 production, accompanied by
changes in activity and composition of the microbial community,
have been related to HRT reductions and OLR increases in anaer-
obic processes.45-47 Moreover, the development of more diverse
and resilient microbial communities, as well as an increase in
the abundance of acetoclastic methanogens, have been reported
when operating methanogenic reactors under dynamic feeding
patterns, as in the case of the proposed feeding strategy of this
work.48,49 Therefore, further studies must still be performed to
assess the impact of alternating feeding on the microbial commu-
nity composition to explain the enhanced acetate uptake and
complete absence of valerate and caproate under this innovative
feeding strategy. Regarding the lower COD removal values com-
pared to other hydrolysates (Table 2), this could be related to
the content of possible uncharacterized inhibitors in agave
bagasse generated after hydrolysis, phenolic compounds
released after lignin decomposition,10 as well as the presence of
slow-degrading VFA such as propionate, isobutyrate and
isovalerate.42
Effect of the feeding strategy on the process stability
The process's stability was continuously monitored by measuring
pH and determining the alkalinity index (Fig. 4). A reduction in pH
or an increase in alkalinity index indicates instability due to the
accumulation of VFA. Such accumulation occurs because acido-
genesis has higher production rates than methanogenesis,
J Chem Technol Biotechnol 2021 © 2021 Society of Chemical Industry (SCI).
www.soci.org
causing a decrease in the medium's buffer capacity, inhibiting
the methanogenic microorganisms and leading to system fail-
ure.50 Despite the changes made from phase I to phase IV, the
pH remained at a value of 7.5 ± 0.1, while the alkalinity index
remained at an average value of 0.29 ± 0.05 – i.e., between the
optimal values recommended for CH4 production.22 The observed
stability was likely related to the nature of the ruminal hydrolysate
mainly composed of VFA, allowing methanogenic microorgan-
isms a direct use of the substrate and higher activity, thus avoid-
ing the undesirable accumulation of VFA. In a certain way, using
pretreatment with ruminal fluid is equivalent to carrying out
anaerobic digestion in a two-stage process, where the first stage
is devoted to hydrolysis, acidification and acetogenesis, while
the second stage is devoted to methanogenesis. In a two-stage
process, acidogenic bacteria and methanogens perform their
functions separately, promoting the degradation rate and stabiliz-
ing the anaerobic biological treatment.46,51
During the feeding strategy, the anaerobic process was sub-
jected to exceptional operating conditions, alternating HRT values
every 4 h with the potential risk of system overloading or granule
disaggregation. Granule disaggregation was already documented
when high OLR (low HRT) of acidic hydrolysates of agave bagasse
were used for hydrogen production.52 The UASB configuration is
designed to operate at short HRT and long SRT values, incorporat-
ing high-activity biomass with a higher loading capacity.53 How-
ever, long HRT values are commonly used for lignocellulosic
biomass hydrolysates (Table 2), while instability conditions and
decreases in CH4 yield have been reported at HRT values between
24 and 10 h.37,40 Despite these demanding conditions, the stable
values of pH and alkalinity index observed under phases III and IV
confirmed that the feeding strategy was robust and can be used
without compromising the process stability.
Practical implications of the alternating feeding strategy
The feeding strategy proposed in this work improved CH4 produc-
tion by enhancing the conversion of VFA to CH4 and maintaining
the system's stability. Similar goals have been achieved by other
methods such as adding conductive materials, supplementing
additives and nutrients, balancing the C/N ratio in feedstocks,
applying two-phase anaerobic digestion and implementing com-
plex control algorithms.12,46,51 For fermentations, the temporary
changes in HRT have been proven to increase hydrogen
production.16 In this sense, the proposed feeding strategy has
7
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 www.soci.org
the following advantages compared to other methods: (i) it is an
easy-to-apply strategy because it only requires to enable the
HRT change in the feed pump; (ii) it does not require the addition
or removal of substances in the process, but can still be used in
combination with them; and (iii) because of its simplicity it could
be applied on a larger scale without relying on the extensive
and expensive online monitoring of complex control algorithms.
Regarding the limitations of this feeding strategy, it must be con-
sidered that (i) it requires prior knowledge of the process regard-
ing the minimal HRT at which washout or reactor instability can
occur, and (ii) special care must be taken when wide influent dis-
turbances are expected. Finally, further analysis regarding the
economic feasibility of implementing the operation strategy
herein proposed is needed.
CONCLUSIONS
The proposed feeding strategy increased CH4 production from
ruminal hydrolysates of agave bagasse by up to 25%. Such
enhanced CH4 production (from 0.76 ± 0.05 to 0.95 ± 0.03 NL
CH4 L−1 d−1) was achieved by alternating HRT values, which
improved VFA conversion without modifying the CH4 yield. The
system stability, determined by alkalinity, was remarkable after
OLR increased from 8 to 13 g COD L−1 d−1 at an average HRT as
short as 15 h. Future work should focus on testing the proposed
feeding strategy on various substrates and assessing the impact
of the alternating HRT on the microbial community composition
to understand better the metabolic changes that trigger CH4 pro-
duction enhancement.
ACKNOWLEDGEMENTS
This research was supported through DGAPA-UNAM (PAPIIT
IN105119 project) and Fondo de Sustentabilidad Energetica
SENER-CONACYT (grant number 247006, Gaseous Biofuels Clus-
ter). The fruitful discussions of Alejandro Vargas and the technical
support of Gloria Moreno, Jaime Pérez and Ángel A Hernández are
acknowledged.
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