Journal of Environmental Management 317 (2022) 115388

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Journal of Environmental Management

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

Research article

Two-phase (acidogenic-methanogenic) anaerobic fixed bed biofilm reactor

enhances the biological domestic sewage treatment: Perspectives for
recovering bioenergy and value-added by-products
Rodrigo B. Carneiro a, b, *, Gisele M. Gomes b, Marcelo Zaiat b, Álvaro J. Santos-Neto a
a
Laboratory of Chromatography (CROMA), Institute of Chemistry of São Carlos, University of São Paulo (USP), 400, Trabalhador São-Carlense Ave., São Carlos, São
Paulo, 13566-590, Brazil
b
Laboratory of Biological Processes (LPB), São Carlos School of Engineering, University of São Paulo (USP), 1100, João Dagnone Ave., Santa Angelina, 13563-120, São
Carlos, São Paulo, Brazil

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

Keywords: The organic matter bioconversion into methane during anaerobic digestion (AD) comprises different steps, the
Anaerobic digestion acidogenic and methanogenic phases being clearly distinct in terms of metabolic activities. In this work, new
Biomethane configurations of anaerobic fixed bed biofilm reactors (AFBBR) were operated under conventional methanogenic
Fermentation
conditions (single phase – SP-AFBBR, M1R), and in a sequential two-phase system, acidogenic reactor followed
Kinetics
Methanoregula
by methanogenic reactor (TP-AFBBR, AcR + M2R), in order to verify the impact of the AD phase separation on
Volatile organic acids the overall system performance in operational, kinetics and microbiological aspects. The results indicated that
feeding the methanogenic reactor with the acidogenic effluent stream provided a shorter operating start-up
period (11 and 32 days for SP and TP-AFBBR, respectively), a greater alkalinity generation (0.14 and 0.41 g-
1
CaCO3⋅g-COD−removed for M1R and M2R, respectively), and the optimization of biomethane production (methane
1
yield of 95 and 154 N-mLCH4⋅g-COD−removed for M1R and M2R, respectively). The COD removal kinetics was also
favored in the TP-AFBBR (k1-COD = 1.4 and 2.9 h− 1 for M1R and M2R, respectively), since the soluble fermen­
tation products were readily bioavailable to the biomass in the reactor. Hydrogenotrophic methanogenesis was
the predominant pathway in the M2R, while the Methanosaeta-driven acetoclastic pathway predominated in the
M1R. The greater diversity of Bacteria and Archaea in M2R denotes a better balance between the species that
degrade volatile organic acids from AcR (i.e. Syntrophorhabdus, Syntrophus and Syntrophobacter) and the
hydrogenotrophic methanogens (Methanoregula, Methanolinea and Methanospirillum) that consume the biodeg­
radation products. The estimated bioenergy generation potential (range of 0.39–0.64 kWh⋅m− 3-sewage consid­
ering the COD removed) for full-scale TP-sewage treatment plants evidences the feasibility of energetic recovery
in the domestic sewage anaerobic treatment.

1. Introduction It is therefore necessary to apply alternative decentralized sewage

Sewage treatment is still deficient in many parts of the world,
aggravating public health problems - waterborne diseases (e.g. diarrhea,
cholera and bacterial dysentery), and environmental problems - aquatic
pollution and ecotoxicological impacts along the food chain (Vezzone
et al., 2021). Wastewater treatment systems are often focused on assist
the needs of the urban population, making it impossible for populations
far from large urban centers to access sewage collection and treatment.
treatment systems that are able to attend this demand (Libralato et al.,
2012). In addition, there is a growing trend to face domestic sewage as a
potential source of value-added products that can be recovered, taking
into account the principles of sustainability and circular bioeconomy,
such as water for reuse, fertilizers (nitrogen, phosphorus, potassium and
sulfur), and bioenergy (methane and hydrogen) (Kong et al., 2021; Li
and Yu, 2016; Stazi and Tomei, 2018; Yang et al., 2022).
Anaerobic digestion (AD) is applied in the biological sewage

* Corresponding author. Laboratory of Chromatography (CROMA), Institute of Chemistry of São Carlos, University of São Paulo (USP), 400, Trabalhador São-
Carlense Ave., São Carlos, São Paulo, 13566-590, Brazil.
E-mail addresses: rodrigocarneiro@sc.usp.br (R.B. Carneiro), gisele.gomes@usp.br (G.M. Gomes), zaiat@sc.usp.br (M. Zaiat), alvarojsn@iqsc.usp.br (Á.J. Santos-
Neto).

https://doi.org/10.1016/j.jenvman.2022.115388
Received 10 March 2022; Received in revised form 23 April 2022; Accepted 21 May 2022
0301-4797/© 2022 Elsevier Ltd. All rights reserved.
R.B. Carneiro et al.
treatment around the world, among other factors, as it is a technology of
lower sludge production and lower operational, maintenance and en­
ergy costs compared to aerobic treatment systems (Foresti et al., 2006).
The AD comprises a series of steps that promote the conversion of
complex organic matter into biogas, composed mainly of methane (CH4)
and carbon dioxide (CO2). These steps include hydrolysis, acidogenesis,
acetogenesis, and methanogenesis, which are catalyzed by different
microbial populations and enzymes (Cohen et al., 1980; Gujer and
Zehnder, 1983). Thus, the process occurs through the association be­
tween hydrolytic, acidogenic, acetogenic, and methanogenic microor­
ganisms. The consumption of hydrogen, formate and acetate by
methanogenic archaea affects the metabolism of other microbial com­
munities. Formation of metabolites by fermentative bacteria is shifted to
more oxidized products, whereas acetogenic bacteria are only able to
metabolize compounds when methanogenic archaea consume hydrogen
and formate efficiently (Stams et al., 2012). Therefore, the good effi­
ciency of the anaerobic digestion depends on a balance between the
production and consumption of the intermediary products from the
fermentation process – short chain volatile organic acids (VOA), solvents
(alcohols and acetone), biohydrogen, and carbon dioxide.
Several factors can affect the AD performance, such as pH, alkalinity,
temperature, macro and micronutrient composition, proportion among
the elements carbon, nitrogen and phosphorus (C:N:P), applied organic
loading rate, hydraulic retention time, food/microorganism ratio (F/M),
inoculum source (Daud et al., 2018; Roopnarain et al., 2021; Sella et al.,
2021). Therefore, some conditions of imbalance or stress, such as
organic overload or the toxic materials input to the reactor, can disfavor
the methanogenic pathway, increasing CO2 and decreasing CH4 content
in the biogas, since methanogenic archaea are more sensitive to load and
pH shocks than fermentative bacteria due to kinetic and thermodynamic
limitations (Hanvajanawong et al., 2022; Mota and Zaiat, 2018).
An alternative to improve anaerobic digestion and decrease the
possibility of methanogenesis collapse is to promote the separation of
the methanogenic step from the acidogenic step in the treatment system.
This can be achieved through a system with anaerobic reactors in series -
acidogenic reactor followed by a methanogenic reactor. Due to the se­
lective pressure imposed by the phase separation, in the first reactor,
microbial reactions of hydrolysis of complex organic matter and gen­
eration of soluble fermentation products (organic acids and solvents)
prevail, while in the second reactor, acetoclastic and hydrogenotrophic
methanogenic archaea prevail, which promote the conversion of these
by-products to biomethane (Fuess et al., 2017; Nabaterega et al., 2021).
Phase separation in anaerobic digestion was identified in the 1980s
as promising for the degradation of high-strength industrial liquid
wastes, aiming at reduction of wastewater treatment plant capital and
operating costs, and higher net energy recovery (Ghosh et al., 1985). For
municipal sewage, initial studies were more focused on optimizing the
sludge stabilization (Fongsatitkul et al., 1995). Van Ginkel et al. (2005)
and Fernandes et al. (2010) demonstrated the possibility of generating
hydrogen from domestic sewage fermentation, reaching 40 and 200
mL-H2⋅g− 1-COD, respectively, which shows a scenario of bioenergetic
diversification from this substrate. Guerrero et al. (2009) observed a
biogas generation potential in the two-phase anaerobic digestion pro­
cess 97% higher than that obtained in the one-phase system treating
synthetic domestic wastewater. Despite the mentioned advantages,
there are no reports of the use of two-phase anaerobic reactors in
full-scale sewage treatment plants, due to the lack of experience with the
process, and difficulties in design, construction, and operation (Mota
and Zaiat, 2018). Furthermore, it is clear that economic issues impose a
barrier to the advancement of this technology, which can be overcome
by the energy valuation of sewage, and taking into account the
value-added products that will be generated.
Anaerobic fixed bed biofilm reactors (AFBBR) have been pointed out
as a promising alternative to conventional anaerobic sewage treatment
systems, since they have the ability to promote an excellent retention of
active biomass in the reaction bed, enabling the maintenance of a high
2
Journal of Environmental Management 317 (2022) 115388
sludge retention time and an inexpressive solids washout in the effluent
stream, which is usual in conventional reactors, e.g. UASB (Up-flow
Anaerobic Sludge Blanket) reactors and septic tanks. Some disadvan­
tages of these reactors are related to clogging of the bed interstices,
channeling and hydraulic short circuit zones, but these problems are
mitigated by applying the ordering of the support material inside the
reaction bed (Carneiro et al., 2020; Mockaitis et al., 2014). The anaer­
obic fixed bed biofilm reactors work as a system of immobilized biomass
on a support bed, which has been presented in different materials, e.g.
polyurethane foam, vegetal carbon, synthetic pumice, ceramic, low- and
high-density polyethylene. The formation of the biofilm in this support
media allows the creation of a favorable environment for the growth and
interaction interspecies of anaerobic microorganisms – fermentative
bacteria and methanogenic archaeas (Garcia et al., 2008; Ratusznei
et al., 2000; Zinatizadeh et al., 2017). Therefore, the application of
AFBBR can lead to an effective bioenergetic recovery from sewage, as
they are more compact units with greater operational stability, and
allow a higher organic load applied without compromising the treat­
ment system (Carneiro et al., 2019, 2020).
Given the scenario presented, this work aims to establish an anaer­
obic acidogenic-methanogenic two-stage treatment system to optimize
the biomethane production in sewage treatment during AD. For this
purpose, a comparison will be made for the first time of single-phase
(methanogenic reactor) and two-phase (acidogenic reactor + meth­
anogenic reactor) systems in anaerobic fixed bed biofilm reactors, in
order to verify the impact of this phase separation on domestic sewage
treatment, clarifying operational, kinetic and microbiological aspects. In
addition, it is intended to highlight the possibility of generating and
using bioenergy from sewage by applying the AFBBR, as well as the
recovery of soluble fermentation products, such as volatile organic
acids, in order to make the process economically sustainable.
2. Material and methods
2.1. Single- and two-phase anaerobic reactors: System configuration and
operating conditions
Fig. S1 shows a layout of the treatment systems used in the study,
which consists of two bench-scale anaerobic fixed bed biofilm reactors
(AFBBR) - a single-phase (SP-AFBBR, methanogenic), and a two-phase
reactor (TP-AFBBR, acidogenic followed by methanogenic). The useful
volume of each reactor is 2 L, and the fixed bed contains as support
material eight vertical strips of polyurethane foam (length of 52 cm,
base of 1 cm2, and mass of 7 g) along the reaction bed. The foam was
selected because it is a low-cost support material, easy to acquire, and it
offers excellent conditions for biomass adhesion and biofilm growth
(Carneiro et al., 2019; Ribeiro et al., 2005). In order to facilitate dis­
cussion of the results and the comparison between the systems, the
methanogenic reactor of the SP-AFBBR was abbreviated as M1R, while
the methanogenic reactor of the TP-AFBBR was abbreviated as M2R, and
the acidogenic reactor is the AcR.
The reactors were kept in a chamber with temperature control at
30 ◦ C, and are fed by peristaltic metering pumps. The hydraulic reten­
tion time (HRT) adopted for the operation of each system is 12 h, ac­
cording to Carneiro et al. (2019). Thus, in the TP-AFBBR system each
reactor (acidogenic or methanogenic) was operated under a 6 h- HRT.
For comparison purposes, both systems (SP- and TP-AFBBR) were
operated with the same applied organic loading rate (OLR) of 2
kg-COD⋅m− 3⋅d− 1, representative for the domestic sewage treatment
(Chernicharo, 2015). The feed substrate consisted of a lab-made sewage
with a theoretical chemical oxygen demand (COD) of 1.0 g-COD⋅L− 1,
whose composition is detailed in Table S1 of Supplementary Material.
The total operating time of the bioreactors was 133 days.
A granular sludge from a UASB reactor in operation treating waste­
water from a poultry slaughterhouse (Avícola Dacar S.A., located in the
city of Tietê, SP, Brazil) was used for the inoculum of the M1R and M2R.
R.B. Carneiro et al.
This inoculum has been successfully applied in the domestic sewage
anaerobic digestion (Carneiro et al., 2019), as it has a high diversity of
metabolic genes from microbial communities involved in the catabolic
cycles of carbon, nitrogen, and phosphorus (Delforno et al., 2017). In the
methanogenic reactors, the adhered biomass remained in contact with
the foam for 4 h before starting to feed them. For the AcR, an acid
pre-treatment (pH 3, HCl 5 mol L− 1) was carried out on the same
inoculum of the methanogenic reactors, prior to the immobilization of
the biomass on the support material. In this case, the biomass remained
in contact with the foam for 24 h before starting to feed the reactor,
according to the methodology described by Penteado et al. (2013). For
the AcR start-up, a higher OLR (24 kg-COD⋅m− 3⋅d− 1) was applied for 2
days, under an HRT of 2 h in order to eliminate the methanogenic
archaea from the reaction bed. Then, the OLR of the AcR returned to the
original design value, and its effluent was collected in a 20 L reservoir to
be the feed substrate for the M2R (Fig. S1). The concentration of volatile
solids in the raw inoculum was 22.9 ± 0.5 g L− 1, and each reactor
presented a concentration of adhered biomass equivalent to 2.93 ± 0.07
g L− 1, 3.14 ± 0.08 g L− 1, and 3.26 ± 0.05 g L− 1, respectively for M1R,
M2R, and AcR.
2.2. Analytical methods
2.2.1. Physicochemical analysis
The physicochemical analysis of each reactor were performed twice
or three times a week for the following parameters - pH, alkalinity, COD,
total carbohydrates (CH) and biogas composition. Analysis of suspended
solids and soluble fermentation products (volatile organic acids - acetic,
propionic, butyric, isobutyric, valeric, isovaleric, caproic, and lactic;
solvents - ethanol, acetone, methanol and n-butanol) in the effluent
streams were performed once a week to monitor the treatment systems
performance. Conventional parameters were analyzed according to the
Standard Methods for the Examination of Water and Wastewater (Baird
et al., 2017) - pH (4500-H+ B, electrometric method); total and volatile
suspended solids (TSS - 2540-E, VSS - 2540-E); COD (5220-D: closed
reflux, colorimetric method). The biogas composition (carbon dioxide,
methane, sulfide, nitrogen and hydrogen) was determined by gas
chromatography with a thermal conductivity detector (GC/TCD), as
detailed in Lebrero et al. (2016) and Perna et al. (2013). The biogas flow
rate (BFR) was measured using a semi-continuous gas meter with a
J-tube hydraulic valve, according to Veiga et al. (1990), and the discrete
data was continuously acquired using an in-house developed software.
According to the number of pulses recorded, the volume of biogas pro­
duced was obtained, following the pre-established J-tube calibration.
The other parameters were determined according to the following
methods: alkalinity, titrimetry – Ripley et al. (1986); total carbohy­
drates, phenol-sulfuric method – Dubois et al. (1956); volatile organic
acids and solvents, gas chromatography – Adorno et al. (2014); lactic
acid, colorimetric method – Taylor (1996).
2.2.2. Microbiological analysis
To carry out the microbiological characterization of the biomass
present in each reactor and compare them with each other and with the
original inoculum, fragments of polyurethane foam were collected in the
reaction bed at the end of the operational period, and the adhered
biomass was detached by washing the foam with PBS (phosphate buff­
ered saline - 137 mmol L− 1 sodium chloride, 10 mmol L− 1 phosphate,
2.7 mmol L− 1 potassium chloride, pH 7.4). The genomic DNA extraction
from the biomass was achieved by applying the FastDNA™ SPIN Kit for
Soil DNA Extraction (MP Biomedicals). The primer sets used for the 16S
rRNA gene were 341F (CCTACGGGNGGCWGCAG (Klindworth et al.,
2013)) and 806R (GGACTACNVGGGTWTCTAAT (Caporaso et al.,
2011)) for the Bacteria and Archaea domains. The sequencing of DNA
samples and the bioinformatics analysis were performed by the com­
pany NGS (Piracicaba, SP, Brazil) adopting Illumina MiSeq technology.
The sequence data were deposited in the NCBI (National Center for
3
Journal of Environmental Management 317 (2022) 115388
Biotechnology Information) database – project access number
PRJNA814683.
To compare the species diversity of the samples, the ecological
indices - Dominance (D), Diversity (Shannon-Wiener - H), and Richness
(Chao-1) - were calculated using the statistical software PAST (PAle­
ontological STatistics, version 4.03, https://www.nhm.uio.no/english/
research/infrastructure/past/). The biomass microscopic evaluation
was performed by using a Leica phase contrast optical microscope
coupled to an Optronics camera with image capture, and the software
Image-Pro Plus 4.5.
2.3. Calculations
The performance assessment of the reactors was monitored in terms
of organic matter removal efficiency, taking into account the COD and
the conversion of total carbohydrates, according to Equation (1). In
addition, the reactors were evaluated in terms of methane production,
for the following response-variables: molar methane flow rate (MMFR,
in N-mmolCH4⋅L− 1⋅d− 1, Equation (2)); volumetric methane production
rate (VMPR, in N-mLCH4⋅L− 1⋅d− 1, Equation (3)); and methane yield
1
(MY, in N-mLCH4⋅g-COD−removed , Equation (4)). Considering the bio­
methane production in each system (SP- and TP-AFBBR), a comparison
was made in terms of the energetic potential of the biogas (EPCH4, in
1
kJ⋅g-COD−removed , Equation (5)).
Cinfluent − Ceffluent
Removal (%) = .100 (1)
Cinfluent
BFR . XCH4 . nbiogas
MMFR = (2)
Vbiogas
BFR . XCH4
VMPR = (3)
Vreactor
BFR . XCH4
MY = ( ) (4)
SFR CODinfluent − CODeffluent
EPCH4 = MY . HVCH4 (5)
where, C represents the COD or CH in mg⋅L− 1; XCH4 is the percentage of
methane in biogas; nbiogas and Vbiogas are the number of moles and the
volume of biogas (L) at standard temperature and pressure conditions
(0 ◦ C, 1 atm); BFR is the biogas flow rate in mL⋅d− 1. SFR is the influent
substrate flow rate of each reactor in L⋅d− 1; and HVCH4 is the heating
value of methane - 40.68 kJ⋅N-mLCH−4 1 (Wang et al., 2018).
The stability of the anaerobic digestion process was also monitored
in terms of the alkalinity generated in each reactor and the relation
between intermediate alkalinity (relative to volatile acids) and partial
alkalinity (relative to bicarbonate) (IA/PA ratio) (Ripley et al., 1986).
The relation between PA produced and COD consumed in each reactor
was used to compare the performance of both methanogenic reactors in
terms of buffering capacity against variations in pH of the medium. In
addition, the organic mass balance in terms of COD was performed to
assess the consistency of the results obtained in each reactor, as detailed
in section S6 of the Supplementary Material.
2.4. Organic matter removal kinetics assessment
The kinetics of organic matter removal was assessed in terms of COD
decay along the spatial profile of the reaction bed, through samples
collected at the side sampling points of each reactor (from P0–P5,
Fig. S1). The experimental data obtained were adjusted by a first-order
kinetic model with residual concentration (Equation (6)), since the flow
pattern in the reactors was assumed to be an ideal plug-flow model,
according to the previous hydrodynamic assays carried out by Carneiro
et al. (2019). In Equation (6), CODHRT is the COD along the reaction bed
for each HRT, COD0 is the COD in the influent stream (HRT = 0), CODR
R.B. Carneiro et al. Journal of Environmental Management 317 (2022) 115388

is the COD residual in the reactor when the value of the reaction rate is monitoring of each reactor (M1R, AcR and M2R), in each considered
null. The parameter k1 stands for the apparent first-order kinetic coef­ system (SP-AFBBR and TP-AFBBR). As can be seen in Fig. 1, the removal
ficient, as it embodies the intrinsic kinetic constant as well as the in­ of organic matter in terms of COD was quite stable throughout the entire
ternal and external mass transfer resistances (Camargo et al., 2002). operational period of the reactors (133 days), reaching stability above
Each sampling point represents a fraction of the total HRT of each 90% at 11 and 32 days for SP and TP-AFBBR, respectively. SP-AFBBR
reactor – from 0 to 12 h in M1R, from 0 to 6 h in AcR and M2R. The VOA presented an overall average COD removal efficiency of 86 ± 16%,
content was also assessed at each sampling point in order to elucidate while TP-AFBBR reached 92 ± 7%, which denoted the importance of the
the main metabolic pathways during the anaerobic digestion in each acidogenic phase during the start-up of the system. After this period, the
bioreactor. The adjustments of the kinetic model obtained were made by COD removal in both systems was statistically the same (95 ± 3%, 96 ±
applying Origin software, version 8.5. 2%, respectively for SP- and TP-AFBBR), demonstrating the stability of
the reactors in the removal of organic matter.
CODHRT = (COD0 − CODR ) . e(− k1 . HRT)
+ CODR (6) Regarding the conversion of total carbohydrates, it was practically
complete in both systems, with the residual concentration in each
reactor below or very close to the detection limit (5 mg L− 1), since the
2.5. Statistical analysis
carbohydrates were easily assimilated by the biomass, and showing that
the hydrolytic step was not an impediment to the good performance of
The statistical differences between the two systems (SP- and TP-
anaerobic digestion. In the TP-AFBBR system, the prior bioconversion of
AFBBR) were calculated by applying Analysis of Variance (ANOVA) of
carbohydrates in the AcR (95 ± 7%) into soluble fermentation products
one factor. The normal distribution of the experimental data was veri­
easily assimilated by acetogenic/methanogenic microorganisms,
fied by Shapiro-Wilks tests. To confirm the significant differences among
notably the volatile organic acids, facilitated their adaptation in the
the bioreactors, a Tukey’s post hoc multiple comparison test was
reactor and enhanced the methanogenic pathway in the M2R.
applied. Significant differences were considered when p-value ≤ 0.05,
The better performance of TP-AFBBR compared to SP-AFBBR was
assuming a 95% confidence interval. All statistical analyzes were carried
also evidenced by the biogas composition in each methanogenic reactor.
out using the PAST software, version 4.03.
The percentage of methane in biogas (XCH4) is significantly higher in
M2R (89 ± 6%) compared to M1R (76 ± 7%), as can be seen in Fig. 2.
3. Results and discussion Furthermore, according to the calculations carried out regarding the
biomethane production, it can be seen from Fig. 3 that this was signif­
3.1. Single-phase (SP-) and two-phase (TP-) AFBBR: comparative icantly higher in the M2R – methane yield (MY) = 95 ± 27 and 154 ± 42
performance assessment 1
N-mLCH4⋅g-COD−removed , for M1R and M2R, respectively. The mass bal­
ance in terms of organic load removal in each reactor (Table S3) denotes
Table 1 presents the main results of the operational performance that the MY results are consistent with the COD removal. The acidogenic
reactor presented a low percentage of methane in the biogas (6.9 ±
Table 1 1.8%, Table S2), however this shows that part of the influent COD was
Performance monitoring parameters of the SP- and TP-AFBBR. Values in bold are converted to methane, and that the acidic pretreatment applied was not
the averages and values in brackets are the standard deviations.
sufficient to eliminate all methanogens microbia from the reaction bed,
Parameter (unit) SP-AFBBR TP-AFBBR which will be further discussed in Section 3.4.
1
M1R AcR M2R AcR + The observed biogas production was 233 and 348 mL⋅L−reactor ⋅d− 1
M2R treating an OLR of 2.0 and 2.6 kg-COD⋅m− 3⋅d− 1, respectively for M1R
OLRinfluent (kg- 2.0 (0.3) 4.0 (0.7) 2.6 (0.6) 2.0 (0.4) and M2R. Kora et al. (2020) evaluated an anaerobic moving bed biofilm
COD⋅m− 3⋅d− 1) reactor (AnMBBR) with sponge cubic form carriers treating municipal
OLRremoved (kg- 1.7 (0.5) 1.4 (0.6) 2.3 (0.6) 1.8 (0.4) wastewater and observed an average biogas production of 173
COD⋅m− 3⋅d− 1) 1
mL⋅L−reactor⋅d− 1 and a COD removal of 69% for an applied OLR of 2.1
HRT (h) 12 6 6 12
CODinfluent (mg⋅L− 1) 1013 (83) 1017 661 (127) 1017
kg-COD⋅m− 3⋅d− 1. A competitive advantage is observed in AFBBR
(105) (105) compared to AnMBBR, evidencing that the conformation and mode of
CODeffluent (mg⋅L− 1) 135 (160) 661 (127) 77 (63) 77 (63) operation of the reaction bed can also influence the efficiency of the
COD Removal (%) 86 (16) 35 (12) 88 (10) 92 (7) anaerobic reactors.
CHinfluent (mg⋅L− 1) 838 (176) 803 (180) 41 (55) 803 (180)
Fig. 4 shows the concentration of volatile organic acids in the
CHeffluent (mg⋅L− 1) 4 (2) 41 (55) 5 (2) 5 (2)
CH Removal (%) 100 (0) 95 (7) 65 (25) 99 (0) effluent from AcR throughout the operation, and the resulting average
pHinfluent 7.73 6.83 5.73 6.83 composition. It is noted that the most relevant acid production routes
(0.19) (0.06) (0.50) (0.06) were the following, in this order: propionic > acetic > isovaleric >
pHeffluent 7.35 5.73 7.40 7.40
butyric > valeric > isobutyric > caproic > lactic. Solventogenic routes
(0.37) (0.50) (0.17) (0.17)
TAinfluent (mg-CaCO3⋅L− 1
) 327 (82) 44 (7) 195 (104) 44 (7)
were not relevant in the AcR, presenting a production of less than 2% in
TAeffluent (mg-CaCO3⋅L− 1
) 500 (100) 195 (104) 337 (127) 337 (127) terms of COD equivalent for the total identified soluble fermentation
PAinfluent (mg-CaCO3⋅L− 1
) 248 (67) 24 (5) 24 (36) 24 (5) products - acetone (0.85%), methanol (1.4%), ethanol (0.07%), butanol
1
PAeffluent (mg-CaCO3⋅L− ) 359 (113) 24 (36) 247 (88) 247 (88) (0.55%).
IA/PAeffluent 0.62 NAa 0.36 0.36
The low OLR applied to AcR (4.0 ± 0.7 kg-COD⋅m− 3⋅d− 1) linked to
(0.85) (0.13) (0.13)
BFR (mL⋅d− 1) 465 (113) NDb 695 (95) 695 (95) the predominance of the propionic pathway might explain the negligible
MMFR (N- 16.2 (4.0) NA 27.9 (4.2) 27.9 (4.2) percentage of biohydrogen in biogas in this reactor (0.53 ± 0.02%,
mmolCH4⋅L− 1⋅d− 1) Table S2). Maintaining the concentration of propionic acid low in the
VMPR (N-mLCH4⋅L− 1⋅d− 1) 182 (45) NA 312 (47) 312 (47) acidogenic reactor would be essential to optimize the bioH2 production,
MY (N-mLCH4⋅g- 95 (27) NA 154 (42) 154 (42)
1
COD−removed )
since the formation of this acid consumes bioH2 in an energetically
1
EPCH4 (kJ⋅g-COD−removed ) 3.9 (1.1) NA 6.3 (1.7) 6.3 (1.7) favorable reaction (ΔG < 0) during the anaerobic digestion, as can be
TSSeffluent (mg⋅L− 1) 42 (14) 69 (16) 42 (15) 42 (15) observed by the following (Saady, 2013).
− 1
VSSeffluent (mg⋅L ) 29 (8) 51 (13) 30 (12) 30 (12)
a
C6 H12 O6 + 2 H2 →2 CH3 CH2 COOH + 2 H2 O ΔG = − 279.4 ​ kJ
NA - Not applicable.
b
ND – Not detected. Another important point regarding the performance of the

4
R.B. Carneiro et al. Journal of Environmental Management 317 (2022) 115388

Fig. 1. Temporal profile of COD removal efficiency in the SP- and TP-AFBBR.

Fig. 2. Temporal variation of the percentage of CH4 and CO2 in biogas fraction in the methanogenic reactors.

Fig. 3. Boxplot of MMFR (a), VMPR (b), MY (c) and EP (d) in the methanogenic reactors.

5
R.B. Carneiro et al.
Fig. 4. Volatile organic acids concentration throughout the AcR-operational period (a) and effluent average composition (b).
bioreactors is the IA/PA ratio. It remained quite stable in both meth­
anogenic reactors (M1R = 0.33 ± 0.07; M2R = 0.34 ± 0.11), except for
the start-up period of the M1R (IA/PA = 1.74 ± 1.46), in which it pre­
sented non-ideal values, which is also consistent with the higher XCO2 in
the biogas (Fig. 2a). According to Ripley et al. (1986), when the AI/AP
ratio starts to become greater than 0.3, there may be a tendency towards
an imbalance in anaerobic digestion, with a direct impact on hydro­
genotrophic/acetoclactic methanogenesis. Nonetheless, it was not
observed after the reactors reached steady-state conditions. It should be
noted that the acidogenic reactor did not receive any additional alka­
linity dosage in order to favor the acidogenic route over the methano­
genic route. Therefore, the M2R did not suffer any negative impact due
to the absence of partial alkalinity (relative to bicarbonate) and unfa­
vorable pH for methanogenesis in the effluent of the AcR (5.7 ± 0.5).
This was also evidenced by the operational results shown so far
(Figs. 1–3), in addition to the higher production of partial alkalinity per
organic load removed (M1R – 0.14 ± 0.05; M2R – 0.41 ± 0.22
1
g-CaCO3⋅g-COD−removed ). Furthermore, the predicted energetic potential
from the bioCH4 production was considerably higher for the M2R, about
1
1.6 times higher (M1R – 3.9 ± 1.1 kJ⋅g-COD−removed ; M2R – 6.3 ± 1.7
− 1
kJ⋅g-CODremoved), showing the feasibility of TP-AFBBR in bioenergy
recovery.
3.2. Organic matter removal kinetics assessment
The COD removal kinetics was evaluated through spatial profiles
along the reaction bed of each reactor. Fig. 5 presents the results ob­
tained from each reactor and for each sampling carried out, and Table 2
clarifies the adjustment data of the kinetic models. It can be seen that the
first-order kinetic model fitted very well to the experimental data, given
the high correlation coefficient (R2 > 0.98) for all adjustments. It was
found that the COD removal in the AcR in the two-phase system enabled
a more favorable removal kinetics in the methanogenic phase, compared
to the single-phase methanogenic reactor (k1-M1R = 1.35 ± 0.30 h− 1;
k1-M2R = 2.87 ± 1.84 h− 1), and also a lower residual concentration (CR-
M1R = 71.8 ± 5.4 mg L− 1; CR-M2R = 48.1 ± 5.1 mg L− 1). The more
Fig. 5. COD removal spatial profiles along the SP- (a) and TP-AFBBR (b). The points refer to the experimental data and the dotted lines indicate the adjustments of
the kinetic model obtained by applying the Origin software (version 8.5).
6
Journal of Environmental Management 317 (2022) 115388
favorable kinetics of organic matter removal in the M2R was also evi­
denced by the higher bioCH4 production of and its percentage in biogas
(Figs. 2 and 3).
The adjusted kinetic model points out to a possibility of reducing
HRT in each reactor without compromising the COD removal efficiency,
since that at the first sampling point of the reaction bed the bioreactors
have already reached removal stability. The ideal plug-flow reactors
feature small intensity dispersion, indicated by the dispersion number
(D/u.L) < 0.01. According to Levenspiel (1999), taking into account the
first order kinetic model applied to the bioreactors, it is obtained
Equation (7). From this equation, applying the COD removal efficiency
obtained for each reactor (M1R and M2R = 92%; AcR = 39%), k1 ac­
cording to Table 2, and D/uL = 0.01, the minimum HRTs that could be
applied in order to ensure the efficiencies reported in the kinetic tests
were estimated, as follows: HRT-M1R = 1.9 h, HRT-M2R = 0.9 h, and
HRT-AcR = 0.4 h. Therefore, this estimated minimum HRT indicates a
reduction of the reaction volume, reducing the predicted implementa­
tion and operating costs for full-scale sewage treatment plants (STP). It is
worth mentioning that the kinetics of organic matter removal can sup­
port the design and sizing of reactors, for optimization of the applied
HRT and estimation of the COD removal in full-scale STPs.
[ ]
− k1 . HRT+(k1 . HRT)2 uL
D
CODR
=e (7)
COD0
Regarding the CH removal in both systems (Fig. S2), it was noted that
the CH bioconversion into volatile organic acids and their subsequent
consumption occurred very quickly in the methanogenic reactors, since
the VOA was not detected along the reaction bed, evidencing their
production and consumption right at the bottom of the reactor. None­
theless, the VOA in the AcR were formed along the spatial profile, with
the maximum value found at the fifth sampling point (P4), as can be seen
in Fig. S3.
R.B. Carneiro et al. Journal of Environmental Management 317 (2022) 115388

Table 2
First-order kinetic expressions estimated for the COD removal in both treatment systems – SP- and TP-AFBBR. Values in bold are the averages and values in brackets are
the standard errors.
Parameter SP-AFBBR TP-AFBBR

M1-R AcR M2R

CODo CODR k1 (h− 1) R2 CODo CODR k1 (h− 1) R2 CODo CODR k1 (h− 1) R2

(mg⋅L− 1) (mg⋅L− 1) (mg⋅L− 1) (mg⋅L− 1) (mg⋅L− 1) (mg⋅L− 1)

1st 889.0 (2.1) 77.5 (0.9) 1.42 0.999 881.2 541.7 0.98 0.986 517.0 (8.5) 43.8 (3.5) 2.93 0.999
sampling (0.09) (28.6) (14.3) (0.23) (1.53)
2nd 1035.0 66.0 (5.3) 1.28 0.999 1001.1 600.3 1.51 0.979 639.0 (8.9) 52.3 (3.7) 2.80 0.999
sampling (12.8) (0.29) (38.6) (17.2) (0.70) (1.03)

3.3. Potential for recovering bioenergy and value-added by-products in
sewage treatment plants
The results presented indicated the competitive advantage of TP-
AFBBR over SP-AFBBR. Considering the energy balance for both re­
actors, there is a greater potential for bioenergy recovery from the TP-
system. In Table 3, an estimation of EPCH4 was made in full-scale
anaerobic sewage treatment plants. It can be noted an expressive po­
tential for bioenergy generation, range from 17 to 75 MWh⋅d− 1
(0.39–0.64 kWh⋅m− 3-sewage considering the COD removed in each
STP) for the TP-system, which can make feasible the phases separation
during anaerobic domestic sewage treatment, since this energy can
contribute to the reduction in costs of maintenance and operation in the
STP, and generate an energy surplus into the municipal electricity net.
Taking into account that the kinetics of organic matter removal (Fig. 5
and Table 2) was more favorable in the TP-system, it can be inferred that
a decrease in HRT could generate an even greater contribution of bioCH4
in the biogas, since the reactor showed no kinetic limitations. Regarding
the AcR, a greater biohydrogen production would be expected when the
HRT is reduced, which would lead to a significant increase in the organic
load applied to the reactor. This bioH2 potential generation in the biogas
would promote an addition of bioenergy into the treatment system.
Given this scenario, the dimensions of a full-scale TP-system could be
greatly reduced compared to the SP-system, since the HRT would be
optimized, which would considerably reduce the costs of implementing
the two-phase STP.
In addition to the bioenergetic recovery potential of the TP-system,
the potential for generating value-added by-products, e.g., volatile
organic acids such as acetic, butyric and propionic, should be high­
lighted. In the TP-system, the efficiency of converting the influent
organic matter into VOA was 31 ± 9%, representing an expressive po­
tential for the production of metabolites considering the full-scale STPs
reported in Table 3: 398–1459, 367–1344, and 194–711 ton⋅year− 1 of
acetic, propionic and butyric acid, respectively (24–39, 22–36, and
12–19 g⋅m− 3-sewage for acetic, propionic and butyric acid, respec­
tively). Applying a lower HRT in AcR would be expected a higher
acidification efficiency, given by the higher organic load applied, as well
as a higher efficiency of the solventogenic pathway by the production of
methanol, ethanol and butanol. These results point out to a usage
diversification from the municipal sewage matrix, meeting the
principles of sustainability and circular bioeconomy.
3.4. Comparative microbiological assessment
The microbial communities’ characterization of the biofilm samples
from each reactor bed and from the original inoculum showed that the
taxonomic diversity was adequately represented, since the plateau was
reached for the rarefaction curves, as can be seen in Fig. S4. The
phylogenetic analyzes of Bacteria and Archaea domains were repre­
sented through a heatmap (Table 4), in which genera with relative
abundance (RA) greater than 1% in at least one of the samples were
selected. Fig. S5 shows the microscopy images of the biomass samples
collected at the end of the operational period of each reactor.
Among methanogenic archaea, only three genera were predominant
(RA > 1%) in the methanogenic bioreactors - Methanosaeta, Methanor­
egula, and Methanolinea. The genus Methanospirillum was also found in
the methanogenic reactors (M1R – RA = 0.46%; M2R – RA = 0.76%). It
was noted that both the hydrogenotrophic and acetoclastic pathway
were present in M1R and M2R. Nonetheless, there is a greater predom­
inance of the hydrogenotrophic pathway in M2R by the greater RA of
archaea that consume H2/CO2 for the bioCH4 production (Methanor­
egula, Methanolinea, and Methanospirillum), possibly due to the prior
fermentation in AcR, which was also evidenced by the lower XCO2 in
M2R biogas compared to M1R (Fig. 2). The genus Methanolinea produces
methane from H2/CO2 and formate, fluoresces at 420 nm wavelength
and requires acetate for their growth, competing with acetoclastic-
Methanosaeta (Fig. S5b) (Battumur et al., 2016; Kotelnikova et al., 1998;
Sakai et al., 2012). Thus, Methanolinea was favored in the M2R, since
acetate was more readily bioavailable in this reactor, due to the con­
version by acidogenic/acetogenic bacteria in the AcR. The genus Meth­
anoregula was detected with a high RA in AcR (13.4%), since this genus
is acid-tolerant and is often associated with acidogenic fermentative
bacteria (Bräuer et al., 2011). It was responsible for the bioCH4 pro­
duction in the AcR biogas (XCH4 = 6.9%), showing that the acid pre­
treatment was not effective in inactivating this archaea.
Regarding the Bacteria domain, the genera Lactococcus and Clos­
tridium_sensu_stricto of the phylum Firmicutes, Paludibacter and Para­
bacteroides of the phylum Bacteroidota were the most abundant in the
AcR. The Clostridium genus is closely associated in fermentative pro­
cesses of bioH2 production, it is a spore-forming bacteria and producer

Table 3
Estimation of energetic potential from biomethane generated in full-scale anaerobic sewage treatment plants around the world (Heffernan et al., 2011), taking into
account the two scenarios (SP- and TP-system) and the average values.
Country Sewage flow-rate (m3⋅d− 1) COD removed (g⋅m− 3) EPCH4 (MWh⋅d− 1) estimated EPCH4 (kWh⋅m− 3-sewage) estimated

SP-system TP-system SP-system TP-system

Brazil 164,000 262 46.2 74.7 0.28 0.46

Brazil 90,000 367 35.5 57.4 0.39 0.64
Brazil 48,000 314 16.2 26.2 0.34 0.55
Brazil 38,000 255 10.4 16.8 0.27 0.44
India 120,000 264 34.1 55.1 0.28 0.46
India 43,000 226 10.5 16.9 0.24 0.39
United Arab Emirates 49,000 360 19.0 30.7 0.39 0.63

7
R.B. Carneiro et al.
Table 4
Heatmap of relative abundance (%) for Archaea and Bacteria domains at genus
level of the samples from each reactor and the original inoculum. The 3-color
gradient scale is represented according to the percentiles P30 (light blue
color), P50 (yellow color), and P70 (orange color). Only genera with RA > 1% in
at least one sample were selected.
of solvents, e.g. butanol, ethanol and acetone (Poehlein et al., 2014). In
Fig. S5a, the presence of endospores or cells in sporulation process can
be observed, and they might be associated with Clostridium genera,
confirming its high RA in AcR. The Lactococcus genus is facultative
anaerobic and can grow from sucrose and starch, which are readily
available in the feed substrate of bioreactors. Its presence may also be
associated with the formation of organic acids in AcR (Fig. 4) (Meucci
et al., 2015). The Paludibacter and Parabacteroides genera are meso­
philic, obligately anaerobic, and acetate- and propionate-producing
fermentative (Qiu et al., 2014; Wang et al., 2019), which explains the
preferred metabolic pathways of propionic and acetic acid production
along the AcR reaction bed (Fig. 4).
For methanogenic reactors, the Smithella genus was the most abun­
dant. It is a VOAs-oxidizing bacteria, able to oxidize propionate to ac­
etate, grows in co-culture with hydrogenotrophic archaea, e.g.
Methanospirillum (Sobieraj and Boone, 2015), which corroborates its
greater relative abundance in the methanogenic phase of each system. It
was noted that some bacterial genera were favored in the M2R, due to
the optimization of the previous volatile acids production in the AcR.
The genera of the phylum Desulfobacterota, i.e. Syntrophorhabdus, Syn­
trophus and Syntrophobacter, showed greater RA in the M2R compared to
M1R (Table 4), since these microbial groups are often associated with
anaerobic degradation of VOA (notably propionic acid) to acetate and
CO2 in the presence of hydrogenotrophic methanogens and the
sulfate-reducing Desulfovibrio (Fig. S5c) (Boone and Bryant, 1980; Chen
et al., 2005; Jackson et al., 1999; Qiu et al., 2008). In contrast, some
genera were found in higher RA in M1R, possibly due to the composition
of the reactor’s nutritional medium. For example, the genera Lentimi­
crobium, Spirochaeta and Blvii28_wastewater-sludge_group grow chemo­
organotrophically on carbohydrates, including starch and sucrose, with
8
Journal of Environmental Management 317 (2022) 115388
formation of volatile organic acids, CO2, and H2 as end products (Dröge
et al., 2006; Su et al., 2014; Sun et al., 2016). The Treponema genus was
also favored in the M1R, as it is a homoacetogenic bacterium which acts
directly on the acetate formation for Methanosaeta genus (Graber and
Breznak, 2004), the most abundant methanogen archaea in this reactor.
The number of operational taxonomic units (OTUs) and the total RA
for the Bacteria and Archaea domains are shown in Table 5. It can be
noted that the number of OTUs and RA for the Archaea domain was
lower in the AcR compared to the other samples, which was expected,
since this reactor selected only acid-tolerant methanogens (Methanor­
egula) and a more restricted group of hydrolytic/fermentative bacteria,
which is evidenced by the higher dominance index (D), but lower di­
versity (H) and richness (Chao-1) indices of species. Comparing the
methanogenic bioreactors, higher H and Chao-1 indices and lower D
index for M2R were observed, which implies a better interspecific
interaction between the fermentation metabolites-producers microor­
ganisms and the consumers of these products, culminating in a more
balanced anaerobic digestion, and favoring methanogenesis in TP-
AFBBR system, as previously discussed in section 3.1. Compared to
the inoculum, M1R and M2R presented higher D and H indices for the
Bacteria and Archaea domains, respectively. The greater diversification
of methanogenic hydrogenotrophic archaea to the detriment of the high
dominance of the acetoclastic genus Methanosaeta in the inoculum evi­
dences a deviation of the methanogenic route during the bioreactors
operation, possibly due to a selective pressure of the feed substrate with
less organic complexity (Table S1) compared to the nutritional medium
of the raw inoculum (Delforno et al., 2017).
4. Conclusions
This study demonstrated that phase separation of anaerobic diges­
tion in anaerobic fixed bed biofilm reactors into acidogenesis and
methanogenesis promotes the enhancement of biomethane production
in domestic wastewater biological treatment systems. The two-phase
anaerobic fixed bed biofilm reactor (TP-AFBBR) proved to be a prom­
ising alternative for the domestic wastewater treatment, since the
methanogenesis in this system was more stable and the biomethane
production was potentiated by feeding the methanogenic reactor from
the acidogenic effluent. Fermentation products, notably volatile organic
acids, facilitated bioconversion and organic matter removal kinetics in
TP-AFBBR (COD removal = 86 and 92% for SP-AFBBR and TP-AFBBR,
respectively). Biomethane production was increased by 1.7-fold in TP-
AFBBR (312 N-mLCH4⋅L− 1⋅d− 1) compared to SP-AFBBR (182 N-
mLCH4⋅L− 1⋅d− 1). Phylogenetic analyzes and ecological indices showed
that M2R presented a greater diversity of Archaea, supporting the results
found with a greater bioCH4 percentage in biogas (76 and 89% for M1R
and M2R, respectively) and alkalinity generated (0.14 and 0.41 g-
1
CaCO3⋅g-COD−removed for M1R and M2R, respectively), thus demon­
strating that anaerobic digestion was favored in TP-AFBBR. Hydro­
genotrophic methanogenesis (Methanoregula, Methanolinea and
Methanospirillum) was the main organic matter bioconversion pathway
in the M2R, in contrast to M1R, which presented a higher relative
abundance of Methanosaeta. The results of this study also highlight the
bioenergetic potential from biomethane in this innovative fixed biofilm
1
treatment system configuration – EPCH4 = 3.9 and 6.3 kJ⋅g-COD−removed
for M1R and M2R, respectively. The estimates of bioenergy generation in
two-phase full-scale sewage treatment plants point out to the possibility
of energy recovery in the STP itself and in the municipal electricity grid,
not to mention the recovery of value-added products, such as acetic,
propionic and butyric acid, diversifying the uses of the domestic sewage
matrix in the context of the circular bioeconomy.
Funding
This work was supported by the São Paulo Research Foundation
(FAPESP - grant numbers 2019/22532-0, 2017/02147-0, and 2015/
R.B. Carneiro et al. Journal of Environmental Management 317 (2022) 115388

Table 5
Operational taxonomic units (OTUs), Relative abundance (RA), and ecological indices of species for Bacteria and Archaea domains of the biomass samples from each
bioreactor and from the original inoculum.
Parameter Inoculum M1R M2R AR

Bacteria Archaea Bacteria Archaea Bacteria Archaea Bacteria Archaea

OTUs 12403 3242 12069 2599 14151 2615 14650 2296

RA (%) 79 21 82 18 84 16 86 14
Dominance (D) 0.042 0.450 0.072 0.354 0.060 0.307 0.131 0.981
Diversity (Shannon – H) 3.677 0.957 3.226 1.194 3.386 1.259 2.526 0.054
Richness (Chao-1) 101 5 67 7 92 5 52 2

06246-7); and the Coordination for the Improvement of Higher Educa­
tion Personnel (CAPES - Finance Code 001).
CRediT authorship contribution statement
Rodrigo B. Carneiro: Conceptualization, Methodology, Formal
analysis, Investigation, Resources, Data curation, Writing – original
draft, Writing – review & editing, Visualization, Project administration,
Funding acquisition. Gisele M. Gomes: Methodology, Investigation,
Visualization. Marcelo Zaiat: Conceptualization, Resources, Writing –
review & editing, Visualization, Supervision, Project administration,
Funding acquisition. Álvaro J. Santos-Neto: Conceptualization, Re­
sources, Writing – review & editing, Visualization, Supervision, Project
administration, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors would like to thank Dr. Eloisa Pozzi and Dr. Isabel K.
Sakamoto for their valuable support with the microbiological analysis;
and Dr. Carolina Sabatini and Dr. Maria Angela for their technical
support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jenvman.2022.115388.
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