Phase Separation in Anaerobic
Digestion: A Potential for Easier
Process Combination?
Eike Janesch, Joana Pereira, Peter Neubauer and Stefan Junne*
Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Berlin, Germany
The flexibilization of bioenergy production has the potential to counteract partly other
fluctuating renewable energy sources (such as wind and solar power). As a weather-
independent energy source, anaerobic digestion (AD) can offer on-demand energy supply
through biogas production. Separation of the stages in anaerobic digestion represents a
promising strategy for the flexibilization of the fermentative part of biogas production.
Segregation in two reactor systems facilitates monitoring and control of the provision of
educts to the second methanogenic stage, thus controlling biogas production. Two-stage
operation has proven to reach similar or even higher methane yields and biogas purities
than single-stage operation in many different fields of application. It furthermore allows
methanation of green hydrogen and an easier combination of material and energy use of
many biogenic raw and residual biomass sources. A lot of research has been conducted in
recent years regarding the process phase separation in multi-stage AD operation, which
includes more than two stages. Reliable monitoring tools, coupled with effluent
recirculation, bioaugmentation and simulation have the potential to overcome the
current drawbacks of a sophisticated and unstable operation. This review aims to
summarize recent developments, new perspectives for coupling processes for energy
and material use and a system integration of AD for power-to-gas applications. Thereby,
cell physiological and engineering aspects as well as the basic economic feasibility are
discussed. As conclusion, monitoring and control concepts as well as suitable separation
technologies and finally the data basis for techno-economic and ecologic assessments
have to be improved.
Keywords: two-stage digestion, feedstock flexibilization, methanation, on-demand production, bioprocess coupling
INTRODUCTION
Flexible biogas production via anaerobic digestion (AD) can complement the energy production by
fluctuating renewable wind and solar energy sources, which are typically affected by seasonality
(Peters et al., 2018). Additionally, the use of biogenic residues for biogas production, such as
agricultural and forestry by-products and industrial as well as municipal residues encourages the
implementation of circular bioeconomy concepts, while facilitating solid waste management. The
competition for the utilization of natural resources, polluting emissions, and feedstock costs are
reduced (Atasoy et al., 2018;Theuerl et al., 2019). In order to increase the potential of existing AD
processes without multiplying the investment costs required for the installation of on-site gas storage,
the fermentation itself can be operated dynamically. This can be achieved by using varying amounts
Edited by:
Sergi Astals,
University of Barcelona, Spain
Reviewed by:
Lucas Vassalle,
Federal University of Minas Gerais,
Brazil
Cesar Huiliñir Curío,
University of Santiago, Chile
*Correspondence:
Stefan Junne
Specialty section:
This article was submitted to
Environmental Chemical Engineering,
a section of the journal
Frontiers in Chemical Engineering
Received: 19 May 2021
Accepted: 18 August 2021
Published: 01 September 2021
Citation:
Janesch E, Pereira J, Neubauer P and
Junne S (2021) Phase Separation in
Anaerobic Digestion: A Potential for
Easier Process Combination?
Front. Chem. Eng. 3:711971.
doi: 10.3389/fceng.2021.711971
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 7119711
REVIEW
published: 01 September 2021
doi: 10.3389/fceng.2021.711971
of feedstock or alternating the feedstock sources. One promising
approach to increase the flexibilization of the production rates is
represented by process-phase separation. Hereby, the ideal
conditions for every process phase can be controlled separately
in each stage. Additionally, a coupling of energy and material use
of various biogenic resources becomes feasible.
The advantages and disadvantages of phase separation in AD
have been described in several research papers and reviews e.g.
from Chatterjee and Mazumder (2019),Van et al. (2020) or
Menzel et al. (2020). The present work aims to complement
previous research on the topic, by highlighting the value-adding
possibilities which can result from coupling the hydrolytic/
acidogenic with the methanogenic stage of AD. The synergies
obtained from phase separation and effluent recirculation in AD
are discussed as means for the achievement of stable, affordable,
and sustainable energy production. Furthermore, the potential
for achieving value-added products beside biogas is highlighted.
Challenges remain though, such as process disturbances caused
by the overgrowth of phase-specific microorganisms brought in
from feedstock, effects of inhibitors and the achievement of
individual suitable feeding rates of substrates and co-factors at
each process stage. Such hurdles can be minimized by improved
process controllability and predictability, which can be achieved
via improved process monitoring, which includes cell viability
measures and, if known, the quick detection of potential
inhibitors. Recent developments in this field are described in
this review as they can support a robust operation in multi-stage
AD. Additionally, a comprehensive summary of the recent
approaches for flexible biogas production coupled with
biological methanation or other bioprocesses is presented.
PHASES OF ANAEROBIC DIGESTION
AD is a complex process, in which various microorganisms are
involved in degrading organic substrates under anaerobic
conditions. In brief, AD is typically described with four
metabolic phases as shown in Figure 1. In the first step –the
hydrolysis - biomass which usually consists of insoluble organic
polymers like proteins, lipids and carbohydrates is degraded by
hydrolytic bacteria into soluble oligo- and monomers
(Kaltschmitt et al., 2016;Rabii et al., 2019). Hydrolysis often
acts as the bottleneck in AD processes as it demands a
combination of enzymes and time to break the complex
molecule structures of the substrate to overcome the steric
hindrance. Mechanical, thermal, chemical, and biological
pretreatment processes, as well as combinations thereof, have
been reviewed and assessed for their potential to increase the
efficiency of AD and shorten process times, e.g. in (Ariunbaatar
et al., 2014). In order to access the high-molecular, sterically
blocked substances, microorganisms use exoenzymes like
hydrolases. Most of these facultative anaerobic or strictly
anaerobic hydrolytic microorganisms are very resilient against
varying chemical and physical conditions. A recent review
focused on the hydrolytic process stage in depth (Menzel
et al., 2020) among other previous reports.
In the second phase of AD, denoted as acidogenesis,
fermentative bacteria degrade the resulting substances from
the hydrolysis into low-molecular intermediates like amino
acids, short-chain carboxylic acids (SCCAs), and alcohols. A
description of the role of microbes within typical digestion
consortia were summarized in Rabii et al. (2019).
In the next step, the acetogenesis, the intermediates from the
acidogenesis are converted into acetic acid. Depending on the
available substances, different ratios of acetic acid, carbon dioxide
and hydrogen are formed by acetogenic bacteria like
Acetobacterium or Clostridium species (Borja, 2011). The
hydrolytic/acidogenic stage, eventually exhibiting acetogenic
conversions, is also called dark fermentation (DF) or acid
fermentation and typically operated with HRT of several days
only (Rosgaard et al., 2007;Menzel et al., 2020;Van et al., 2020). A
pH-value of about 5.0 up to 6.0 is optimal, while a lower pH-value
can lead to increased retention time; alkaline pH values may totally
inhibit the process (Zhang et al., 2005;Moestedt et al., 2016).
FIGURE 1 | Stages in an anaerobic digestion process (LCCA, long chain carboxylic acids; SCCA, short chain carboxylic acids).
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 7119712
Janesch et al. Phase Separation in Anaerobic Digestion
In the final step of AD, strictly anaerobic methanogenic
archaea transform hydrogen together with acetic acid and
carbon dioxide into methane. In comparison to this
conversion, that is restricted to a small group of
microorganisms inside the archaea domain, the
hydrogenotrophic methanogenesis is conducted by a wide
variety of species as reviewed recently (Kaltschmitt et al., 2016;
Castellano-Hinojosa et al., 2018;Rabii et al., 2019). In this case,
the reduction of carbon dioxide with hydrogen leads to the
formation of methane. Growth of acetogens and methanogens
is favored by mesophilic conditions and neutral pH-values. Since
the anaerobic archaea show the slowest growth among most of
the microbes in AD, they are the most fragile towards varying
conditions and the presence of inhibitors like ammonia. Thus, in
the widespread use of single-stage AD applications, the physical
and chemical parameters of the system are adjusted in favor to
keep these organisms vital (Van et al., 2020). Typical
representatives of archaea in biogas fermenters are described
in more detail by Wirth et al. (2012).
POTENTIALS OF PHASE SEPARATION
Reactor types used in AD are typically continuously operated
stirred tank reactors (STRs), upflow anaerobic sludge blankets
(UASB) and plug-flow reactors (PFRs); the first two reactor
designs are typically used for multi-stage AD. A lot of AD
applications with UASBs can be found in tropical countries.
These reactors benefit from their potential to handle high
organic loading rates and their longevity and robustness due
to the lack of moving parts like stirrers (Bischofsberger, 2005).
In the dominant single-stage stirred tank biogas reactors, all
process phases are conducted in parallel. Beside the advantage of
an intercellular hydrogen transfer, also some disadvantages of
such a system exist: high loads of organic substrate or a sudden
change in the feedstock composition can lead to acidification,
when the hydrolytic and acidogenic or acetogenic reactions
dominate and are conducted faster than the acid consumption
by the acetoclastic organisms. This can lead to severe failures in
fermentation and considerable down times (Moeller and
Zehnsdorf, 2016). Although no detailed data about this has
been published, the dominance of STRs over plug-flow
reactors PFRs, in which a separation of phases is achieved due
to the nature of the low axial mixing, is also caused by higher
investment costs of the latter reactor technology. It is still not fully
proven, however, if a PFR concept exhibits a higher robustness
against flexible feedstock loads than a STR design due to the many
possibilities of feedstock and operational conditions in the
numerous published studies. Multi-stage AD applications,
however, aim to combine some of the advantages of stirred
tank and plug-flow concepts.
Theoretically, all four phases of AD can be divided and
conducted in different reactors. Most of the hydrolytic and
acidogenic bacteria, however, share similar optimal growth
conditions. Generally, these are represented by mesophilic or
thermophilic microbes, whose maximum growth is reached at
slightly acidic pH-values of between 4.5 and 6.0. Aceto- and
methanogens prefer neutral pH-values between 6.5 and 7.5. Thus,
they differ in their nutritional demands, growth kinetics and
temperature preferences compared to the hydrolytic and
acidogenic microorganisms (Van et al., 2020). In multi-stage
AD processes, it becomes feasible to adjust the process
parameters individually in each part of the reactor. Therefore,
such a process design does not compromise as much between the
different growth requirements of the microbes as in single-stage
AD. In a typical multi-stage AD, the first stage comprises mostly
of hydrolytic and acidogenic microorganisms while the
subsequent stages contain mainly acetogenic and
methanogenic microorganisms. By monitoring and controlling
the pH-value, it is possible to separate these phases from each
other with a low effort (Chatterjee and Mazumder, 2019;Van
et al., 2020).
Phase separation has proven to be beneficial to produce
hydrogen-enriched biogas (10–30% v/v) with a profitable
process (Dahiya et al., 2018). An AD process with phase
separation can facilitate on-demand biogas production because
the products of the hydrolysis can be added dynamically to the
methanogenic stage. This way, the methane production can be
enhanced for a certain time, e.g. by about 60% (Linke et al., 2015).
Leftover nutrients in the digestate can be used as biofertilizer,
containing nitrogen in inorganic form (often over 60% (w/w) of
total nitrogen), phosphorus, and potassium, characterized by a
high bioavailability (Chojnacka et al., 2020). A separate
hydrolytic/acidogenic stage can save costs as other feedstock
pre-treatment becomes useless (Blank and Hoffmann, 2011).
The phyla Proteobacteria, Bacteroidetes, Firmicutes and
Chloroflexi can typically be found in AD broths (Jie et al.,
2014;Liu et al., 2014;Wu et al., 2016). These bacteria possess
a higher natural resilience against disturbances and fluctuating
process conditions than methanogens (Kaltschmitt et al., 2016).
When the first stage of an AD application is not dependent on the
viability of methanogens due to separated phases, the whole
process can gain more stability against a varying organic
loading rate (OLR) and chemical inhibitors like oxygen,
ammonia, and toxins, that are brought into the process by
feedstock feeding. While different fermentation variants exist,
like acetic-, butyric-, ethanol- or mixed-hydrolysis, Menzel et al.
(2020) consider acetic and butyric fermentation as most suitable
for multi-stage AD applications. This type of fermentation is
favored under thermophilic conditions at pH-values of between
5.0 and 6.0. Bacteria of the genus Clostridium release acetic and
butyric acid and yields a biodegradability of up to 95% for the
treatment of FW and rice straw in CSTR (Sträuber et al., 2012;
Chen et al., 2015). The metabolic activity of hydrolytic and
acidogenic microorganisms is only inhibited at higher
concentrations of SCCAs beyond 10 g/L or more; much higher
concentrations than it is usually the case for methanogens (Zhang
et al., 2017;Chatterjee and Mazumder, 2019).
AD is typically operated under either mesophilic (35–39°C) or
thermophilic conditions (50–60°C). While thermophilic AD
provides shorter HRT, digestion under mesophilic conditions
is commonly considered as a slower, but rather stable process
(Van et al., 2020) with lower energy costs (Fernández-Rodríguez
et al., 2016). A combination of hydrolysis and acidogenesis in a
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 7119713
Janesch et al. Phase Separation in Anaerobic Digestion
separate stage offers the possibility of temperature-gradients
between the stages. This is of particular importance for the
treatment of residues with high solid-contents like organic
fractions of municipal solid waste (OFMSW) (Fernández-
Rodríguez et al., 2016). Thermophilic operation, on the other
side, promotes the deactivation of infectious pathogens as present
in biogenic residues (Zhao and Liu, 2019). According to Pandey
and Soupir (2011), the inactivation rate of Escherichia coli, as a
representative for pathogenic organisms is 15-fold higher at 52°C
than at 37°C. Fernández-Rodríguez et al. (2016) showed an
application with up to 34% higher removal rates of VS and
increased methane production in a two-phase AD operated
with temperature combinations. By this a compromise
between a purely mesophilic or thermophilic operation is made.
In order to achieve new opportunities for a flexible feedstock
load and for demand-driven biogas production, research has been
performed on multi-stage AD, especially in two-stage concepts.
Several strategies and scenarios have been described, in which
existing plant infrastructure is re-engineered to increase the
production flexibility within multi-stage concepts. Other
publications have reviewed some of these aspects regarding the
OFMSW (Chatterjee and Mazumder, 2019) and food waste (FW)
(Srisowmeya et al., 2020), as well as for process configurations
(Van et al., 2020) and mixed gas production(Hans and Kumar,
2019). Rajendran and co-authors focused on techno-economic
assessments and related investments and operational costs under
the consideration of mono-digester plants (Rajendran et al.,
2020).
Typical characteristics of multi-stage AD are summarized in
Table 1. The following chapters will further describe recent
advances in more detail, including potential strategies to
increase process robustness with monitoring and control tools,
and the systemic integration into energy and biomaterial
production grids.
TWO-STAGE ANAEROBIC DIGESTION
Two-stage AD is the most widely studied design within multi-
phase processes. Recent work focused on operational
optimization and the application of feedstock that is difficult
to be degraded. Li et al. (2017) published results for an OFMSW
treatment with a two-phase AD application. A setup of three 20 L
STRs was used, the first two for the combination of hydrolysis and
acidogenesis, similar to the first stage of a two-stage anaerobic
digester. While the single-stage operation achieved a biogas
production of 540 ml/g-VS, the two-stage process reached
710 ml/g-VS at a 30% higher OLR. The results of this study
show that two-stage AD is not necessarily better than a single-
stage AD in terms of substrate-specific biogas production yields,
but might offer the possibility of higher loading and production
rates. The net energetic production of a two-stage anaerobic
digester for a substrate mixture consisting of whey and
glycerine was compared to a single-stage system by Lovato
et al. (2020). The authors concluded that the energy
production from the two-stage AD (7.0 MJ/kg-COD
removed
)
makes it profitable. Ghanimeh et al. (2019) investigated the
biogas production performance of a single- (STR, 9 L) and a
two-stage thermophilic anaerobic digester. The two-stage digester
showed better overall degradation rates and better biogas quality:
at an average OLR between 2.0 and 2.4 g-VS/(L·d), the reduction
of total COD was nearly 80% higher in the multi-stage process
compared to the mono-digester. The methane content of the
biogas reached 45 and 54% in the single- and two-stage process
design, respectively (Ghanimeh et al., 2019).
Two-stage AD has the potential to produce desulfurized
biogas without a subsequent (external) desulfurization. The
first stage of the AD application is controlled to favor the
coexistence of acidogenic and sulfate reducing bacteria. A
sulfate reduction in the first stage consequently reduces the
hydrogen sulfide (H
2
S) production in the methanogenic stage
(Tijani et al., 2018). Yun et al. (2017) were able to reduce the
H
2
S-content in the biogas in a two-stage AD system (STR, UASB)
by nearly 90%, compared to a conventional single-stage digester.
The first stage of their application acted as a sulfidogenic-
acidogenic-reactor with a sulfate reduction of 70% at an
optimum pH-value of 5.5. As a model substrate, the authors
used glucose combined with sodium sulfate (Yun et al., 2017).
Similar results were described by Tijani et al. (2018), who
examined the desulfurization of biogas in a two-stage system
consisting of a shear-loop anaerobic bed and an anaerobic
PFR for the treatment of palm oil mill waste. They considered
TABLE 1 | Characteristics of stage separation in anaerobic digestion.
Application Advantages Disadvantages Typical process arrangement
Single-stage •Low costs •Risk of quick acidification Hydrolysis/acidogenesis/acetogenesis/
methanogenesis
•Applicable for very high/low TS-
contents
•Repression of methanogens by inhibitors from the substrate
(e.g. NH
3
)
•Established process •Risk of bacterial overgrowth
•No recovery of side products
Two-stage •Recovery of side products •High costs 1. Stage: hydrolysis/acidogenesis
•Temperature-phased •Not efficient for high TS-contents 2. Stage: acetogenesis/methanogenesis
•Disturbed intercellular hydrogen exchange
Three-stage •Recovery of side products •Very high costs and operational effort 1. Stage: hydrolysis/acidogenesis
•High hydrolysis efficiency •Lack of research on pilot scale 2. Stage: acidogenesis
•Shock load resistant •Disturbed intercellular hydrogen exchange 3. Stage: acetogenesis/methanogenesis
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 7119714
Janesch et al. Phase Separation in Anaerobic Digestion
a pH-value of 5.4 as optimal for the sulfidogenic-acidogenic stage.
The first stage of their application was able to reduce the sulfate by
75%, and the biogas from the methanogenic stage was suitable for
electricity production without H
2
S removal (Tijani et al., 2018).
Flores-Cortés et al. (2021) used a two-stage anaerobic nitrate-
reducing bioreactor to desulfurize H
2
S-rich biogas; 95% of H
2
S
was oxidized.
As described before, depending on the substrate, two-stage
operation can, beside other benefits, reach higher energy
production, methane yields and methane ratios in the biogas
within shorter HRT than single-staged processes in different
pilot-scale studies, e.g. for the thermophilic treatment of the
OFMSW (Ghanimeh et al., 2019) or the digestion of food
waste (FW) (Gioannis et al., 2017). There is, however, still
room for improvement. One major challenge is the
interspecies-hydrogen transfer. The hydrogen produced by the
acidogens is released in a different tank than the one with the
hydrogen-consuming methanogens, that make use of it. As
hydrogen has a very low solubility in water, it has to be
ensured that enough hydrogen is dissolved in the second stage
of two-stage AD or hydrogen has to be released in the off-gas,
thus lowering the carbon dioxide binding capacity of the
biological methanation. High-pressure two-stage AD
represents an approach to enrich biogas with methane,
without the need for external purification. Merkle et al. (2017)
investigated the influence of high pressure up to 50 bar on a two-
stage AD application, consisting of a leach bed reactor and an
anaerobic filter, fed with maize and grass silage. The
methanogenic reactor was operated at 37°C, an OLR of
between 4.2 and 4.4 g-COD/(L·d), pH-values of about 6.6 and
pressures of 10, 25 and 50 bar. While the specific methane yield
decreased from 0.33 to 0.04 L/(g-COD) with an increased
pressure from 10 to 50 bar, the methane content in the biogas
increased by 11% (to finally 90%) under these conditions. As a
result, the biogas could be transferred directly into the gas grid
without additional purification or pressurization (Merkle et al.,
2017). Lemmer et al. (2015) have also reported an increase in the
methane content from 70 to 77% when increasing the pressure
from 1 to 9 bar in a two-stage AD composed by a leach bed
reactor for a separate acidogenesis at 55°C, and a methanogenic
stage at 37°C at an OLR of 5 g-COD/(L·d) of maize and grass
silage. The application of high pressure in two-stage AD systems
leads, however, to a decreasing pH-value, which consequently
effects the methane production negatively (Lemmer et al., 2015).
Thus, Lemmer et al. (2015) tested, if a higher ammonium content
in the methanogenic stage can counteract this issue by rising the
buffer capacity. While the methane content of the biogas rose, the
specific biogas yield decreased.
Furthermore and most important, concepts for an easy and
cheap achievement of a two-stage process at existing biogas plants
have to be accomplished, since the yearly numbers of new plants is
slowing down (European Biogas Association, 2019). The “ReBi 2.0”
(variable biogas production) from the Fraunhofer IEE represents a
concept for upgrading existing biogas plants into variable biogas
producers, which relies on the use of effluents enriched with
SCCAs in the methanogenesis. After a hydrolytic stage, a phase
separator enables the storage of the SCCAs-rich liquid in a storage
tank. The solid effluents are digested in a conventional fermenter
for continuous biogas production. The easily degradable SCCAs in
turn are transferred to a fixed bed reactor, depending on the
current demand of biogas. Experiments in a large-scale facility
provided promising results with amounts of up to 70% (v/v) of
methane in the biogas (Fraunhofer IEE, 2018).
THREE-STAGE PROCESSES
Research on three-stage applications has proven, that the process
conditions of AD can be further optimized. There exist different
concepts for the separation of the process phases between the three
stages. The first one is to separate the acetogens from the
methanogens as they possess different nutritional requirements,
resulting in an order of 1) hydrolysis/acidogenesis, 2) acetogenesis
and 3) methanogenesis (Kim et al., 2006;Kim et al., 2008). As the
interspecies hydrogen transfer is a critical factor in multi-stage AD,
the second concept is to rather split up hydrolysis and acidogenesis
than to separate the acetogens and methanogens. This leads to the
process sequence 1) hydrolysis, 2) acidogenesis, and 3)
acetogenesis/methanogenesis, as described for several cases
(Zhang et al., 2017;Chatterjee and Mazumder, 2019;Van et al.,
2020). A review of Chatterjee and Mazumder (2019) in contrast,
refers to the stage separation order 1) hydrolysis, 2) acidogenesis/
acetogenesis and 3) methanogenesis, mainly due to the adverse
effect of mixing, which is required for the homogenization of waste
in the first stage.
One of the first studies on three-stage AD originate from Kim
et al. (2000). Under mesophilic conditions, FW was treated in a
STR for hydrolysis/acidogenesis and two UASB for acidogenesis/
acetogenesis/methanogenesis. Production rates of up to 700 ml
biogas/(g VS) with a methane content of 72% were achieved. The
enhanced degradation of three-stage AD was confirmed by COD
reduction rates of over 90%. Back in 2005, Salsali et al. showed
that three-stage AD for the treatment of waste activated sludge
can provide higher methane yields than two-stage digesters
(Salsali et al., 2005). A maximum of over 650 ml biogas/(g VS)
were reached in their mesophilic application, operated with three
STRs. A three-stage AD application with a focus on an enhanced
hydrolysis in the first stage was compared to a single- and two-
stage process design by Zhang et al. (2017). Their FW-fed digester
consisted of three chambers, responsible for hydrolysis,
acidogenesis and methanogenesis in a sequential arrangement.
The enhanced hydrolysis decreased the reduction of VS by half
compared to the two-stage digester (44 vs 83% w/w). In addition,
the methane yield of the three-stage application increased by up
to 54%; the methane content of the biogas rose to nearly 70% (v/
v), assumingly due to an intensified hydrolysis compared to the
two-stage process (Zhang et al., 2017). Similar results were
described by Zhang et al. (2020). The authors used a three-
stage thermophilic reactor to treat a mixture of FW and
horticultural waste. Methane yields were (with about 0.42 L/g
VS) 31–45% higher than those in one- and two-stage reactors.
The reduction of VS (63%) increased by over 60% compared to
the single-stage system (Zhang et al., 2020). The anaerobic
treatment of tofu whey wastewater (COD: 15.9 g/L) in a three-
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 7119715
Janesch et al. Phase Separation in Anaerobic Digestion
stage reactor was described by Rani et al. (2020). The authors used
a system which consisted of three 10 L packed bed reactors. Due
to the phase separation, which was indicated by the respective
pH-values, the system was able to resist hydraulic shock loads
(Rani et al., 2020). It was possible to maintain the biogas
production rate (20 L/d) as well as the pH value (around 5.0
in the first two reactors and around 7.0 in the third reactor) and
the COD at the outflow after the organic load was increased 12-
TABLE 2 | Examples for one-, two- and three-stage phase separation applications in anaerobic digestion.
Feedstock Reactor
type
1st,
2nd,
3rd
TS [%] OLR
[g-VS/(L·d)]
HRT [d] pH Biogas
yield [ml-CH
4
/
(g-VS)]
Temp Reference
WAS STR 4.2 - 15 7 240 mesophilic Heng et al. (2021)
Ensiled OFMSW Batch
reactor
12–28 - 25 6.0–8.2 431 mesophilic Castellón-Zelaya and
González-Martínez (2021)
MSW and fly ash STR 9.72 - 45 7.2–7.5 268–287 mesophilic Markphan et al. (2020)
OFMSW STR 1.75 1.35 25 7.94 404 thermophilic Schievano et al. (2012)
Sewage sludge STR 4.0–5.2 2.1–2.5 15 - 260–306 mesophilic Salsali et al. (2005)
Fruit waste micro
digester
0.25 - 30 7.5–8.5 610
a
mesophilic Chanakya and Shwetmala
(2017)
Sugar molasses Packed
bed
- 34 0.55–0.74 4.5–5.2 324
b
mesophilic Detman et al. (2017)
UASB - 6.71 6.5–7.6 mesophilic
Oily FW STR 3.57 14.2 3 5.36 450 thermophilic
mesophilic
Wu et al. (2015)
STR 2 2.6 12 7.59
MSW STR 3.9 13 7.5 5.5–6 540 mesophilic Li et al. (2017)
STR - 3.8 15 - mesophilic
Chicken manure glas vessel 3.7 2.2 2 6.3–6.8 554 mesophilic Dalkılıc and Ugurlu (2015)
glas vessel 10 7.7–8 thermophilic
FW, grass, chicken
manure
batch
reactor
20 4 30 4.5–7.5 113 mesophilic Li W. et al. (2018)
batch
reactor
6.6–7.5 mesophilic
slaughterhouse blood
waste
tank reactor 2.8 1.2 11.1 6–6.2 384
b
mesophilic Wang et al. (2018)
tank reactor 0.4–1 24.2 7–7.5 mesophilic
Ethanol wastewater SBR 3.17 15–21
c
- 5.5 686
b
mesophilic Jiraprasertwong et al. (2018)
SBR - - 6–7 mesophilic
SBR - - 7.2–7.5 mesophilic
Cassava wastewater UASB - 5–18
c
- 5.5 328
b
mesophilic Jiraprasertwong et al. (2019)
UASB - 6.8 mesophilic
UASB - - mesophilic
FW STR 17.5 22–22.8 2 5–5.5 650–700 mesophilic (each
stage)
Kim et al. (2000)
UASB 3 25–27.4 2 5–5.5
UASB 16 12–18.8 12 7.6–7.9
WAS STR 4.6 2.13–2.53 (each
stage)
5 (each
stage)
8 (each
stage)
559–664 mesophilic (each
stage)
Salsali et al. (2005)
STR 3.5
STR -
WAS, waste activated sludge; OFMSW, organic fraction of municipal solid waste; FW, food waste; MSW, municipal solid waste; STR, stirred tank reactor; UASB, upflow anaerobic sludge
blanket; SBR, sequencing batch reactor.
a
[ml-CH
4
/(g-TS)].
b
[ml-CH
4
/(g-COD)].
c
[g-COD/(L·d)].
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 7119716
Janesch et al. Phase Separation in Anaerobic Digestion
fold for 1 h (0.33 vs 4.0 L/h). The ratio of alkalinity to volatile
SCCAs, which rose during the shock load, was reduced and
stabilized within 8 h after the shock load. Superior process
performance has also been reported for a three-stage anaerobic
system using sequencing batch reactors, when compared to both
single- and two-stage anaerobic processes treating several types of
wastewaters (Jiraprasertwong et al., 2018). With an optimum
COD loading rate of 18 kg/(m
3
·d), the process achieved 686 ml
CH
4
/g-COD, with an energy yield of 22.5 kJ/g-COD, and an
overall COD removal of 92%. The system performance has been
related to the high microbial concentrations reached in the
bioreactors, high alkalinity, and adequate pH, with values of
5.5, 6.0–7.0, and 7.5 in the first, second, and third stages,
respectively. Several typical residual resources contain
lignocellulose. For the treatment of such feedstock, a separate
hydrolytic stage, which relied on white rot fungi, was able to
degrade lignin enzymatically (Wan and Li, 2012;Meegoda et al.,
2018).
Further examples for AD applications with one, two and three
stages and their respective process conditions are summarized in
Table 2.
EFFLUENT RECIRCULATION IN
MULTI-STAGE AD
Effluents from each stage can be recirculated and added to every
tank in phase-separated AD. Thus, high degradation rates can
be achieved, when e.g. incompletely degraded substances from
the methanogenesis undergo hydrolysis twice, enhancing
energy recovery up to 9% in two-stage systems (Chatterjee
and Mazumder, 2019). Previous work has reported the
dilution of high solid feedstocks and pH control, achieved by
recirculating the alkaline effluent obtained in the second stage,
as main benefits in two-stage AD (Van et al., 2020). In fact, this
preserves nutrients and microorganisms in the system, allowing
for the use of higher OLR, e.g. discussed by Menzel et al. (2020).
By recirculating the liquid fraction of the AD effluent after solid-
liquid separation, a wash out of fluidized microorganisms to the
next stage will decrease. Thereby, the retention times of the
microbes is elongated, which also improves process stability and
performance, as observed by Jiraprasertwong et al. (2018) in
their three-stage AD system. Effluent recirculation can thus be
used as a tool to stabilize and control AD processes, which is
typically seen as a hurdle for their commercialization at large-
scale. Recent studies have proven that recirculation strategies
are indeed beneficial to increase process stability and
performance for e.g., in the digestion of toxic citrus wastes,
while increasing methane yields by 79% (Wikandarietal.,2018).
Qin et al. (2019) have also reported more stability in the long-
term operation of a two-phase process with effluent
recirculation for the AD of food and paper waste. The study
suggested that hydrogen-producers were recirculated to the
first-stage after proliferation in the second stage, which
contributed to the production of 79 L-H
2
/kg-VS and 329 L-
CH
4
/kg-VS. Wang et al. (2020) have also demonstrated an
improved hydrogen and methane production resulting from
effluent recirculation in their two-stage AD experiments using
FW combined with cow dung, while additionally reducing the
amount of alkali addition for pH control in the hydrogen-
reactor. Paillet et al. (2021) reported that the performance of
hydrogen production from anaerobic OFMSW degradation
couldbeimprovedby330%,upto17.2ml/g-VS,when
applying a strategy based on effluent recirculation and
systematic heat shock treatment. Compared to a single-stage
reactor, Ding et al. (2021) described an increased energy yield of
18% and a higher possible FW loading (up to 20% of the
working volume), along with stable biogas production, in a
FW-fed two-stage AD system with liquid recirculation from the
methanogenic into the hydrolytic stage. The recirculation of the
effluent from the second reactor also led to a higher robustness
of the system against side effects through high OLR (e.g.
acidification) compared to the single-stage application.
Moreover, methane yields of nearly 400 ml/g-VS, that is over
90% of the methane production potential, were reported.
Recirculation of the liquid phase can stabilize the microbial
consortium and adds another possibility for process control.
Wu et al. (2015) investigated the performance of two
temperature-phased (thermophilic-mesophilic) double-STR
digesters for the treatment of FW and compared it to a
mesophilic single-stage application. In the two-stage digester
without recirculation, the processes of hydrolysis and
acidogenesis were inhibited by a pH drop below 4.0 in the
first stage. Thus, the results for the methane production were
very similar to the mono-digester, around 440 to 450 ml-CH
4
/
(g-VS). The methane ratio in the produced biogas from the
second digester was 61% (Wu et al., 2015). The two-stage AD
application with recirculation from the second into the first
stageachievedtokeepthepH-valueinthefirst digester at an
optimal level of 5.0–5.5. In consequence, the microorganisms in
the first digester produced biogas with a hydrogen share of 30%
(v/v). The portion of methane in the biogas rose to 70% (v/v).
The particulate COD was reduced from 66.7% in the substrate to
10.3% (w/w) in the effluent. In comparison, in the single-stage
digester and the two-stage AD application without recirculation,
the COD remained at 14.6% (w/w).
Effluent recirculation in two-stage AD processes, however,
must be carefully monitored and controlled, as methane
production may be favored at a pH-value that is higher than
6.0, hindering biohydrogen production (Micolucci and
Uellendhal, 2018). This issue is avoided in three-stage AD
systems with separated hydrolysis and acidogenesis, as the
recirculation of methanogenic sludge into the first stage does
not interfere with hydrogen synthesis in the acidogenic reactor
(Jiraprasertwong et al., 2018).
MONITORING AND CONTROL
A reliable monitoring and control system is a pre-requisite for
phase separation in AD. A suitable monitoring system is required
to orchestrate the exchange of the gas, and in particular the liquid
phase in between the stages to prevent cell starvation or product
inhibition.
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 7119717
Janesch et al. Phase Separation in Anaerobic Digestion
Sensors for AD need a resistance against chemical and physical
conditions like low pH-values and an undefined multiple-
component matrix in the broth (Bockisch et al., 2019).
Naturally, an important process parameter for AD is the pH-
value. The response time of the system’s pH-value to changing
conditions inside is not by all means very fast due to the buffer
capacity of the broth (Li et al., 2014). An accumulation of acids
inside a tank does not necessarily mean an immediate decrease of
the pH-value. Any drop in the pH-value exhibits severe
imbalances, which cannot be rapidly counteracted and
typically need several days or even weeks to recover (Pfeiffer
et al., 2020). In that case, the OLR has to be decreased
significantly, even the addition of anti-foam agents might be
needed due to increased cell lysis rates and protein excretion
(Moeller and Zehnsdorf, 2016). Controlling the pH-value,
however, is the most suitable tool for separating the different
groups of microorganisms between the reactors of multi-stage
AD. At a low pH-value of between 5.0 and 6.0, the presence of
methanogens in the acidogenic phase is prevented. The pH-value
can be measured with on line monitoring methods like classical
voltametric pH electrodes (AgCl), fluorescence based sensors or
ion selective field effect transistors (Jimenez-Jorquera et al., 2010;
Bockisch et al., 2014;Janzen et al., 2015). Fluctuations of the pH-
value caused by an organic overload of the AD system can be
corrected by reducing the substrate feed. Acidified digesters can
be treated additionally with sodium bicarbonate in a bypass or
between the stages of multi-stage AD to rise the pH-value
(Burgstaler et al., 2010).
For all AD applications, especially for FW-treating digesters,
the amount of produced SCCAs is a proper parameter to observe
the process functionality (Pfeiffer et al., 2020). An adequate
amount of the effluent from the acidogenesis needs to be
transferred to the second (or third) stage in order to prevent
starvation in the methanogenic stage without the risk of its
acidification. SCCAs can be monitored on- and off line with
near or mid-wavelength infrared spectroscopy sensors (Falk et al.,
2015;Nespeca et al., 2017). Infrared spectroscopy (Nespeca et al.,
2017) or gas chromatography for the detection of volatile
compounds (Zheng et al., 2020) has proven to be a suitable
strategy for identifying concentration changes of acids in a short
time scale (Björnsson et al., 2001), however this is rarely applied
in practice so far.
Alkalinity is also considered as an early-warning indicator that
can effectively predict disturbances in AD processes. Several
studies have demonstrated that enhanced SCCA production
could be obtained from sewage sludge under alkaline
conditions, typically using NaOH to maintain pH values
around 10 (Kurahashi et al., 2017;Liu et al., 2018). Controlled
alkalinity at a pH in the range of between 10 and 11 during the
AD of sewage sludge in a full-scale reactor (30 m³) inhibited
methane production, while enhancing SCCA accumulation,
particularly acetic acid which accounted for 58% (w/w) of the
total SCCAs (Liu et al., 2018). A thorough review about process
stability for AD of FW has suggested that a ratio of intermediate
to partial alkalinity (IA/PA) lower or equal to 0.3 should be used,
along with total alkalinity (TA) concentrations of between 13 and
15 g/L (Li et al., 2018). Despite the proposed thresholds, these
depend on substrate quality and the type of operation, and are less
reliable under variable feedstock or operating conditions (Wu
et al., 2019).
Cell polarizability measurements can provide a value that
reflects the metabolic activity of cells that is related to cell
physiology. This is especially helpful if different consortia exist
in multiple stages while the measurement of intermediates, and
thus the production rates, is not easily feasible or cell stress that
usually leads to a reduction of the polarizability, shall be
prevented quickly. This can be starvation due to a lack of
nutrient supply or an oversupply of nutrients. Frequency-
dispersed anisotropic polarizability (FDAP) can be used as an
at-line control to evaluate cell viability and activity in each stage.
Such electrooptical measurements have been successfully used to
monitor DF using mixed cultures (Gómez-Camacho et al., 2020),
which exposes a different metabolic activity on the dependence of
acid accumulation. The same study also revealed the suitability of
flow cytometry for DF cultures, in which a typical live-dead
staining can be conducted, which is otherwise difficult in cultures
of one-stage AD, probably due to agglomerate formation between
acidogens and methanogens.
Ammonia acts as an inhibitor for specific enzymatic reactions.
It has been shown previously that biogas plants suffer from
microbial inhibition and methane losses when using N-rich
substrates (Morozova et al., 2020). While concentrations above
200 mg/L are considered to hinder AD significantly, according to
Chen et al. (2008), lower amounts are beneficial for stabilizing AD
processes, by buffering the system (Nsair et al., 2020). Ammonia
concentrations of 14 mg/L can already have an impact on
methane production, as described by Nsair et al. (2020).
Therefore, in multi-stage AD applications, the monitoring of
ammonia concentrations has a particular importance for the
methanogenic stage. Ammonia can be measured with
potentiometric sensors, electronic tongues or luminescent
ammonia sensors (Nery and Kubota, 2016;Urriza-Arsuaga
et al., 2019). A study comparing off line and on line
measurements concluded that the NH
4
electrode had high
accuracy, but was strongly affected by sodium and potassium,
so that it was not of an equal accuracy (Zhou and Boyd, 2016).
The performance of AD can also benefit from a microbial
adaptation, e.g. through a gradually N-increasing feeding rate,
in which the viability and activity of the microbial community is
investigated (Morozova et al., 2020). An issue remains for
measurements at the typically high concentrations above
50 mg/L, which hinders often on line applications as it is not
easily feasible to dilute the broth in an automatic manor.
In order to use the hydrogenotrophic methane production
pathway, methanogenic archaea are dependent on dissolved
hydrogen in the liquid phase of an AD application. The
hydrogen solubility in water is low. At 25°C and a pressure of
1 bar, hydrogen saturated water contains 1.6 mg hydrogen/L
(Kaye and Laby, 1992). Nevertheless, an accumulation of
hydrogen during AD can inhibit the growth of certain
acidogenic bacteria (e.g. Clostridium). While the threshold
depends on the microbial consortia, hydrogen partial pressures
above 10
–5
to 10
–3
bar are described as having negative effects,
especially on butyrate and propionate degradation (Lowe et al.,
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 7119718
Janesch et al. Phase Separation in Anaerobic Digestion
1993;Mutungwazi et al., 2021). In the worst case, this can lead to a
collapse of the AD system and the biogas production through
acidification (Huck et al., 2013).
In multi-stage AD, the interspecies-hydrogen transfer between
acidogens and methanogens is interrupted due to their spatial
separation. The phase-transfer from gas to liquid can thus act as
the bottleneck of methanation (Díaz et al., 2020). Monitoring of the
process stability simply by observing the gas phase may result in
problems like reactor overloads, as the reaction to system changes in
the headspace tends to be slow compared to the response in the
liquid phase (Björnsson et al., 2000). Björnsson et al. (2001) showed,
that the dissolved hydrogen concentration reacts faster to changes in
OLR or other system fluctuations, for example the accumulation of
SCCAs. In their experiments, the authors used an on-line Teflon
membrane for sampling, combined with a semiconductor sensor,
which is highly hydrogen specific(Björnsson et al., 2001).
Acetogenic bacteria are highly dependent on the hydrogen
uptake by the archaea, as the accumulation of dissolved hydrogen
leads the inhibition of their degradation of SSCA like butyrate and
propionate (Harper and Pohland, 1986). In consequence, these
acids can be enriched and cause further system instabilities. It was
shown that ethanol accumulated up to 0.08 g/L under a hydrogen
headspace pressure of 2 bar during anaerobic digestion. The
profile of acids was partially altered (Sarkar et al., 2017). This
example demonstrates that hydrogen plays a crucial regulatory
role in acidogenesis and acetogenesis; a partial pressure of
hydrogen below 10
–4
bar is required to maintain the
thermodynamical feasibility of the acid synthesis (Pavlostathis,
2011). Thus, the monitoring of the dissolved hydrogen shows, if
the different groups of microorganisms act at equilibrium
(Björnsson et al., 2001). The monitoring of dissolved hydrogen
is typically coupled to the use of membranes in order to increase
the hydrogen selectivity of a measurement unit. These
membranes tend to be overgrown by microbial communities
and their biofilms in AD applications. Researchers at the Kurt-
Schwabe-Institute (Meinsberg, Germany) are developing a new
approach for the on-line measurement of dissolved hydrogen
based on the potentiostatic coulometry. The system relies on a
gas-liquid extraction without membranes (Zosel et al., 2011).
Optimization work for the application in AD is ongoing.
BIOLOGICAL METHANATION OF
HYDROGEN
Demand-oriented energy production is a main issue in a
sustainable society, which relies on fluctuating energy
resources. During the ongoing transformation of the energy
supply systems, a temporary surplus or shortage of electrical
power occurs with the necessary expansion of renewable
resources like wind and solar power (Sensfuss and Pfluger,
2014;Peters et al., 2018). For instance, feed-in management
measures led to the curtailment of 5.4 TWh from renewables
in Germany in 2018, 97% of it originated from wind plants
(Bundesnetzagentur, 2019). Energy curtailment created costs of
1.5 billion Euro to Germany’s electricity consumers in 2017
(International Energy Agency, 2020). Flexible biogas
production has the potential to partly buffer this residual load
in rural areas up to a certain degree (Lecker et al., 2017). In recent
years, the concept of power-to-gas has gained a broad interest
(Peters et al., 2018). Surpluses of electrical energy from renewable
sources can be used to produce hydrogen from water by
electrolysis. In combination with carbon dioxide, this
hydrogen can react chemically to methane through the
Sabatier reaction (Lecker et al., 2017;Rachbauer et al., 2017).
CO2+4H2→CH4+2H2O
This process is called chemical methanation. To store the
chemical energy as gas withing the gas distribution grid is much
easier compared to storing electricity. The German gas grid, for
example, can store up to 260 TWh of energy on its own (Lecker
et al., 2017).
In biogas reactors, hydrogenotrophic methanogens (archaea) like
methanococcales or methanobacterium convert hydrogen into
methane through the Sabatier reaction (Bassani et al., 2015;
Rachbauer et al., 2017). The hydrogen for biological methanation
originate from the electrolysis of water, the required carbon dioxide
can be taken from the AD process (Peters et al., 2018). Thus, the
concept is suited for a multi-stage AD: the first stage provides the
necessary carbon dioxide for the methanation and simultaneously
provide SCCAs as a carbon source for archaea. With the lack of
intermediates from fermentative bacteria, a single-stage methanation
would have to rely on an additional supply with organic carbon and
different trace elements.
Through the addition of hydrogen to AD applications,
hydrogenotrophic methanogenesis is amplified and a shift in
consortia of methanogenic archaea is observable (Rachbauer
et al., 2017;Okoro-Shekwaga et al., 2019). A higher quantity
of hydrogenotrophic methanogens leads to a higher quality of the
biogas due to the exhaustion of the carbon dioxide, e.g.
originating from the previous AD processes, during
methanogenesis. Therefore, a utilization of external hydrogen
in biogas plants can decrease the costs for the enrichment of
biogas, if used as biomethane (Lecker et al., 2017).
Hydrogenotrophic methanogenesis in multi-stage AD with
addition of hydrogen has been investigated by several researchers.
Bassani et al. (2015) described that hydrogen addition to a two-
stage AD-reactor (STR) fed with cattle manure increased the
content of methane in the biogas from 70% to nearly 90% (v/v)
under mesophilic conditions through hydrogen inflow. The HRT
was 25 days in the first and 33 days in the second reactor, with a
total OLR of 0.6 g-VS/(L·d). The hydrogen was added to the
second reactor through a diffuser at a rate of 192 ml/(L·d), to meet
the stoichiometric needs for methanation (four moles of
hydrogen per mole of carbon dioxide). Similar results were
achieved by Luo et al. (2012). The authors demonstrated that
the methane production rate of a cattle manure (3% (w/w) of TS,
of which 2.6% VS) increased more than 20% due to the
continuous addition of hydrogen. According to Peters et al.
(2018), the methane fraction in the biogas can achieve up to
95% through hydrogenotrophic methanogenesis. Km values of
anaerobic cell suspensions are in the range of between 5 and
10 μM (Robinson and Tiedje, 1982). The addition of hydrogen to
AD applications results in a rising partial pressure, which can
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 7119719
Janesch et al. Phase Separation in Anaerobic Digestion
even lead to inhibition as described in a previous section. If it is
converted fast enough, it creates no change in substrate
consumption though as shown by Luo and Angelidaki (2013).
The possibilities of biogas upgrading by pulse hydrogen addition
to AD fed with straw and sludge from a biogas plant was
investigated by Agneessens et al. (2017). An OLR of 0.77 g-
VS/(L·d) and a HRT of 20 days were chosen as process
conditions. The experiments showed a fast adaption and
hydrogen assimilation of the microbial community, an
increase of hydrogenotrophic methanogens as
Methanobacterium was detected. The methane production (up
to 0.44 L/(L·d)) was reduced at a carbon dioxide gas
concentration below 12% (v/v) (Agneessens et al., 2017),
which shows the importance to maintain a sufficient
availability to restrict it to become rate limiting.
Methanobacterium was one of the prevalent archaea found by
Li et al. (2020) on the conversion of hydrogen to methane in a
two-stage anaerobic digester fed with rice-straw. It was possible to
increase the methane yield (by 45%) and the methane share in the
biogas (by 101%) through addition of hydrogen to a STR - up-
flow reactor combination (Li et al., 2020). The concept of
hydrogen addition to upgrade the produced biogas was also
investigated in a two-stage thermophilic reactor (STR and up-
flow reactor) digesting cattle manure and potato-starch by
Corbellini et al. (2018). The hydrogen was injected in the first
reactor and channeled to the second one, together with the off-
gas. 98% of the hydrogen was metabolized into methane.
Within the large group of methanogenic archaea, countless
numbers of species exist. Some of these species are especially
persistent against the stress caused by the process conditions
inside of an AD reactor or the chemical substances inflowing with
the feed. The concept of enriching exactly these microorganisms
with bioaugmentation is used since nearly 20 yr (Nzila, 2017).
The concept is especially suitable for multi-stage AD, where the
different organism groups can be augmented more individually. It
was observed by Ács et al. (2019), as they changed the conditions
inside a mesophilic lab-scale reactor, inoculated with the effluent
of an industrial biogas plant (for pig manure and plant silage), to
those of a power-to-methane system, by sole hydrogen feeding.
The diversity of the archaea community was significantly
reduced, while Methanobacterium remained the predominant
genus (Ács et al., 2019). Zhang et al. (2018) described an
example of how to use syntrophic relationships of anaerobic
digesters in combination with bioaugmentation. They added
exoelectrogenic Geobacter sulfurreducens to a batch culture of
methanogens. Methanosaetaceae and Methanobacteriaceae
benefited strongly from the augmentation, as they occurred in
direct vicinity of the new species. With this new bioaugmentation
strategy, the methane production rate was increased by nearly
80%. A rearrangement of the methanogenic consortium inside an
AD application is especially helpful for the treatment of nutrient-
poor substrates, or those which have a high amount of inhibitors
like ammonia, as seen by the experimental results from Li Y. et al.
(2018) and Fotidis et al. (2017).
An alternative to the direct addition of hydrogen to full AD is a
single methanogenic stage or bio-electrochemical
methanogenesis, which could benefit from carbon dioxide
supply of other AD stages, but would be operated separately
from the broth streams. The utilization of power-to-gas has also
been examined for bio-electro-methanogenesis with pure or
mixed methanogenic cultures, combining electrochemical
operation with microbial conversion to produce methane from
carbon dioxide. Cathodes inside the bioreactors act as electron
donators for the methanogenic reduction of carbon dioxide or as
in-situ hydrogen producers (Geppert et al., 2019;Kracke et al.,
2019). In-situ hydrogen production in bio-electrochemical
methanogenesis offer a flexibilization of energy production
through power-to-gas applications, as they can be used with
single-stage, pure methanogenic species in defined cultures and
without external hydrogen supply. Kracke et al. (2020)
investigated the power-to-gas electromethanogenesis of
Methanococcus maripaludis in stirred bio-electrochemical
reactors. Carbon dioxide for the biological methanation was
continuously added. Volumetric methane production rates of
nearly 1.4 L/(L·d) were achieved. The methanogens, that were
exposed to cathodes showed slightly higher metabolization
efficiency than a control group, which was supplied with
external hydrogen. The efficiency of Methanococcus
maripaludis in terms of electro-methanogenesis was confirmed
by Mayer et al. (2019), who compared the electrosynthesis of
methane of different archaea strains in a fed-batch, two-chamber
pilot-scale reactor (H-cell), and by Enzmann et al. (2019), using
carbon dioxide fed bubble columns with carbon layer cathodes.
Kobayashi et al. (2017) used a thermophilic (55°C), high-pressure
system with a mixed culture of methanogens for the bio-
electrochemical synthesis of methane. The archaea in the
system were dominated by Methanothermobacter (Kobayashi
et al., 2017). At even higher temperatures (60°C), but with
atmospheric pressure, Song et al. (2019) did similar
experiments with a mixed culture of methanogens.
The overall efficiency of a process nexus (electrolysis,
methanation and conversion into usable energy) is barely
above 50%, depending on the pressure and the temperature
(Bernacchi et al., 2014). Nevertheless, the conversion of
hydrogen into methane represents a promising technology for
the prospective increase of energy supply from fluctuating
sources. While this concept can be implemented directly into
existing digestion processes, research results exist only from pilot
scale applications yet and therefore need further investigation on
the large scale (Lecker et al., 2017;Theuerl et al., 2019).
MODELLING OF MULTI-STAGE AD
PROCESSES
Mathematical models are an important tool to understand and
predict the degradation processes in AD. They are especially
helpful for designing and operating multi-stage reactor concepts,
as they estimate the flow rates between and feeding rates of the
individual stages. Naturally, first mechanistic models were
obviously established for single-stage AD processes.
Blumensaat and Keller (2005) adapted the “Anaerobic
Digestion Model No. 1 (ADM1)”to a two-stage AD process.
The authors were able to predict the process performances of
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 71197110
Janesch et al. Phase Separation in Anaerobic Digestion
both stages individually. Muha et al. (2013), simulated the share
of reactions in a leach bed reactor and an anaerobic filter.
According to the model, all steps of AD took place in both
reactors except for the hydrolysis: about 40% of the acidogenesis
and nearly 75% of the methanogenesis occurred in the second
stage. Vega De Lille et al. (2016) were able to predict the COD
course from domestic wastewater in a two-stage AD system,
consisting of a sequencing batch reactor and a fixed bed reactor.
The authors used an adjusted ADM1, which showed good results
for the degradation of varying feedstock. Kamyab and Zilouei
(2021) simulated the biogas production from a two-stage mixed
UASB reactor for the treatment of baker’s yeast wastewater. It
predicted, that the efficiency of the COD removal rises from 11 to
36% with an increased input concentration between 1.5 and
4.1 mg-COD/cm³. Simultaneously, the methane production
rate decreases from 1.2 L/g-COD at 1.5 mg-COD/cm³ to
almost zero at 4.1 mg-COD/cm³. Bayen and Gajardo (2019)
used the anaerobic digestion model AM2 for simulating biogas
production it in a two-stage system consisting of a separate
acidogenic and methanogenic stage. The model expressed the
optimal flow rates within the system, e.g. for the treatment of a
substrate with a high release of SCCAs.
Mathematical models for AD are often developed for a certain
scope of application. Therefore it can be useful to adapt models
from a different application for one or all stages of AD, as
described by Postawa (2018). He used a model originally
designed for high pressure AD, to predict the biogas
production in a temperature phased two-stage AD and
compared the results with literature data. While not fully
optimised, the model predicted the biogas production well for
time periods of 40 days in AD. The recent results from the
modelling of multi-stage AD processes show that stage
separation becomes predictable. It might be even easier to
simulate separate stages as the number of parallel reactions
and influencing factors is reduced. If sufficient data under
dynamic operation is available, either the production dynamics
can be predicted or process disturbances can be identified with
suitable monitoring tools. On a system level, the potential yields
and value generation can be simulated.
ECONOMIC COMPETITIVENESS AND
ALTERNATIVE PRODUCTS
Due to the higher investment and operational costs, and although
promising as described previously, multi-stage digesters cannot
compete economically for all types of feedstock. The investment
costs for single- and two-stage biogas plants with a capacity of
100 kW have been estimated as 7000 €/kW (Carlini et al., 2017)
and 7400 €/kW (Renda et al., 2016), respectively. Organic waste
residues with a dry matter content below 3% like sewage sludge
exhibit similar methane yields (0.5 L/(L
digestate
·d) when treated
either in single-stage AD (STR) or two-stage processes (Schievano
et al., 2012;Van et al., 2020). For the transfer between the stages of
AD, a sufficient mixing is a premise. Thus, multi-stage AD is
typically operated with STRs. Feedstock with a high TS-content
(>15–20%) is difficult to apply in these reactors due to the high
viscosity (Van et al., 2020). According to this study, great
amounts of water and energy as well as long HRT are needed
to treat such feedstock in two-stage digesters. Therefore, dry
single-stage digesters are recommended in this case.
Beside the benefits for biogas production, phase separation in
AD allows the recovery of value-added side products from some
of the stages, e.g. from DF. Two-stage AD processes for the
combined production of biohydrogen and biomethane from
organic wastes can yield a biohythane gas containing 10–15%
H
2
, 50–55% CH
4
,and30–40% CO
2
. The upgrade of the SCCA-
enriched liquid effluent generated in the first stage can lead to
higher energy recovery and higher degradation efficiency than
in traditional biogas production processes (O-Thong, 2018). Li
et al. (2020) tested a semi-continuous two-stage AD system for
biohythane production from cornstalk. The hydrogen
production in the first-stage reached 25 ml/g-TS, the
hydrogen content in the biohythane gas was 18.47%. In the
second stage UASB, a methane yield of 95 ml/g-TS was
additionally achieved by using the liquid effluent generated
in the first stage. Ghimire et al. (2020) investigated the role
of temperature in a two-stage DF and AD process using FW, and
observed that mesophilic (34°C) DF provided a hydrogen yield
of 53.5 ml H
2
/g-VS, while thermophilic conditions (55°C)
favored methane yields in AD, achieving 307.5 ml CH
4
/g-VS.
Yan et al. (2020) assessed the performance of a two-stage system
using leach bed reactors for the AD of several types of FW, and
achieved a hydrogen production of 61 ml/g-VS. By applying the
produced leachate and acidogenic off-gas in the second stage,
the authors reached a methane production of 420 ml/g-VS.
Chen et al. (2021) used a temperature phased AD of rice
straw and pig manure for biohythane production. In a
mesophilic-thermophilic reactor concept, up to 16.7 ml-H
2
/
g-VS and 197.7 ml-CH
4
/g-VS were measured, which implies
aratioof1mlH
2
per 12 ml CH
4
.
Based on the DF of several biodegradable wastes, it is
estimated that two-thirds of the carbon input can be
fermented into SCCAs such as acetic, propionic, butyric,
isovaleric, valeric and caproic acid, among others (Slezak et al.,
2017). Although extensive reviews exist on the topic, SCCAs
production via AD is sensitive to several operational factors,
namely feedstock, pH-value, temperature, organic loading rate,
alkalinity, and retention time, respectively, which typically depict
a synergetic effect on the microbial consortia (Atasoy et al., 2018).
Before SCCAs gain an economic value during AD, they have to be
concentrated and separated from the effluent (Kleerebezem et al.,
2015). Gioannis et al. (2017) described the production of SCCAs
in a single- and two-stage FW-fed STR. SCCAs concentrations of
5 g/L were detected in the acidogenesis stage. Bioaugmentation
has been successfully applied to enhance SCCAs production,
increasing butyric and caproic acid yields by up to 300% with
Clostridia,Sphingobacteriales,Desulfobacteraceae and Bacillus
species (Reddy et al., 2018). Further investigation is required
to maximize acid concentration while keeping the microbial
consortia vital and eventually combine the concomitant
biomass growth with the enrichment of species that have a
high value as fertilizer, e.g. for soil recovery or as biofungicide
producers like Bacillus sp.
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 71197111
Janesch et al. Phase Separation in Anaerobic Digestion
CONCLUSION
Phase separation in AD provides a great potential for the
flexibilization of AD applications, which is beneficial for a
demand-driven biogas production. With more research on up-
scaling, this approach is likely to be become applicable while
using similar infrastructure as conventional AD processes.
Monitoring concepts coupled with dynamic effluent
recirculation as also discussed in this work, allow for an
increasing system robustness and stability. A robust and stable
system also facilitates the use of feeding strategies with alternating
feedstock, making it possible to integrate AD into regional
material cycles. Strategies such as bioaugmentation help the
adaptation of microbial communities that are most suitable for
the conditions and requirements of the specific AD application.
Finally, additional benefits from the separation of SCCAs
between stages of a multi-phase AD can couple biomass
energy and material use. As one example, among others, the
use of SCCAs as feed for the production of long-chain
polyunsaturated fatty acids in subsequent microalgae mono-
cultivation processes shall be mentioned (Chalima et al., 2020;
Patel et al., 2021). They have a high value in nutrition and serve as
a replacement of fish oil and fish meal. Such a process can, beside
the value addition, represent a more sustainable production of
these compounds for food and feed application (Bartek et al.,
2021). Nevertheless, research and development to increase the
cost-efficiency of coupled systems and reduce efforts for
separation is a pre-requisite so that such process combinations
will be applied in industrial scale. Finally, data for techno-
economic and sustainability assessments are required to
identify suitable feedstock-product combinations within the
manyfold options that a multi-stage AD theoretically offers to
its users.
AUTHOR CONTRIBUTIONS
EJ collected data, performed literature research, and wrote the
main body of the manuscript. JP supported data collection
and manuscript preparation and review. PN supported
supervision of the manuscript preparation and review. SJ
supported data collection, supervised manuscript
preparation and review.
FUNDING
The authors acknowledge funding of the German Federal
Ministry for Economics within the framework program
Biomass energy use, grant no. 03EI5409A.
REFERENCES
Ács, N., Szuhaj, M., Wirth, R., Bagi, Z., Maróti, G., Rákhely, G., et al. (2019).
Microbial Community Rearrangements in Power-To-Biomethane Reactors
Employing Mesophilic Biogas Digestate. Front. Energ. Res. 7, 132.
doi:10.3389/fenrg.2019.00132
Agneessens, L. M., Ottosen, L. D. M., Voigt, N. V., Nielsen, J. L., de Jonge, N.,
Fischer, C. H., et al. (2017). In-situ Biogas Upgrading with Pulse H 2 Additions:
The Relevance of Methanogen Adaption and Inorganic Carbon Level.
Bioresour. Technol. 233, 256–263. doi:10.1016/j.biortech.2017.02.016
Ariunbaatar, J., Panico, A., Esposito, G., Pirozzi, F., and Lens, P. N. L. (2014).
Pretreatment Methods to Enhance Anaerobic Digestion of Organic Solid
Waste. Appl. Energ. 123, 143–156. doi:10.1016/j.apenergy.2014.02.035
Atasoy, M., Owusu-Agyeman, I., Plaza, E., and Cetecioglu, Z. (2018). Bio-based
Volatile Fatty Acid Production and Recovery from Waste Streams: Current
Status and Future Challenges. Bioresour. Technol. 268, 773–786. doi:10.1016/
j.biortech.2018.07.042
Bartek, L., Strid, I., Henryson, K., Junne, S., Rasi, S., and Eriksson, M. (2021). Life
Cycle Assessment of Fish Oil Substitute Produced by Microalgae Using Food
Waste. Sustainable Prod. Consumption 27, 2002–2021. doi:10.1016/
j.spc.2021.04.033
Bassani, I., Kougias, P. G., Treu, L., and Angelidaki, I. (2015). Biogas Upgrading via
Hydrogenotrophic Methanogenesis in Two-Stage Continuous Stirred Tank
Reactors at Mesophilic and Thermophilic Conditions. Environ. Sci. Technol. 49,
12585–12593. doi:10.1021/acs.est.5b03451
Bayen, T., and Gajardo, P. (2019). On the Steady State Optimization of the Biogas
Production in a Two-Stage Anaerobic Digestion Model. J. Math. Biol. 78,
1067–1087. doi:10.1007/s00285-018-1301-3
Bernacchi, S., Weissgram, M., Weissgram, M., Wukovits, W., and Herwig, C.
(2014). Process Efficiency Simulation for Key Process Parameters in Biological
Methanogenesis. AIMS Bioeng. 1, 53–71. doi:10.3934/bioeng.2014.1.53
Bischofsberger, W. (2005). Anaerobic Technology [in German]. Berlin: Springer.
Björnsson, L., Murto, M., and Mattiasson, B. (2000). Evaluation of Parameters for
Monitoring an Anaerobic Co-digestion Process. Appl. Microbiol. Biotechnol. 54,
844–849. doi:10.1007/s002530000471
Björnsson, L., Murto, M., Jantsch, T. G., and Mattiasson, B. (2001). Evaluation of
New Methods for the Monitoring of Alkalinity, Dissolved Hydrogen and the
Microbial Community in Anaerobic Digestion. Water Res. 35, 2833–2840.
doi:10.1016/S0043-1354(00)00585-6
Blank, A., and Hoffmann, E. (2011). Upgrading of a Co-digestion Plant by
Implementation of a Hydrolysis Stage. Waste Manag. Res. 29, 1145–1152.
doi:10.1177/0734242X11423954
Blumensaat, F., and Keller, J. (2005). Modelling of Two-Stage Anaerobic Digestion
Using the IWA Anaerobic Digestion Model No. 1 (ADM1). Water Res. 39,
171–183. doi:10.1016/j.watres.2004.07.024
Bockisch, A., Biering, J., Päßler, S., Vonau, W., Junne, S., and Neubauer, P. (2014).
In Situ Investigation of the Liquid Phase in Industrial Yeast Fermentations with
Mobile Multiparameter Sensors. Chem. Ingenieur Technik 86, 1582.
doi:10.1002/cite.201450231
Bockisch, A., Kielhorn, E., Neubauer, P., and Junne, S. (2019). Process Analytical
Technologies to Monitor the Liquid Phase of Anaerobic Cultures. Process
Biochem. 76, 1–10. doi:10.1016/j.procbio.2018.10.005
Borja, R. (2011). “Biogas Production,”in Comprehensive Biotechnology. Editors
M. Butler and M. Moo-Young (Saint Louis, MO: Elsevier), 785–798.
doi:10.1016/b978-0-08-088504-9.00126-4
Bundesnetzagentur (2019). Monitoring Report 2019 - Key Findings and Summary.
Bonn.
Burgstaler, J., Blumenthal, J., Wiedow, D., Godlinski, F., and Kanswohl, N. (2010).
Possibilities to Regulate the pH Value of Acidified Biogas Fermenters and the
Effects on the Biogas Yield. Available at: http://nbn-resolving.de/urn:nbn:de:gbv:
253-201012-dn047464-1 (Accessed March 4, 2021).
Carlini, M., Mosconi, E., Castellucci, S., Villarini, M., and Colantoni, A. (2017). An
Economical Evaluation of Anaerobic Digestion Plants Fed with Organic Agro-
Industrial Waste. Energies 10, 1165. doi:10.3390/en10081165
Castellano-Hinojosa, A., Armato, C., Pozo, C., González-Martínez, A., and
González-López, J. (2018). New Concepts in Anaerobic Digestion Processes:
Recent Advances and Biological Aspects. Appl. Microbiol. Biotechnol. 102,
5065–5076. doi:10.1007/s00253-018-9039-9
Castellón-Zelaya, M. F., and González-Martínez, S. (2021). Silage of the Organic
Fraction of Municipal Solid Waste to Improve Methane Production. Water Sci.
Technol. 83, 2536–2548. doi:10.2166/wst.2021.148
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 71197112
Janesch et al. Phase Separation in Anaerobic Digestion
Chalima, A., Taxeidis, G., and Topakas, E. (2020). Optimization of the Production
of Docosahexaenoic Fatty Acid by the Heterotrophic Microalga
Crypthecodinium Cohnii Utilizing a Dark Fermentation Effluent. Renew.
Energ. 152, 102–109. doi:10.1016/j.renene.2020.01.041
Chanakya, H., and Shwetmala, K. (2017). Anaerobic Two Stage Degradation
Pattern of Fermentable Components of Municipal Solid Waste. Glob. Nest J.
19, 706–715. doi:10.30955/gnj.002377
Chatterjee, B., and Mazumder, D. (2019). Role of Stage-Separation in the
Ubiquitous Development of Anaerobic Digestion of Organic Fraction of
Municipal Solid Waste: A Critical Review. Renew. Sustainable Energ. Rev.
104, 439–469. doi:10.1016/j.rser.2019.01.026
Chen, Y., Cheng, J. J., and Creamer, K. S. (2008). Inhibition of Anaerobic Digestion
Process: A Review. Bioresour. Technol. 99, 4044–4064. doi:10.1016/
j.biortech.2007.01.057
Chen, X., Yuan, H., Zou, D., Liu, Y., Zhu, B., Chufo, A., et al. (2015). Improving
Biomethane Yield by Controlling Fermentation Type of Acidogenic Phase in
Two-phase Anaerobic Co-digestion of Food Waste and Rice Straw. Chem. Eng.
J. 273, 254–260. doi:10.1016/j.cej.2015.03.067
Chen, H., Huang, R., Wu, J., Zhang, W., Han, Y., Xiao, B., et al. (2021). Biohythane
Production and Microbial Characteristics of Two Alternating Mesophilic and
Thermophilic Two-Stage Anaerobic Co-digesters Fed with Rice Straw and Pig
Manure. Bioresour. Technol. 320, 124303. doi:10.1016/j.biortech.2020.124303
Chojnacka, K., Moustakas, K., and Witek-krowiak, A. (2020). Bio-based Fertilizers:
A Practical Approach Towards Circular Economy. Bioresour. Technol. 295,
122223. doi:10.1016/j.biortech.2019.122223
Corbellini, V., Kougias, P. G., Treu, L., Bassani, I., Malpei, F., and Angelidaki, I.
(2018). Hybrid Biogas Upgrading in a Two-Stage Thermophilic Reactor. Energ.
Convers. Manage. 168, 1–10. doi:10.1016/j.enconman.2018.04.074
Dahiya, S., Kumar, A. N., Shanthi Sravan, J., Chatterjee, S., Sarkar, O., and Mohan,
S. V. (2018). Food Waste Biorefinery: Sustainable Strategy for Circular
Bioeconomy. Bioresour. Technol. 248, 2–12. doi:10.1016/j.biortech.2017.07.176
Dalkılıc, K., and Ugurlu, A. (2015). Biogas Production from Chicken Manure at
Different Organic Loading Rates in a Mesophilic-Thermopilic Two Stage
Anaerobic System. J. Biosci. Bioeng. 120, 315–322. doi:10.1016/
j.jbiosc.2015.01.021
Detman, A., Chojnacka, A., Błaszczyk, M., Kaźmierczak, W., Piotrowski, J., and
Sikora, A. (2017). Biohydrogen and Biomethane (Biogas) Production in the
Consecutive Stages of Anaerobic Digestion of Molasses. Pol. J. Environ. Stud.
26, 1023–1029. doi:10.15244/pjoes/68149
De Gioannis, G., Muntoni, A., Polettini, A., Pomi, R., and Spiga, D. (2017). Energy
Recovery from One- and Two-Stage Anaerobic Digestion of Food Waste. Waste
Manage. 68, 595–602. doi:10.1016/j.wasman.2017.06.013
Díaz, I., Fdz-Polanco, F., Mutsvene, B., and Fdz-Polanco, M. (2020). Effect of
Operating Pressure on Direct Biomethane Production from Carbon Dioxide
and Exogenous Hydrogen in the Anaerobic Digestion of Sewage Sludge. Appl.
Energ. 280, 115915. doi:10.1016/j.apenergy.2020.115915
Ding, L., Chen, Y., Xu, Y., and Hu, B. (2021). Improving Treatment Capacity and
Process Stability via a Two-Stage Anaerobic Digestion of Food Waste
Combining Solid-State Acidogenesis and Leachate Methanogenesis/
recirculation. J. Clean. Prod. 279, 123644. doi:10.1016/j.jclepro.2020.123644
Enzmann, F., Gronemeier, D., and Holtmann, D. (2019). Process Stability
Examinations of Bioelectromethanogenesis Using a Pure Culture of M.
Maripaludis. Biochem. Eng. J. 151, 107321. doi:10.1016/j.bej.2019.107321
European Biogas Association (2019). EBA Statistical Report 2019. Brussels.
Falk, H. M., Reichling, P., Andersen, C., and Benz, R. (2015). Online Monitoring of
Concentration and Dynamics of Volatile Fatty Acids in Anaerobic Digestion
Processes with Mid-infrared Spectroscopy. Bioproc. Biosyst. Eng. 38, 237–249.
doi:10.1007/s00449-014-1263-9
Fernández-Rodríguez, J., Pérez, M., and Romero, L. I. (2016). Semicontinuous
Temperature-Phased Anaerobic Digestion (TPAD) of Organic Fraction of
Municipal Solid Waste (OFMSW). Comparison with Single-Stage Processes.
Chem. Eng. J. 285, 409–416. doi:10.1016/j.cej.2015.10.027
Flores-Cortés, M., Pérez-Trevilla, J., de María Cuervo-López, F., Buitrón, G., and
Quijano, G. (2021). H2S Oxidation Coupled to Nitrate Reduction in a Two-
Stage Bioreactor: Targeting H2S-Rich Biogas Desulfurization. Waste Manage.
120, 76–84. doi:10.1016/j.wasman.2020.11.024
Fotidis, I. A., Treu, L., and Angelidaki, I. (2017). Enriched Ammonia-Tolerant
Methanogenic Cultures as Bioaugmentation Inocula in Continuous
Biomethanation Processes. J. Clean. Prod. 166, 1305–1313. doi:10.1016/
j.jclepro.2017.08.151
Fraunhofer IEE (2018). Final Report on the ReBi 2.0 Project: Regulation of the Gas
Production of Biogas Plants (ReBi) for Demand-Oriented, Controlled Generation
of Electricity from Biogas: Transfer of Results from the Controlled Biogas
Production from the Technical Scale to the Demonstration Plant. Goettingen.
Geppert, F., Liu, D., Weidner, E., and Heijne, A. t. (2019). Redox-flow Battery
Design for a Methane-Producing Bioelectrochemical System. Int. J. Hydrogen
Energ. 44, 21464–21469. doi:10.1016/j.ijhydene.2019.06.189
Ghanimeh, S., Al-Sanioura, D., Saikaly, P. E., and El-Fadel, M. (2019). Comparison
of Single-Stage and Two-Stage Thermophilic Anaerobic Digestion of SS-
OFMSW during the Start-Up Phase. Waste Biomass Valor. 11, 6709–6716.
doi:10.1007/s12649-019-00891-8
Ghimire, A., Luongo, V., Frunzo, L., Lens, P. N. L., Pirozzi, F., and Esposito, G.
(2020). Biohythane Production from Food Waste in a Two-Stage Process:
Assessing the Energy Recovery Potential. Environ. Technol. 1, 1–7. doi:10.1080/
09593330.2020.1869319
Gómez-Camacho, C. E., Pellicer Alborch, K., Bockisch, A., Neubauer, P., Junne, S.,
and Ruggeri, B. (2020). Monitoring the Physiological State in the Dark
Fermentation of Maize/Grass Silage Using Flow Cytometry and Electrooptic
Polarizability Measurements. Bioenerg. Res. doi:10.1007/s12155-020-10184-x
Hans, M., and Kumar, S. (2019). Biohythane Production in Two-Stage Anaerobic
Digestion System. Int. J. Hydrogen Energ. 44, 17363–17380. doi:10.1016/
j.ijhydene.2018.10.022
Harper, S. R., and Pohland, F. G. (1986). Recent Developments in Hydrogen
Management During Anaerobic Biological Wastewater Treatment. Biotechnol.
Bioeng. 28, 585–602. doi:10.1002/bit.260280416
Heng, G. C., Isa, M. H., Lock, S. S. M., and Ng, C. A. (2021). Process Optimization
of Waste Activated Sludge in Anaerobic Digestion and Biogas Production by
Electrochemical Pre-treatment Using Ruthenium Oxide Coated Titanium
Electrodes. Sustainability 13, 4874. doi:10.3390/su13094874
Huck, C., Poghossian, A., Wagner, P., and Schöning, M. J. (2013). Combined
Amperometric/field-Effect Sensor for the Detection of Dissolved Hydrogen.
Sensors Actuators B: Chem. 187, 168–173. doi:10.1016/j.snb.2012.10.050
International Energy Agency (2020). Germany 2020 Energy Policy Review. Paris.
Janzen, N. H., Schmidt, M., Krause, C., and Weuster-Botz, D. (2015). Evaluation of
Fluorimetric pH Sensors for Bioprocess Monitoring at Low pH. Bioproc.
Biosyst. Eng. 38, 1685–1692. doi:10.1007/s00449-015-1409-4
Jie, W., Peng, Y., Ren, N., and Li, B. (2014). Volatile Fatty Acids (VFAs)
Accumulation and Microbial Community Structure of Excess Sludge (ES) at
Different pHs. Bioresour. Technol. 152, 124–129. doi:10.1016/
j.biortech.2013.11.011
Jimenez-Jorquera, C., Orozco, J., and Baldi, A. (2010). ISFET Based Microsensors
for Environmental Monitoring. Sensors 10, 61–83. doi:10.3390/s100100061
Jiraprasertwong, A., Vichaitanapat, K., Leethochawalit, M., and Chavadej, S.
(2018). Three-Stage Anaerobic Sequencing Batch Reactor (ASBR) for
Maximum Methane Production: Effects of COD Loading Rate and Reactor
Volumetric Ratio. Energies 11, 1543. doi:10.3390/en11061543
Jiraprasertwong, A., Maitriwong, K., and Chavadej, S. (2019). Production of Biogas
from Cassava Wastewater Using a Three-Stage Upflow Anaerobic Sludge
Blanket (UASB) Reactor. Renew. Energ. 130, 191–205. doi:10.1016/
j.renene.2018.06.034
Kaltschmitt, M., Hartmann, H., and Hofbauer, H. (2016). Energy from Biomass [in
German]. Berlin, Heidelberg: Springer Berlin Heidelberg.
Kamyab, B., and Zilouei, H. (2021). Investigating the Efficiency of Biogas
Production Using Modelling Anaerobic Digestion of baker’s Yeast
Wastewater on Two-Stage Mixed-UASB Reactor. Fuel 285, 119198.
doi:10.1016/j.fuel.2020.119198
Kaye, G. W. C., and Laby, T. H. (1992). Tables of Physical and Chemical Constants
and Some Mathematical Functions. Harlow, UK: Longman, 477.
Kim, S. W., Park, J. Y., Kim, J. K., Cho, J. H., Chun, Y. N., Lee, I. H., et al. (2000).
Development of a Modified Three-Stage Methane Production Process Using
Food Wastes. ABAB 84-86, 731–742. doi:10.1385/ABAB:84-86:1-9:731
Kim, J. K., Oh, B. R., Chun, Y. N., and Kim, S. W. (2006). Effects of Temperature
and Hydraulic Retention Time on Anaerobic Digestion of Food Waste. J. Biosci.
Bioeng. 102, 328–332. doi:10.1263/jbb.102.328
Kim, J. K., Han, G. H., Oh, B. R., Chun, Y. N., Eom, C.-Y., and Kim, S. W. (2008).
Volumetric Scale-Up of a Three Stage Fermentation System for Food Waste
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 71197113
Janesch et al. Phase Separation in Anaerobic Digestion
Treatment. Bioresour. Technol. 99, 4394–4399. doi:10.1016/
j.biortech.2007.08.031
Kleerebezem, R., Joosse, B., Rozendal, R., and van Loosdrecht, M. C. M. (2015).
Anaerobic Digestion without Biogas?. Rev. Environ. Sci. Biotechnol. 14,
787–801. doi:10.1007/s11157-015-9374-6
Kobayashi, H., Nagashima, A., Kouyama, M., Fu, Q., Ikarashi, M., Maeda, H., et al.
(2017). High-pressure Thermophilic Electromethanogenic System Producing
Methane at 5 MPa, 55°C. J. Biosci. Bioeng. 124, 327–332. doi:10.1016/
j.jbiosc.2017.04.001
Kracke, F., Wong, A. B., Maegaard, K., Deutzmann, J. S., Hubert, M. A., Hahn, C.,
et al. (2019). Robust and Biocompatible Catalysts for Efficient Hydrogen-
Driven Microbial Electrosynthesis. Commun. Chem. 2. doi:10.1038/s42004-
019-0145-0
Kracke, F., Deutzmann, J. S., Gu, W., and Spormann, A. M. (2020). In Situ
Electrochemical H2 Production for Efficient and Stable Power-To-Gas
Electromethanogenesis. Green. Chem. 22, 6194–6203. doi:10.1039/d0gc01894e
Kurahashi, K., Kimura, C., Fujimoto, Y., and Tokumoto, H. (2017). Value-adding
Conversion and Volume Reduction of Sewage Sludge by Anaerobic Co-
digestion with Crude Glycerol. Bioresour. Technol. 232, 119–125.
doi:10.1016/j.biortech.2017.02.012
Lecker, B., Illi, L., Lemmer, A., and Oechsner, H. (2017). Biological Hydrogen
Methanation - A Review. Bioresour. Technol. 245, 1220–1228. doi:10.1016/
j.biortech.2017.08.176
Lemmer, A., Chen, Y., Lindner, J., Wonneberger, A. M., Zielonka, S., Oechsner, H.,
et al. (2015). Influence of Different Substrates on the Performance of a Two-
Stage High Pressure Anaerobic Digestion System. Bioresour. Technol. 178,
313–318. doi:10.1016/j.biortech.2014.09.118
Li, L., He, Q., Wei, Y., He, Q., and Peng, X. (2014). Early Warning Indicators for
Monitoring the Process Failure of Anaerobic Digestion System of Food Waste.
Bioresour. Technol. 171, 491–494. doi:10.1016/j.biortech.2014.08.089
Li, W., Guo, J., Cheng, H., Wang, W., and Dong, R. (2017). Two-phase Anaerobic
Digestion of Municipal Solid Wastes Enhanced by Hydrothermal Pretreatment:
Viability, Performance and Microbial Community Evaluation. Appl. Energ. 189,
613–622. doi:10.1016/j.apenergy.2016.12.101
Li, Z., Wachemo, A. C., Yuan, H., Korai, R. M., and Li, X. (2020). Improving
Methane Content and Yield from Rice Straw by Adding Extra Hydrogen into a
Two-Stage Anaerobic Digestion System. Int. J. Hydrogen Energ. 45, 3739–3749.
doi:10.1016/j.ijhydene.2019.07.235
Li, L., Peng, X., Wang, X., and Wu, D. (2018). Anaerobic Digestion of Food Waste:
A Review Focusing on Process Stability. Bioresour. Technol. 248, 20–28.
doi:10.1016/j.biortech.2017.07.012
Li, W., Loh, K.-C., Zhang, J., Tong, Y. W., and Dai, Y. (2018). Two-stage Anaerobic
Digestion of Food Waste and Horticultural Waste in High-Solid System. Appl.
Energ. 209, 400–408. doi:10.1016/j.apenergy.2017.05.042
Li, Y., Li, L., Sun, Y., and Yuan, Z. (2018). Bioaugmentation Strategy for Enhancing
Anaerobic Digestion of High C/N Ratio Feedstock with Methanogenic
Enrichment Culture. Bioresour. Technol. 261, 188–195. doi:10.1016/
j.biortech.2018.02.069
Linke, B., Rodríguez-Abalde, Á., Jost, C., and Krieg, A. (2015). Performance of a
Novel Two-phase Continuously Fed Leach Bed Reactor for Demand-Based
Biogas Production from maize Silage. Bioresour. Technol. 177, 34–40.
doi:10.1016/j.biortech.2014.11.070
Liu, Y., Li, X., Kang, X., Yuan, Y., and Du, M. (2014). Short Chain Fatty Acids
Accumulation and Microbial Community Succession during Ultrasonic-
Pretreated Sludge Anaerobic Fermentation Process: Effect of Alkaline
Adjustment. Int. Biodeterior. Biodegrad. 94, 128–133. doi:10.1016/
j.ibiod.2014.07.004
Liu, H., Han, P., Liu, H., Zhou, G., Fu, B., and Zheng, Z. (2018). Full-scale
Production of VFAs from Sewage Sludge by Anaerobic Alkaline Fermentation
to Improve Biological Nutrients Removal in Domestic Wastewater. Bioresour.
Technol. 260, 105–114. doi:10.1016/j.biortech.2018.03.105
Lovato, G., Albanez, R., Ruggero, L. S., Stracieri, L., Ratusznei, S. M., and
Domingues Rodrigues, J. A. (2020). Energetic Feasibility of a Two-Stage
Anaerobic Digestion System Compared to a Single-Stage System Treating
Whey and Glycerin. Biochem. Eng. J. 161, 107653. doi:10.1016/
j.bej.2020.107653
Lowe, S. E., Jain, M. K., and Zeikus, J. G. (1993). Biology, Ecology, and
Biotechnological Applications of Anaerobic Bacteria Adapted to
Environmental Stresses in Temperature, pH, Salinity, or Substrates.
Microbiol. Rev. 57, 451–509. doi:10.1128/mr.57.2.451-509.1993
Luo, G., and Angelidaki, I. (2013). Co-Digestion of Manure and Whey for In Situ
Biogas Upgrading by the Addition of H(2): Process Performance and Microbial
Insights. Appl. Microbiol. Biotechnol. 97, 1373–1381. doi:10.1007/s00253-012-
4547-5
Luo, G., Johansson, S., Boe, K., Xie, L., Zhou, Q., and Angelidaki, I. (2012).
Simultaneous Hydrogen Utilization and In Situ Biogas Upgrading in an
Anaerobic Reactor. Biotechnol. Bioeng. 109, 1088–1094. doi:10.1002/bit.24360
Lukitawesa, R., Wikandari, R., Millati, R., Taherzadeh, M. J., and Niklasson, C.
(2018). Effect of Effluent Recirculation on Biogas Production Using Two-Stage
Anaerobic Digestion of Citrus Waste. Molecules 23, 3380. doi:10.3390/
molecules23123380
Markphan, W., Mamimin, C., Suksong, W., Prasertsan, P., and O-Thong, S. (2020).
Comparative Assessment of Single-Stage and Two-Stage Anaerobic Digestion
for Biogas Production from High Moisture Municipal Solid Waste. PeerJ 8,
e9693. doi:10.7717/peerj.9693
Mayer, F., Enzmann, F., Lopez, A. M., and Holtmann, D. (2019). Performance of
Different Methanogenic Species for the Microbial Electrosynthesis of Methane
from Carbon Dioxide. Bioresour. Technol. 289, 121706. doi:10.1016/
j.biortech.2019.121706
Meegoda, J., Li, B., Patel, K., and Wang, L. (2018). A Review of the Processes,
Parameters, and Optimization of Anaerobic Digestion. Ijerph 15, 2224.
doi:10.3390/ijerph15102224
Menzel, T., Neubauer, P., and Junne, S. (2020). Role of Microbial Hydrolysis in
Anaerobic Digestion. Energies 13, 5555. doi:10.3390/en13215555
Merkle, W., Baer, K., Lindner, J., Zielonka, S., Ortloff, F., Graf, F., et al. (2017).
Influence of Pressures up to 50 Bar on Two-Stage Anaerobic Digestion.
Bioresour. Technol. 232, 72–78. doi:10.1016/j.biortech.2017.02.013
Micolucci, F., and Uellendhal, H. (2018). Two-Stage Dry Anaerobic Digestion
Process Control of Biowaste for Hydrolysis and Biogas Optimization. Chem.
Eng. Technol. 41, 717–726. doi:10.1002/ceat.201700467
Moeller, L., and Zehnsdorf, A. (2016). Process Upsets in a Full-Scale Anaerobic
Digestion Bioreactor: Over-acidification and Foam Formation during Biogas
Production. Energ Sustain. Soc. 6, 30. doi:10.1186/s13705-016-0095-7
Moestedt, J., Nordell, E., Hallin, S., and Schnürer, A. (2016). Two-stage Anaerobic
Digestion for Reduced Hydrogen Sulphide Production. J. Chem. Technol.
Biotechnol. 91, 1055–1062. doi:10.1002/jctb.4682
Morozova, I., Nikulina, N., Oechsner, H., Krümpel, J., and Lemmer, A. (2020).
Effects of Increasing Nitrogen Content on Process Stability and Reactor
Performance in Anaerobic Digestion. Energies 13, 1139. doi:10.3390/
en13051139
Muha, I., Zielonka, S., Lemmer, A., Schönberg, M., Linke, B., Grillo, A., et al. (2013).
Do Two-phase Biogas Plants Separate Anaerobic Digestion Phases? - A
Mathematical Model for the Distribution of Anaerobic Digestion Phases
Among Reactor Stages. Bioresour. Technol. 132, 414–418. doi:10.1016/
j.biortech.2012.12.031
Mutungwazi, A., Ijoma, G. N., and Matambo, T. S. (2021). The Significance of
Microbial Community Functions and Symbiosis in Enhancing Methane
Production During Anaerobic Digestion: A Review. Symbiosis 83, 1–24.
doi:10.1007/s13199-020-00734-4
Nery, E. W., and Kubota, L. T. (2016). Integrated, Paper-Based Potentiometric
Electronic Tongue for the Analysis of Beer and Wine. Anal. Chim. Acta 918,
60–68. doi:10.1016/j.aca.2016.03.004
Nespeca, M. G., Rodrigues, C. V., Santana, K. O., Maintinguer, S. I., and de Oliveira,
J. E. (2017). Determination of Alcohols and Volatile Organic Acids in
Anaerobic Bioreactors for H 2 Production by Near Infrared Spectroscopy.
Int. J. Hydrogen Energ. 42, 20480–20493. doi:10.1016/j.ijhydene.2017.07.044
Nsair, A., Onen Cinar, S., Alassali, A., Abu Qdais, H., and Kuchta, K. (2020).
Operational Parameters of Biogas Plants: A Review and Evaluation Study.
Energies 13, 3761. doi:10.3390/en13153761
Nzila, A. (2017). Mini Review: Update on Bioaugmentation in Anaerobic Processes
for Biogas Production. Anaerobe 46, 3–12. doi:10.1016/j.anaerobe.2016.11.007
Okoro-Shekwaga, C. K., Ross, A. B., and Camargo-Valero, M. A. (2019). Improving
the Biomethane Yield from Food Waste by Boosting Hydrogenotrophic
Methanogenesis. Appl. Energ. 254, 113629. doi:10.1016/j.apenergy.2019.113629
O-Thong, S. (2018). “Biohythane Production from Organic Wastes by Two-Stage
Anaerobic Fermentation Technology,”in Advances In Biofuels And Bioenergy.
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 71197114
Janesch et al. Phase Separation in Anaerobic Digestion
Editor C. Mamimin (Rijeka, Croatia: IntechOpen), 83–116. doi:10.5772/
intechopen.74392
Paillet, F., Barrau, C., Escudié, R., Bernet, N., and Trably, E. (2021). Robust
Operation Through Effluent Recycling for Hydrogen Production from the
Organic Fraction of Municipal Solid Waste. Bioresour. Technol. 319, 124196.
doi:10.1016/j.biortech.2020.124196
Pandey, P. K., and Soupir, M. L. (2011). Escherichia coli Inactivation Kinetics in
Anaerobic Digestion of Dairy Manure Under Moderate, Mesophilic and
Thermophilic Temperatures. AMB Express 1, 18. doi:10.1186/2191-0855-1-18
Patel, A., Mahboubi, A., Horváth, I. S., Taherzadeh, M. J., Rova, U.,
Christakopoulos, P., et al. (2021). Volatile Fatty Acids (VFAs) Generated by
Anaerobic Digestion Serve as Feedstock for Freshwater and Marine Oleaginous
Microorganisms to Produce Biodiesel and Added-Value Compounds. Front.
Microbiol. 12, 614612. doi:10.3389/fmicb.2021.614612
Pavlostathis, S. G. (2011). “Kinetics and Modeling of Anaerobic Treatment and
Biotransformation Processes,”in Comprehensive Biotechnology. Editor
M. Moo-Young (Burlington, MA: Academic Press), 385–397. doi:10.1016/
b978-0-08-088504-9.00385-8
Peters, L., Uhlenhut, F., Biernacki, P., and Steinigeweg, S. (2018). Aktueller Stand
der Flexibilisierungskonzepte von Biogasanlagen zur Abdeckung der
Residuallast. Chem. Ingenieur Technik 90, 36–46. doi:10.1002/cite.201700101
Pfeiffer, W., Nguyen, V. T., Neumann, J., Awe, D., and Tränckner, J. (2020).
Operation and Control of a Full-Scale Biogas Plant Treating Wastewater from
the Cleaning of Car Tanks. Chem. Eng. Technol. 43, 84–94. doi:10.1002/
ceat.201900398
Postawa, K. (2018). Novel Solutions in Modeling of Anaerobic Digestion Process -
Two-phase AD Models Development and Comparison. Int. J. Chem. Reactor
Eng. 16. doi:10.1515/ijcre-2017-0139
Qin, Y., Li, L., Wu, J., Xiao, B., Hojo, T., Kubota, K., et al. (2019). Co-production of
Biohydrogen and Biomethane from Food Waste and Paper Waste via
Recirculated Two-phase Anaerobic Digestion Process: Bioenergy Yields and
Metabolic Distribution. Bioresour. Technol. 276, 325–334. doi:10.1016/
j.biortech.2019.01.004
Rabii, A., Aldin, S., Dahman, Y., and Elbeshbishy, E. (2019). A Review on
Anaerobic Co-digestion with a Focus on the Microbial Populations and the
Effect of Multi-Stage Digester Configuration. Energies 12, 1106. doi:10.3390/
en12061106
Rachbauer, L., Beyer, R., Bochmann, G., and Fuchs, W. (2017). Characteristics of
Adapted Hydrogenotrophic Community During Biomethanation. Sci. Total
Environ. 595, 912–919. doi:10.1016/j.scitotenv.2017.03.074
Rajendran, K., Mahapatra, D., Venkatraman, A. V., Muthuswamy, S., and
Pugazhendhi, A. (2020). Advancing Anaerobic Digestion Through Two-
Stage Processes: Current Developments and Future Trends. Renew.
Sustainable Energ. Rev. 123, 109746. doi:10.1016/j.rser.2020.109746
Rani, W., Sriwuryandari, L., Priantoro, E. A., and Sintawardani, N. (2020). Shock-
adaptation in a Three-Stage Anaerobic Reactor Treating Tofu Whey
Wastewater. IOP Conf. Ser. Earth Environ. Sci. 483, 012033. doi:10.1088/
1755-1315/483/1/012033
Reddy, M. V., Hayashi, S., Choi, D., Cho, H., and Chang, Y.-C. (2018). Short Chain
and Medium Chain Fatty Acids Production Using Food Waste Under Non-
augmented and Bio-Augmented Conditions. J. Clean. Prod. 176, 645–653.
doi:10.1016/j.jclepro.2017.12.166
Renda, R., Gigli, E., Cappelli, A., Simoni, S., Guerriero, E., and Romagnoli, F.
(2016). Economic Feasibility Study of a Small-Scale Biogas Plant Using a Two-
Stage Process and a Fixed Bio-Film Reactor for a Cost-Efficient Production.
Energ. Proced. 95, 385–392. doi:10.1016/j.egypro.2016.09.042
Robinson, J. A., and Tiedje, J. M. (1982). Kinetics of Hydrogen Consumption by
Rumen Fluid, Anaerobic Digestor Sludge, and Sediment. Appl. Environ.
Microbiol. 44, 1374–1384. doi:10.1128/aem.44.6.1374-1384.1982
Rosgaard, L., Andric, P., Dam-Johansen, K., Pedersen, S., and Meyer, A. S. (2007).
Effects of Substrate Loading on Enzymatic Hydrolysis and Viscosity of
Pretreated Barley Straw. Appl. Biochem. Biotechnol. 143, 27–40. doi:10.1007/
s12010-007-0028-1
Salsali, H., Parker, W., and Sattar, S. (2005). Influence of Staged Operation of
Mesophilic Anaerobic Digestion on Microbial Reduction. Proc. Water Environ.
Fed. 2005, 4571–4586. doi:10.2175/193864705783866676
Sarkar,O.,Butti,S.K.,andVenkataMohan,S.(2017).AcidogenesisDriven
by Hydrogen Partial Pressure Towards Bioethanol Production Through
Fatty Acids Reduction. Energy 118, 425–434. doi:10.1016/
j.energy.2016.12.017
Schievano, A., Tenca, A., Scaglia, B., Merlino, G., Rizzi, A., Daffonchio, D., et al.
(2012). Two-stage vs Single-Stage Thermophilic Anaerobic Digestion:
Comparison of Energy Production and Biodegradation Efficiencies. Environ.
Sci. Technol. 46, 8502–8510. doi:10.1021/es301376n
Sensfuss, F., and Pfluger, B. (2014). Optimized Pathways Towards Ambitious
Climate protection in the European Electricity System. Karlsruhe.
Slezak, R., Grzelak, J., Krzystek, L., and Ledakowicz, S. (2017). The Effect of Initial
Organic Load of the Kitchen Waste on the Production of VFA and H 2 in Dark
Fermentation. Waste Manage. 68, 610–617. doi:10.1016/j.wasman.2017.06.024
Song, H., Choi, O., Pandey, A., Kim, Y. G., Joo, J. S., and Sang, B.-I. (2019).
Simultaneous Production of Methane and Acetate by Thermophilic Mixed
Culture from Carbon Dioxide in Bioelectrochemical System. Bioresour.
Technol. 281, 474–479. doi:10.1016/j.biortech.2019.02.115
Srisowmeya, G., Chakravarthy, M., and Nandhini Devi, G. (2020). Critical
Considerations in Two-Stage Anaerobic Digestion of Food Waste - A
Review. Renew. Sustainable Energ. Rev. 119, 109587. doi:10.1016/
j.rser.2019.109587
Sträuber, H., Schröder, M., and Kleinsteuber, S. (2012). Metabolic and Microbial
Community Dynamics during the Hydrolytic and Acidogenic Fermentation in
a Leach-Bed Process. Energ Sustain. Soc. 2, 13. doi:10.1186/2192-0567-2-13
Theuerl, S., Herrmann, C., Heiermann, M., Grundmann, P., Landwehr, N.,
Kreidenweis, U., et al. (2019). The Future Agricultural Biogas Plant in
Germany: A Vision. Energies 12, 396. doi:10.3390/en12030396
Tijani, H., Yuzir, A., and Abdullah, N. (2018). Producing Desulfurized Biogas
Using Two-Stage Domesticated Shear-Loop Anaerobic Contact Stabilization
System. Waste Manage. 78, 770–780. doi:10.1016/j.wasman.2018.06.045
Urriza-Arsuaga, I., Bedoya, M., and Orellana, G. (2019). Tailored Luminescent
Sensing of NH3 in Biomethane Productions. Sensors Actuators B: Chem. 292,
210–216. doi:10.1016/j.snb.2019.04.109
Van, D. P., Fujiwara, T., Leu Tho, B., Song Toan, P. P., and Hoang Minh, G. (2020).
A Review of Anaerobic Digestion Systems for Biodegradable Waste:
Configurations, Operating Parameters, and Current Trends. Environ. Eng.
Res. 25, 1–17. doi:10.4491/eer.2018.334
Vega De Lille, M., Forstner, J., Groß, F., Benning, R., and Delgado, A. (2016).
Modeling the Two-Stage Anaerobic Digestion of Domestic Wastewater with the
Development of a Monitoring Application. Braz. J. Chem. Eng. 33, 801–815.
doi:10.1590/0104-6632.20160334s20150150
Wan, C., and Li, Y. (2012). Fungal Pretreatment of Lignocellulosic Biomass.
Biotechnol. Adv. 30, 1447–1457. doi:10.1016/j.biotechadv.2012.03.003
Wang, S., Hawkins, G. L., Kiepper, B. H., and Das, K. C. (2018). Treatment of
Slaughterhouse Blood Waste Using Pilot Scale Two-Stage Anaerobic Digesters
for Biogas Production. Renew. Energ. 126, 552–562. doi:10.1016/
j.renene.2018.03.076
Wang, Y., Wang, Z., Zhang, Q., Li, G., and Xia, C. (2020). Comparison of Bio-
Hydrogen and Bio-Methane Production Performance in Continuous Two-
phase Anaerobic Fermentation System Between Co-digestion and Digestate
Recirculation. Bioresour. Technol. 318, 124269. doi:10.1016/
j.biortech.2020.124269
Wirth, R., Kovács, E., Maróti, G., Bagi, Z., Rákhely, G., and Kovács, K. L. (2012).
Characterization of a Biogas-Producing Microbial Community by Short-Read
Next Generation DNA Sequencing. Biotechnol. Biofuels 5, 41. doi:10.1186/
1754-6834-5-41
Wu, L.-J., Kobayashi, T., Li, Y.-Y., and Xu, K.-Q. (2015). Comparison of Single-
Stage and Temperature-Phased Two-Stage Anaerobic Digestion of Oily Food
Waste. Energ. Convers. Manage. 106, 1174–1182. doi:10.1016/
j.enconman.2015.10.059
Wu, Q.-L., Guo, W.-Q., Zheng, H.-S., Luo, H.-C., Feng, X.-C., Yin, R.-L., et al.
(2016). Enhancement of Volatile Fatty Acid Production by Co-fermentation of
Food Waste and Excess Sludge Without pH Control: The Mechanism and
Microbial Community Analyses. Bioresour. Technol. 216, 653–660.
doi:10.1016/j.biortech.2016.06.006
Wu, D., Li, L., Zhao, X., Peng, Y., Yang, P., and Peng, X. (2019). Anaerobic
Digestion: A Review on Process Monitoring. Renew. Sustainable Energ. Rev.
103, 1–12. doi:10.1016/j.rser.2018.12.039
Yan, B. H., Selvam, A., and Wong, J. W. C. (2020). Bio-hydrogen and Methane
Production from Two-phase Anaerobic Digestion of Food Waste Under the
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 71197115
Janesch et al. Phase Separation in Anaerobic Digestion
Scheme of Acidogenic Off-Gas Reuse. Bioresour. Technol. 297, 122400.
doi:10.1016/j.biortech.2019.122400
Yun, Y.-M., Sung, S., Shin, H.-S., Han, J.-I., Kim, H.-W., and Kim, D.-H. (2017).
Producing Desulfurized Biogas Through Removal of Sulfate in the First-Stage of
a Two-Stage Anaerobic Digestion. Biotechnol. Bioeng. 114, 970–979.
doi:10.1002/bit.26233
Zhang, B., Zhang, L.-L., Zhang, S.-C., Shi, H.-Z., and Cai, W.-M. (2005). The
Influence of pH on Hydrolysis and Acidogenesis of Kitchen Wastes in Two-
phase Anaerobic Digestion. Environ. Technol. 26, 329–340. doi:10.1080/
09593332608618563
Zhang, J., Loh, K.-C., Li, W., Lim, J. W., Dai, Y., and Tong, Y. W. (2017). Three-
stage Anaerobic Digester for Food Waste. Appl. Energ. 194, 287–295.
doi:10.1016/j.apenergy.2016.10.116
Zhang, S., Chang, J., Liu, W., Pan, Y., Cui, K., Chen, X., et al. (2018). A Novel
Bioaugmentation Strategy to Accelerate Methanogenesis via Adding Geobacter
Sulfurreducens PCA in Anaerobic Digestion System. Sci. Total Environ. 642,
322–326. doi:10.1016/j.scitotenv.2018.06.043
Zhang, L., Kuroki, A., Loh, K.-C., Seok, J. K., Dai, Y., and Tong, Y. W. (2020).
Highly Efficient Anaerobic Co-digestion of Food Waste and Horticultural
Waste Using a Three-Stage Thermophilic Bioreactor: Performance
Evaluation, Microbial Community Analysis, and Energy Balance
Assessment. Energ. Convers. Manage. 223, 113290. doi:10.1016/
j.enconman.2020.113290
Zhao, Q., and Liu, Y. (2019). Is Anaerobic Digestion a Reliable Barrier for
Deactivation of Pathogens in Biosludge? Sci. Total Environ. 668, 893–902.
doi:10.1016/j.scitotenv.2019.03.063
Zheng, G., Liu, J., Shao, Z., and Chen, T. (2020). Emission Characteristics and
Health Risk Assessment of VOCs from a Food Waste Anaerobic Digestion
Plant: A Case Study of Suzhou, China. Environ. Pollut. 257, 113546.
doi:10.1016/j.envpol.2019.113546
Zhou, L., and Boyd, C. E. (2016). Comparison of Nessler, Phenate, Salicylate and Ion
Selective Electrode Procedures for Determination of Total Ammonia Nitrogen in
Aquaculture. Aquaculture 450, 187–193. doi:10.1016/j.aquaculture.2015.07.022
Zosel, J., Schelter, M., Oelßner, W., and Zimmermann, P. (2011). Final Report of
the Joint Project: Process Optimization of Biogas Production by Means of
Innovative Measuring and Control Technology for the Detection of Dissolved
Hydrogen as a Microbial Key Intermediat (BINERWA). Available at: https://
www.fnr-server.de/ftp/pdf/berichte/22009407.pdf (Accessed April 8, 2021).
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Publisher’s Note: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations, or those of
the publisher, the editors and the reviewers. Any product that may be evaluated in
this article, or claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Copyright © 2021 Janesch, Pereira, Neubauer and Junne. This is an open-access
article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with
these terms.
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 71197116
Janesch et al. Phase Separation in Anaerobic Digestion
