Bioresource Technology 234 (2017) 310–319

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Optimization of hydrogen dispersion in thermophilic up-flow reactors

for ex situ biogas upgrading
Ilaria Bassani a, Panagiotis G. Kougias a,⇑, Laura Treu a,b, Hugo Porté a, Stefano Campanaro c,
Irini Angelidaki a
a
Department of Environmental Engineering, Technical University of Denmark, Kgs. Lyngby DK-2800, Denmark
b
Department of Agronomy, Food, Natural Resources, Animals and Environment (DAFNAE), University of Padua, viale dell’Università 16, 35020 Legnaro (PD), Italy
c
Department of Biology, University of Padua, Via U. Bassi 58/b, 35121 Padova, Italy

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Ex situ biogas upgrading to 96% CH4 is

achieved in thermophilic up-flow
reactors.

The totality of H2 and CO2 is converted
to CH4 reaching 0.25 LCH4/LH2 CH4
yield.

Higher gas recirculation and diffusers’
pore size improved gas-liquid contact.

H2 selectively enhances
hydrogenotrophic methanogens and
syntrophic bacteria.

a r t i c l e i n f o a b s t r a c t

Article history: This study evaluates the efficiency of four novel up-flow reactors for ex situ biogas upgrading converting
Received 11 January 2017 externally provided CO2 and H2 to CH4, via hydrogenotrophic methanogenesis. The gases were injected
Received in revised form 6 March 2017 through stainless steel diffusers combined with alumina ceramic sponge or through alumina ceramic
Accepted 8 March 2017
membranes. Pore size, input gas loading and gas recirculation flow rate were modulated to optimize
Available online 12 March 2017
gas-liquid mass transfer, and thus methanation efficiency. Results showed that larger pore size diffusion
devices achieved the best kinetics and output-gas quality converting all the injected H2 and CO2, up to
Keywords:
3.6 L/LREACTORd H2 loading rate. Specifically, reactors’ CH4 content increased from 23 to 96% and the
Ex situ biogas upgrading
Gas-liquid mass transfer rate
CH4 yield reached 0.25 LCH4/LH2. High throughput 16S rRNA gene sequencing revealed predominance of
Ceramic sponge bacteria belonging to Anaerobaculum genus and to uncultured order MBA08. Additionally, the massive
Ceramic membrane increase of hydrogenotrophic methanogens, such as Methanothermobacter thermautotrophicus, and syn-
16S rRNA gene sequencing trophic bacteria demonstrates the selection-effect of H2 on community composition.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction

The reduction of greenhouse gases emissions, such as CO2, is of

great importance for a sustainable industrial development and to
move towards cleaner energy production and utilization. Conse-
⇑ Corresponding author at: Department of Environmental Engineering, Technical
quently, several technologies have been developed to remove, con-
University of Denmark, Bld 113, 2800 Lyngby, Denmark.
vert or store CO2 (Mikkelsen et al., 2010). In this context, biological
E-mail address: panak@env.dtu.dk (P.G. Kougias).

http://dx.doi.org/10.1016/j.biortech.2017.03.055
0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
 I. Bassani et al. / Bioresource Technology 234 (2017) 310–319
carbon fixation emerged as a very promising alternative to existing
methods, offering the advantage of working in mild conditions of
temperature and pressure and without the use of chemical sub-
stances (Alitalo et al., 2015; Lee et al., 2012). Biological carbon fix-
ation besides capturing CO2, can also convert it into different
products, such as CH4.
Anaerobic digestion (AD) of biomass is an effective method for
bioenergy production. The resulting biogas consists of 50–70%
CH4 and 30–50% CO2 and it is commonly burned in a combined
heat and power unit (CHP) to produce electricity and heat. Nowa-
days, there is an increasing interest to expand the utilization
potentials of biogas by a process called biogas upgrading. Through
this process, the CH4 content in the biogas is increased by remov-
ing or transforming the contained CO2 (and Supplementary due to
removal of other impurities, such as water, hydrogen sulfide and
siloxanes) (Kougias et al., 2017). Thereafter, the upgraded biogas
can be injected into the natural gas grid or can be used as fuel
for vehicles (Deng and Hägg, 2010).
Moreover, H2 production from renewable sources, such as wind
and solar power, is gaining greater importance in the context of
alternative energy development. In fact, in northern EU countries,
such as Denmark, >26% of the electricity produced from wind mills
is a temporary surplus and although some of it is exported to
neighbouring countries, the potential of wind mills is not fully uti-
lized (Carton and Olabi, 2010; Sharman, 2005; Sovacool, 2013). H2
generation from water electrolysis using off-peak electricity sur-
plus from wind and/or solar power can solve the limitations of
variable wind power production and electricity storage (Levene
et al., 2007). Nevertheless, because of its low density, H2 presents
limitations linked to its transportation and management
(Granovskii et al., 2006). Therefore, the opportunity of coupling
the H2 with the CO2 present in the biogas and their subsequent
conversion to CH4 is a promising effective and cheaper alternative
technology to upgrade the biogas to higher CH4 content. In fact,
although information about biological upgrading energy require-
ments is not currently available, previous studies affirm that, dur-
ing this process, the electricity consumed for conversion and
storage, with the exception of electrolysis process (4–5 kW h/
m3 H2), is considered to be negligible, especially when compared
with surplus electricity (Götz et al., 2016; Jürgensen et al., 2014).
Moreover, the heat generate from biomethanation can be used to
heat a biogas digester (e. g. 420 kW for a 5 MW plant) (Götz
et al., 2016).
Simplicity and robustness of biological biogas upgrading tech-
nology is mainly attributable to the effectiveness of the heteroge-
neous microbial consortium involved in AD process. In fact,
biomethanation efficiency is strongly determined by syntrophic
relations between bacteria and/or archaea co-existing in anaerobic
digesters’ microbial communities (Kougias et al., 2016b). During ex
situ biogas upgrading process, biogas and H2 are injected into an
anaerobic reactor, where the CO2 contained in the biogas is cou-
pled with H2, to be converted to CH4, by a consortium of mixed
microbial species, via hydrogenotrophic methanogenesis (Kougias
et al., 2017). Previous studies on H2 assisted biogas upgrading
showed that H2 addition affected microbial community composi-
tion enhancing hydrogenotrophic methanogenesis and bacterial
syntrophic interactions with methanogenic archaea (Bassani
et al., 2015). Nevertheless, due to the fact that the microbial pro-
cesses occur exclusively in aqueous environment, the injected
gases must be dissolved in the reactor liquid phase in order to be
utilized by microorganisms. However, H2 gas-liquid mass transfer
rate is known to be very low, strongly limiting H2 availability for
methanogens (Bassani et al., 2015, 2016; Luo et al., 2012; Luo
and Angelidaki, 2012). H2 gas transfer coefficient ðkL a) is mainly
determined and can be modulated by changing parameters such
311
as mixing speed (Kramer and Bailey, 1991; Luo and Angelidaki,
2012), gas recirculation (Guiot et al., 2011) and H2 diffusion device
(Díaz et al., 2015; Luo and Angelidaki, 2013). Previous studies on
H2 assisted biogas upgrading reported kL a ranging from 1 to
10⁄103 day1, depending on applied diffusion device and selected
operating conditions (i.e. mixing speed, gas recirculation flow rate)
(Díaz et al., 2015; Luo et al., 2012; Luo and Angelidaki, 2013).
The current study aimed at maximizing H2 uptake by methano-
gens, by increasing the H2 gas-liquid mass transfer rate, so as to
improve the efficiency of ex situ biogas upgrading. This was
achieved by designing and operating four novel up-flow configura-
tions, in which the gas was introduced to the reactors through dif-
ferent gas distribution devices, i.e. stainless steel diffusers and
ceramic sponge or ceramic membrane. Moreover, different pore
sizes of the distribution devices, input gas flow rate ðQ IN Þ and gas
recirculation rate ðQ RC Þ were tested to improve H2 and CO2 conver-
sion to CH4. Finally, the microbial profile at the beginning and at
the end of the process was analyzed via high throughput 16S rRNA
amplicon sequencing, to elucidate the microbial community
change driven by the applied operating conditions.
2. Materials and methods
2.1. Inoculum characteristics and feedstock preparation
The digestate used as nutrient feedstock was obtained from
Snertinge biogas plant (Denmark). After arrival to the laboratory,
the digestate was filtered through a 2 mm net to remove large par-
ticles and stored at 55 °C at anaerobic conditions in 5 L bottles. The
bottles were kept in the incubator for one month in order to be
completely degassed. The chemical composition and trace ele-
ments content of the digestate are shown in Tables S1 and S2.
Before usage, the digestate was diluted with water and 1 M HCl
to reduce the pH from average 8.48 ± 0.11 to 6.91 ± 0.19 and main-
tain reactors’ pH values within the range for methanogenesis
(Weiland, 2010). The ratio of digestate, 1 M HCl and water was
1:0.7:0.3. The reactors were inoculated with 600 mL of undiluted
degassed digestate and 250 mL of active enriched hydrogeno-
trophic inoculum obtained from an upgrading biogas reactor
(Bassani et al., 2015). The purpose of the enriched culture was to
provide active hydrogenotrophic methanogens and thus shorten
the overall adaptation period. 50 mL of fresh diluted digestate were
daily replaced from the bottom of the reactor.
2.2. Reactor’s setup and operation
A detailed reactors’ description is provided in Supplementary
Information (SI) (Fig. S1). Each reactor setup was composed of an
up-flow reactor with 850 mL working volume. Reactors were
maintained at thermophilic conditions (55 ± 1 °C) by circulating
hot water through a water jacket around the reactor’s glass walls.
Reactors’ mixing was ensured by continuous gas recirculation. A
gas mixture composed of H2, CH4 and CO2 with ratio 62:23:15
(AGA A/S, Denmark) was continuously provided to the reactors.
In reactor 1 (R1) and reactor 2 (R2) the gas mixture was injected
through 3 stainless steel diffusers with pore size 0.5 lm and
2 lm, respectively. Moreover, R1 and R2 were filled with inert alu-
mina ceramic sponge, to reduce H2 bubbles’ size and extend gas
retention time in the liquid increasing the contact with reactors’
liquid phase. Conversely, in reactor 3 (R3) and reactor 4 (R4), the
gas mixture was injected through an Al2O3 ceramic membrane
with pore size 0.4 lm and 1.2 lm, respectively. The experiment
was divided into 6 periods, where, either the gas retention time
(GRT) was reduced by increasing input gas flow rate ðQ IN Þ ([L/LRd]
expressed as per litter reactor (LR)) or gas recirculation rate ðQ RC Þ
312 I. Bassani et al. / Bioresource Technology 234 (2017) 310–319

[L/LRh] was increased, in order to optimize CO2 and H2 conversion CH4 yield per H2 loaded [LCH4/LH2] was determined by Eq. (2):
efficiencies (gCO2 and gH2 ) (Table 1). Q RC was initially set to, on
P CH4
average, 42 times Q IN and then increased to 82 times as previously Y CH4 ¼ ð2Þ
H2used;T
described (Kougias et al., 2017).
where H2used;T represented the theoretical H2 utilization rate [LH2/LRd]
2.3. Calculations (i.e. considering that the 2% extra H2 is remained unutilized according
to the stoichiometry of hydrogenotrophic methanogenesis) and is
The residual biogas produced mainly from content of volatile calculated as described by Eq. (3):
fatty acids (VFA) in the digestate used (essentially acetate and pro-
pionate) was calculated according to the following conversion H2used;T ¼ Q H2 ;IN  Q H2 ;OUT;T ð3Þ
reactions and subtracted from output CH4 and CO2 rates ðQ CH4 ;OUT
and Q CO2 ;OUT expressed as [L/LRd]) where Q H2 ;IN represents the H2 loading rate [LH2/LRd] and Q H2 ;OUT;T
the theoretical H2 output rate [LH2/LRd].
Acetate CH3 COOH ! CH4 þ CO2 H2 utilization efficiency [%] was calculated as described by Eq. (4):
H2used
Propionate CH3 CH2 COOH þ 0:5 H2 O ! 1:75 CH4 þ 1:25 CO2 gH2 ¼  100 ð4Þ
H2used;T
Similarly, as it will be further discussed, extra H2 was found to
be coupled with a portion of extra CO2 produced from VFA degra- where H2used represented the actual H2 utilization rate [LH2/LRd], cal-
dationðQ CO2 ;VFA , [LCO2/LRd]) generating additional CH4. Therefore, culated as follows (Eq. (5)):
H2 utilized for this reaction has been added to the H2 output rate
H2used ¼ Q H2 ;IN þ Q H2 ;VFA  Q H2 ;OUT ð5Þ
(Q H2 ;OUT , [LH2/LRd]) to provide unambiguous results when defining
ex situ biogas upgrading mass balance. Indeed, a source of extra H2 where Q H2 ;VFA is the H2 utilized in VFA reactions [LH2/LRd].
was represented by the input gas mixture. This mixture was com- CO2 conversion efficiency [%] was calculated based on the CO2
posed of CO2 and H2 according to the stoichiometry of hydrogeno- fraction contained in the gas phase, as described by Eq. (6):
trophic methanogenesis reaction (1:4, equivalent to 15 and 60% of
the total input gas volume), 23% CH4, in order to simulate typical CO2used
gCO2 ¼  100 ð6Þ
biogas composition, and 2% extra H2 as ground gas, which was Q CO2 ;IN
expected to remain unutilized.
where CO2used is the CO2 utilization rate [LCO2/LRd] and was calcu-
Based on these calculations, CH4 production rate derived from
lated as follows (Eq. (7)):
ex situ biogas upgrading was determined as follows (Eq. (1)):
PCH4 ¼ Q CH4 ;OUT  Q CH4 ;IN  Q CH4 ;VFA ð1Þ CO2used ¼ Q CO2 ;IN  Q CO2 ;VFA  Q CO2 ;OUT ð7Þ

where Q CH4 ;IN represents the CH4 loading rate [LCH4/LRd] and Q CH4 ;VFA where Q CO2 ;IN represents the CO2 loading rate [LCO2/LRd].
is the CH4 production rate derived from VFA degradation Finally H2 gas transfer coefficient [day1] was calculated as
[LCH4/LRd]. described by Eq. (8):

Table 1
Reactors’ performances under steady state conditions.

Period GRT [h] Q RC [L/LR.h] Reactor Q IN [L/LR.d] Q OUT;T [L/LR.d] Q OUT [L/LR.d] Output gas composition P CH4 [LCH4/LRd] Y CH4 [LCH4/LH2] gH2 [%] gCO2 [%]
CH4 [%] CO2 [%] H2 [%]
I 15 2.88 R1 1.60 ± 0.01 0.64 ± 0.01 0.543 ± 0.06 90.8 ± 1.4 5.1 ± 0.3 4.0 ± 1.7 0.08 ± 0.05 0.09 ± 0.05 100.0 ± 0.1 89.6 ± 1.4
R2 1.59 ± 0.01 0.64 ± 0.01 0.53 ± 0.05 93.1 ± 2.5 4.4 ± 0.6 2.5 ± 1.9 0.09 ± 0.03 0.09 ± 0.03 100.0 ± 0.1 91.1 ± 1.6
R3 1.60 ± 0.01 0.64 ± 0.01 0.54 ± 0.05 89.5 ± 0.6 6.4 ± 0.4 4.1 ± 0.7 0.09 ± 0.05 0.09 ± 0.05 100.0 ± 0.1 86.2 ± 1.4
R4 1.50 ± 0.01 0.60 ± 0.01 0.59 ± 0.03 92.0 ± 1.3 5.9 ± 1.9 2.1 ± 1.0 0.17 ± 0.03 0.19 ± 0.04 100.0 ± 0.1 85.5 ± 4.5
II 15 5.75 R1 1.58 ± 0.03 0.63 ± 0.01 0.55 ± 0.03 93.7 ± 1.2 4.7 ± 0.6 1.6 ± 0.7 0.11 ± 0.02 0.11 ± 0.02 100.0 ± 0.1 90.7 ± 2.1
R2 1.57 ± 0.02 0.63 ± 0.01 0.54 ± 0.06 94.9 ± 0.8 4.0 ± 0.2 1.2 ± 0.6 0.10 ± 0.05 0.11 ± 0.06 100.0 ± 0.1 92.6 ± 0.5
R3 1.58 ± 0.05 0.63 ± 0.02 0.55 ± 0.05 95.1 ± 0.1 3.2 ± 0.2 1.7 ± 0.2 0.12 ± 0.06 0.13 ± 0.07 100.0 ± 0.1 93.3 ± 0.9
R4 1.42 ± 0.04 0.60 ± 0.01 0.56 ± 0.01 96.3 ± 0.2 2.8 ± 0.4 0.8 ± 0.3 0.21 ± 0.02 0.24 ± 0.03 100.0 ± 0.1 92.9 ± 1.5
III 7 5.75 R1 3.42 ± 0.02 1.37 ± 0.01 1.24 ± 0.03 93.1 ± 1.2 3.0 ± 0.8 3.9 ± 0.9 0.33 ± 0.03 0.16 ± 0.02 100.0 ± 0.1 94.5 ± 1.5
R2 3.43 ± 0.02 1.37 ± 0.01 1.28 ± 0.07 93.9 ± 0.6 2.6 ± 0.5 3.5 ± 0.6 0.38 ± 0.07 0.18 ± 0.04 100.0 ± 0.1 95.4 ± 0.9
R3 3.50 ± 0.03 1.40 ± 0.01 1.28 ± 0.05 93.3 ± 0.7 2.3 ± 0.4 4.5 ± 0.9 0.35 ± 0.05 0.17 ± 0.02 99.9 ± 0.2 96.6 ± 1.9
R4 3.16 ± 0.04 1.26 ± 0.02 1.18 ± 0.05 96.0 ± 0.6 1.4 ± 0.5 2.6 ± 0.4 0.37 ± 0.04 0.19 ± 0.02 100.0 ± 0.1 97.9 ± 0.8
IV 7 10.04 R1 3.43 ± 0.01 1.37 ± 0.01 1.23 ± 0.05 94.8 ± 0.1 2.4 ± 0.4 2.8 ± 0.3 0.35 ± 0.04 0.17 ± 0.02 100.0 ± 0.1 95.5 ± 1.2
R2 3.32 ± 0.02 1.34 ± 0.01 1.25 ± 0.06 94.8 ± 0.9 2.6 ± 0.3 2.6 ± 0.7 0.39 ± 0.07 0.19 ± 0.03 100.0 ± 0.1 95.3 ± 0.9
R3 3.46 ± 0.02 1.39 ± 0.01 1.35 ± 0.08 90.4 ± 1.5 2.7 ± 0.2 6.9 ± 1.4 0.40 ± 0.07 0.19 ± 0.03 98.7 ± 0.7 95.6 ± 0.8
R4 3.17 ± 0.04 1.27 ± 0.02 1187 ± 0.06 95.9 ± 0.5 2.0 ± 0.3 2.2 ± 0.3 0.37 ± 0.06 0.20 ± 0.03 100.0 ± 0.1 97.4 ± 1.4
V 4 10.04 R1 5.93 ± 0.01 2.37 ± 0.01 2.36 ± 0.04 90.4 ± 2.3 2.7 ± 0.4 7.0 ± 2.1 0.72 ± 0.05 0.20 ± 0.01 98.6 ± 1.2 97.7 ± 1.1
R2 5.87 ± 0.01 2.35 ± 0.01 2.28 ± 0.08 91.6 ± 2.4 2.3 ± 0.4 6.1 ± 2.0 0.69 ± 0.08 0.20 ± 0.02 99.0 ± 0.9 98.8 ± 1.0
R3 6.02 ± 0.01 2.41 ± 0.01 2.43 ± 0.08 87.6 ± 0.7 2.7 ± 0.4 9.6 ± 0.6 0.70 ± 0.07 0.19 ± 0.02 96.8 ± 0.5 97.8 ± 1.2
R4 5.54 ± 0.01 2.26 ± 0.01 2.22 ± 0.09 91.6 ± 0.5 2.0 ± 0.3 6.4 ± 0.6 0.73 ± 0.08 0.22 ± 0.03 99.1 ± 0.5 99.3 ± 0.8
VI 4 20.14 R1 5.93 ± 0.03 2.37 ± 0.01 2.37 ± 0.12 92.7 ± 1.9 2.4 ± 0.7 4.9 ± 1.5 0.79 ± 0.11 0.22 ± 0.03 99.6 ± 0.7 97.7 ± 1.7
R2 5.86 ± 0.02 2.34 ± 0.01 2.32 ± 0.16 96.0 ± 0.5 1.4 ± 0.3 2.6 ± 0.5 0.82 ± 0.15 0.23 ± 0.04 100.0 ± 0.1 100.0 ± 1.0
R3 6.04 ± 0.06 2.42 ± 0.02 2.30 ± 0.17 92.5 ± 0.6 2.0 ± 0.5 5.6 ± 0.6 0.70 ± 0.15 0.19 ± 0.04 99.7 ± 0.3 99.3 ± 1.3
R4 5.59 ± 0.01 2.22 ± 0.01 2.16 ± 0.03 94.1 ± 1.2 1.7 ± 0.3 4.2 ± 0.9 0.71 ± 0.02 0.21 ± 0.01 100.0 ± 0.1 99.9 ± 0.6

Q OUT represents the total gas output rate and Q OUT;T the total theoretical gas output rate.
 I. Bassani et al. / Bioresource Technology 234 (2017) 310–319
rt
kL a ¼
22:4ðH2gTh  H2l Þ
where rt is the H2 gas-liquid mass transfer rate, which can be iden-
tified with the H2 utilization rate ðH2used Þ, described by Eq. (5), 22.4
[L/mol] is the gas volume to mole ratio (1 mol gas corresponds to
22.4 L at STP), and H2gTh [M] and H2l [M] are the H2 concentrations
in the gas and in the liquid phase, respectively.
2.4. Analytical methods
The output biogas was recorded in daily basis by an automated
gas meter with a 100 mL reversible cycle and registration
(Angelidaki et al., 1992). Total solids (TS), volatile solids (VS),
Ammonium nitrogen (NH4-N) and Total Kjeldahl Nitrogen (TKN)
were measured according to the Standard Methods for Examina-
tion of Water and Wastewater (APHA, 2005). Liquid samples from
the reactors were collected for pH and VFA analysis every second
day. The pH was measured immediately after the collection to
avoid the CO2 removal from the liquid phase, by digital PHM210
pH meter connected to the Gel pH electrode (pHC3105-8;
Radiometer analytical). VFA samples were determined as previ-
ously described by (Kougias et al., 2015). The biogas composition
was measured every second day by GC-TCD (Mikrolab, Aarhus A/
S, Denmark) as previously described (Kougias et al., 2015). Dis-
solved H2 was measured from liquid samples as described in SI.
Trace elements analysis was performed on initial digestate and
reactors’ liquid samples during steady state operation of period I,
as described in SI.
2.5. Microbial community composition
Samples were obtained at the beginning of the experiment and
during reactors’ steady state operation of the last period to ensure
representative process conditions and microbial community stabil-
ity. Genomic DNA was extracted from reactors’ liquid samples, in
triplicates, with PowerSoilÒ DNA Isolation Kit (MO BIO Laborato-
ries, Carlsbad, CA). Minor modifications were introduced to the pro-
cedure in order to improve the quality of the genomic DNA
recovered as described by Bassani et al. (2015). Quality and quan-
tity of the DNA extracted were determined using NanoDrop (Ther-
moFisher Scientific, Waltham, MA) and QubitTM fluorimeter (Life
Technologies, Carlsbad, CA). 16S rRNA gene V4 hypervariable region
was amplified with universal primers (Caporaso et al., 2012) and
sequenced using Illumina MiSeq sequencing technology. The
obtained reads were submitted to the NCBI sequence read archive
database (SRA) with accession number SRP091653. Detailed sample
IDs are listed in Table S3. Analysis of 16S rRNA gene sequences has
been performed as described by Kougias et al. (2017). A further
alignment was performed using BLASTN against NCBI 16S rRNA
database as described by Bassani et al. (2015). A predictive metage-
nomic approach investigating KEGG and COG functional categories
was applied to the identified genera, as described in SI. Heat maps
showing relative abundance and fold change of most relevant oper-
ational taxonomic units (OTUs) were drawn using Multiexperiment
Viewer software (MeV) (Howe et al., 2010).
2.6. Statistical analysis
Descriptive statistics were carried out for all data and mean val-
ues and standard deviations were calculated. The comparison of
quantitative variables (e.g. CH4 content in the output gas) between
the different tested configurations to determine significant differ-
ences (p < 0.05) was achieved by conducting one-way analysis of
variance (ANOVA) using Graphpad Prism 5 program (Graphpad
Software, Inc., San Diego, CA). Moreover, regarding the microbial
313
community, statistical analysis to identify significantly abundance
ð8Þ
differences for each microbe among the samples, was carried out
as previously described by Tsapekos et al. (2016).
3. Results and discussion
At the beginning of Period I, the pH of the reactors was
increased due to the removal of the dissolved CO2 in the liquid
media. The addition of HCl to the daily feeding, in order to decrease
the pH values and to maintain them within the optimum range for
methanogenesis (Weiland, 2010), became effective allowing the
reduction of pH levels during the following experimental periods
(Table 2, and Fig. S2a).
Moreover, although the digestate used to inoculate the reactors
had been degassed for one month to avoid any residual biogas pro-
duction, during the whole experimental period; additional biogas
was produced from degradation of residual organic matter in the
digestate, mainly acetate and propionate. Thus, a remarkable VFA
reduction was recorded until Period IV, where VFA levels decreased
to 0.3 g/L from 1.5 g/L (Period I) (Table 2 and Fig. S2b and S3).
Moreover, it was assumed that extra H2 would be utilized to convert
the CO2 from the biogas produced from acetate and propionate
degradation. This assumption was documented by the H2 utilization
efficiency ðgH2 Þ which exceeded 100% during the most of the exper-
imental period. Considering both additional CH4 production and CO2
conversion (i.e. from biogas production and upgrade due to VFA
degradation), it was calculated that, on average, 0.04 LCH4/LRd were
produced (Table S4).
Finally, the increase in total micronutrients’ content indicates
that the daily provided digestate represented a complete source
of nutrients and its provision rate was in surplus and could ensure
microbial growth and metabolism (Table S2). Therefore, for future
applications, lower digestate flow rate as nutrient source could be
provided.
3.1. Overview of biogas upgrading process performances
The beginning of the experiment mainly represented a start-up
period, during which microbial community adapted to the new
operation conditions. Nevertheless, microorganisms rapidly
started to convert H2 and CO2 to CH4 and thus, in Period I, the
CH4 content increased from 23%, in the input gas, to >91% in the
output flow, then stabilizing around 96% in period VI. Approxi-
mately 2–4% H2 remained unutilized and was recorded in the out-
put gas (Table 1 and Fig. 1). Moreover, despite the fact that the
experiment started with low CO2 conversion efficiency (gCO2 Þ, it
rapidly increased within the first 10 days stabilizing to 88% at
the end of Period I. Interestingly, gCO2 continued to increase along
all periods, in all the configurations, achieving >99% in Period VI
(Table 1 and Fig. 2a).
As expected, in Periods I and II, in most of the reactor configu-
rations, low CH4 productions rates (P CH4 ) and CH4 yields ðY CH4 )
were observed (Table 1 and Fig. 2c and d). Their concomitance with
high gH2 suggested that the input H2, although efficiently utilized,
was not totally exploited for CH4 production, but rather for other
microbial reactions, such cell maintenance and growth purposes
(Kleerebezem and Stams, 2000). This argument, together with
the reduction of micronutrients known to be essential for cells
growth, supports the hypothesis of an initial acclimation of the
microbial community (Kayhanian and Rich, 1995) (Table S2). After
such adaptation phase, Y CH4 gradually increased reaching values
closer to the stoichiometric maximum of 0.25 LCH4/LH2 (Table 1
and Fig. 2d). This trend is more visible in Fig. S4, where the stoi-
chiometric difference between H2 utilization and CH4 production
rates is plotted against time. Values recorded remained higher than
314 I. Bassani et al. / Bioresource Technology 234 (2017) 310–319

Table 2
Reactors’ pH, VFA, dissolved H2 concentration (H2l) and H2 transfer coefficient (kLa) under steady state conditions.

Period Reactor pH TVFA [g/L] Acetate [g/L] Propionate [g/L] H2l [M] kL a [day1]
6
I R1 8.77 ± 0.05 1.77 ± 0.13 1.11 ± 0.10 0.22 ± 0.02 (0.05 ± 0.01)*10 5.76*103
R2 8.75 ± 0.03 1.52 ± 0.11 1.00 ± 0.06 0.15 ± 0.02 (0.06 ± 0.01)*106 10.08*103
R3 8.78 ± 0.07 1.36 ± 0.08 0.88 ± 0.06 0.12 ± 0.01 (0.19 ± 0.07)*106 3.27*103
R4 8.82 ± 0.09 1.62 ± 0.08 1.07 ± 0.08 0.16 ± 0.02 (0.46 ± 0.19)*106 18.34*103
II R1 8.64 ± 0.07 1.46 ± 0.13 0.96 ± 0.08 0.20 ± 0.01 (0.08 ± 0.02)*106 17.03*103
R2 8.66 ± 0.07 1.22 ± 0.13 0.79 ± 0.14 0.14 ± 0.02 (0.31 ± 0.09)*106 18.64*103
R3 8.81 ± 0.04 1.36 ± 0.22 0.90 ± 0.19 0.15 ± 0.03 (0.35 ± 0.12)*106 5.74*103
R4 8.94 ± 0.05 1.65 ± 0.21 1.15 ± 0.17 0.18 ± 0.02 (0.47 ± 0.03)*106 8.62*103
II I R1 8.44 ± 0.06 0.38 ± 0.08 0.14 ± 0.08 0.22 ± 0.01 (0.29 ± 0.10)*106 5.58*103
R2 8.44 ± 0.07 0.45 ± 0.15 0.24 ± 0.13 0.19 ± 0.02 (0.27 ± 0.02)*106 10.07*103
R3 8.57 ± 0.15 1.11 ± 0.24 0.76 ± 0.15 0.19 ± 0.01 (0.18 ± 0.13)*106 2.52*103
R4 8.55 ± 0.10 1.56 ± 0.18 1.06 ± 0.16 0.22 ± 0.01 (0.34 ± 0.13)*106 8.51*103
IV R1 8.33 ± 0.10 0.27 ± 0.04 0.06 ± 0.03 0.17 ± 0.02 (0.36 ± 0.02)*106 2.85*103
R2 8.27 ± 0.11 0.29 ± 0.06 0.12 ± 0.04 0.14 ± 0.02 (0.33 ± 0.07)*106 5.48*103
R3 8.29 ± 0.13 0.24 ± 0.08 0.11 ± 0.04 0.04 ± 0.02 (0.30 ± 0.04)*106 3.27*103
R4 8.30 ± 0.13 0.63 ± 0.12 0.40 ± 0.09 0.13 ± 0.02 (0.27 ± 0.12)*106 13.08*103
V R1 8.20 ± 0.11 0.36 ± 0.07 0.09 ± 0.03 0.14 ± 0.02 (0.24 ± 0.10)*106 7.72*103
R2 8.18 ± 0.06 0.18 ± 0.06 0.09 ± 0.02 0.05 ± 0.02 (0.19 ± 0.05)*106 7.93*103
R3 8.15 ± 0.05 0.34 ± 0.05 0.23 ± 0.02 0.05 ± 0.01 (0.70 ± 0.18)*106 3.79*103
R4 8.12 ± 0.10 0.44 ± 0.06 0.29 ± 0.05 0.07 ± 0.01 (0.16 ± 0.05)*106 5.99*103
VI R1 8.11 ± 0.15 0.17 ± 0.04 0.08 ± 0.02 0.06 ± 0.02 (0.52 ± 0.10)*106 9.32*103
R2 8.03 ± 0.12 0.11 ± 0.07 0.07 ± 0.05 0.02 ± 0.03 (0.23 ± 0.06)*106 15.21*103
R3 8.13 ± 0.17 0.31 ± 0.04 0.22 ± 0.05 0.05 ± 0.01 (0.29 ± 0.03)*106 7.50*103
R4 8.09 ± 0.14 0.18 ± 0.07 0.13 ± 0.05 0.03 ± 0.01 (0.22 ± 0.01)*106 7.47*103

zero during most of the experimental time indicating that the
input H2 was partially utilized for microbial maintenance.
In summary, the reported results indicate that by applying the
proposed reactor configurations efficient biogas upgrading was
achieved in terms of kinetics of CH4 production, conversion effi-
ciency and consequent output gas quality, generating biogas with
96% CH4 content, which could be employed as transportation fuel
(Deng and Hägg, 2010). The analyzed biochemical parameters
demonstrate that the biological conversion of CO2 and H2 to CH4
was efficiently performed as during the whole experimental per-
iod, no VFA accumulation was recorded (Table 2 and Fig. S2b). This
practically means that the injected CO2 and H2 were exclusively
utilized by hydrogenotrophic methanogens to generate methane
and not by homoacetogenic bacteria that could produce acetate
(Table 2 and Fig. S3a).
A further test was conducted in order to clarify the direct
impact of H2 mass transfer rate on upgrading performances. There-
fore, based on dissolved and gaseous H2 concentrations, H2 transfer
coefficient ðkL a) has been calculated, according to Eq. (8), and the
results are shown in Table 2. As expected, dissolved H2 concentra-
tion (H2l Þ was markedly lower compared to H2 concentration in the
gas phase ðH2gTh Þ (i.e. on average 55 times lower). Nevertheless,
compared to previous studies (Díaz et al., 2015; Luo et al., 2012;
Luo and Angelidaki, 2013), remarkable kL a values were achieved
ranging from 2.5⁄103 day1 and 18.6⁄103 day1. The outcomes
confirm the importance of this parameter to ensure H2 availability
for microorganisms and enlighten the effectiveness of applied
reactor configuration and process parameters for ex situ biogas
upgrading process.
3.2. Effect of diffusion device and pore size on upgrading performances
From the analysis of operational parameters, it was demon-
strated that the pore size of the diffusion devices influenced the
upgrading process, although its contribution was not statistically
significant in all the applied conditions. In order to avoid any con-
fusion in the interpretation of the results and the implementation
of the proposed concepts under full-scale conditions, it should be
noted that the characterization of pore sizes as ‘‘large” or ‘‘small”
refers to a diameter range between 0.4 lm and 2 lm. Thus, it
was found that larger pore size resulted in higher output gas qual-
ity compared to the smaller pore diameter. On average, reactors in
which the gas mixture was distributed through larger pore size dif-
fusers (R2 and R4) reached CH4 content 94.2 ± 1.8%, compared to
92.0 ± 2.2% achieved by smaller pore size diffusers (R1 and R3).
Accordingly, at the end of the experiment (Period VI), CH4 con-
tent stabilized to 94 and 96% in R4 and R2, respectively, compared
to >92% of R1 and R3 (Table 1 and Fig. 1). Similarly, despite all con-
figurations performed at very high gCO2 during the whole experi-
ment (on average 95%), reactors with larger pore size diffusers
(R2 and R4) showed the best performances (on average 96%). Like-
wise, H2 was efficiently consumed during the whole period, with
R4 showing the highest gH2 (>99%) (Table 1 and Fig. 2b). This out-
come is of interest because smaller pore size devices are expected
to deliver smaller bubbles, increasing gas-liquid contact and, there-
fore, availability of substrates for microorganisms. A possible
explanation for the obtained results could be attributed to the
higher mixing of reactor’s liquid phase provided by larger pore size
devices compared with smaller ones (Merchuk et al., 1998). In fact,
proper mixing speed is known to increase gases kL a improving the
contact between gases and reactor’s liquid media (Kramer and
Bailey, 1991; Luo and Angelidaki, 2012). Specifically, when the
bubbles, formed at the pores of the diffusers, circulate in a vigor-
ously agitated gas-liquid dispersion, they generate a turbulent
flow, where shear forces acting on the bubbles break them up into
smaller ones (Villadsen et al., 2011). Therefore, in this experiment,
larger pore size devices presented a positive impact on the gas-
liquid transfer as the intense mixing resulted in a more efficient
gas bubble break, improving the gas-liquid contact. This result
was confirmed by measuring the dissolved H2 and consequently
calculating the kL a coefficient. The outcome of this test proved that
larger pore size diffusion devices, in particular R2, were able to uti-
lize H2 providing the highest kL a (on average 11.2⁄103 day1)
(Table 2). Coherently with parameters previously investigated the
 I. Bassani et al. / Bioresource Technology 234 (2017) 310–319 315

Fig. 1. Biogas composition of (a) R1, (b) R2, (c) R3 and (d) R4. Continuous lines present gases content at the output of the reactors, while dashed lines illustrate input gas
composition.

highest average PCH4 and Y CH4 were shown by R4, while R2 reported importance for upgrading performances, unexpectedly, diffusion
the highest values at the end of the experiment (0.82 LCH4/LR d device (diffusers and ceramic sponge vs ceramic membrane) did
(p < 0.05) and 0.23 LCH4/LH2, respectively) (Table 1 and not show significant performance distinctions throughout the
Fig. 2c and d). Finally, while diffusers’ pore size resulted of great experiment.
316 I. Bassani et al. / Bioresource Technology 234 (2017) 310–319

Fig. 2. Reactors’ CO2 conversion efficiency (gCO2 ) (a), H2 utilization efficiency (gH2 ) (b), CH4 production rate (P CH4 ) (c) and CH4 yield ðY CH4 ) (d).

3.3. Effect of gas recirculation and input gas flow rate on upgrading
performances
Notably, in all the reactors increase of gas recirculation rate
ðQ RC Þ led to a marked improvement of upgrading performances,
resulting in significant higher CH4 content and/or PCH4 in several
operating condition and reactor configurations Moreover, in accor-
dance with previous studies (Guiot et al., 2011), kL a increased with
higher Q RC ; for example, 36% higher kL a was recorded from period
V to period VI (Table 2). These results demonstrate the positive
effect of recirculation on gas-liquid mass transfer rate extending
gas-liquid contact time and therefore enhancing H2 availability
for microorganisms (Guiot et al., 2011).
Nevertheless, change in microbial community living conditions
and, more specifically, excessive input gas flow rate (Q IN Þ could
affect negatively the process resulting in lower performance effi-
ciencies (Kleerebezem and Stams, 2000). In this experiment, a
slight decline of performances, which could have been due to
insufficient H2 solubilisation at this Q IN , has been recorded only
in Period V, when the highest Q IN was provided. The drop was
totally recovered in Period VI, thanks to the application of higher
Q RC (Table 1 and Fig. 1 and 2b).
In conclusion, the obtained results indicate that larger pore size
devices together with a proper gas recirculation flow managed to
achieve the most efficient biogas upgrading due to the optimal
mixing speed and gas retention time. Finally, it was shown that
the application of a ceramic sponge material having very high sur-
face area (65 m2/reactor) was able to increase the contact between
H2 bubbles and liquid and extend the permanence of gases in the
liquid media, therefore increasing H2 kL a.
 I. Bassani et al. / Bioresource Technology 234 (2017) 310–319 317

3.4. Microbial community composition
16S rRNA gene sequencing results are summarized in Table S3.
Rarefaction curves and Principal Component Analysis (PCoA)
clearly showed a shift in microbial composition between the initial
inoculum used and the reactors content at the end of the experi-
ment, with replicate samples clustering together (Fig. S5). More-
over, rarefaction curves showed an increased species richness in
the reactors after H2 addition. The results reported are focused
on most abundant members of the microbial community having
relative abundance >0.5%.
In accordance with previous studies, Firmicutes was the most
represented phylum accounting for the 32% of the microbial com-
munity after H2 addition (Campanaro et al., 2016). Interestingly,
Synergistetes resulted the second most abundant phylum, which
relative abundance doubled reaching >26% at the end of the exper-
iment. In particular, most abundant OTUs, accounting between 10
and 15% of the community were assigned to Anaerobaculum mobile
(98% identity; increasing < 3-folds; p < 0.05) and family Synergis-
taceae (Dataset S1, Figs. 3 and 4). The occurrence of these microor-
ganisms, together with the increment of other 2 OTUs (19 and 40-
folds, respectively, p < 0.05) assigned to the same phylum, has been
previously observed in reactors used for biogas upgrading (Bassani
et al., 2015; Treu et al., 2016b) and is consistent with their role and
resilience in AD process (Campanaro et al., 2016; Werner et al.,
2011). Moreover, according to the predictive metagenomic analy-
sis investigating KEGG and COG functional categories, Anaerobacu-
lum is involved in amino acids and carbohydrates metabolism
presenting an enriched number of genes for glycolysis, glycerol
utilization, pentose and amino acids degradation and peptides
transport, compared to the other genera analyzed (Dataset S2). In
addition, its predominance and concomitant increase with Methan-
othermobacter thermautotrophicus, together with the presence of
genes involved in Wood-Ljungdahl pathway, indicates a possible
role of Anaerobaculum in syntrophic acetate oxidation (SAO) with
this methanogen (Dataset S2).
Notably, the second most abundant OTU, accounting for 15%
of the community, was assigned to the newly discovered order
MBA08 (class Clostridia). In addition, BLASTn search against NCBI
database revealed a similarity to the newly identified Hydrogenis-
pora ethanolica with 90% identity. Nevertheless, being the sequence
identity score lower than the threshold for genera classification
(Dataset S1), the taxonomy of this OTU remains uncertain, and
defined only at class level as Clostridia sp.1, indicating this microor-
ganism as a new species. The remarkable abundance of Clostridia
sp.1 in the initial inoculum points out a possible important func-
tion of this bacterium in AD process. Moreover, its high abundance
at the end of the experiment was in accordance with a recent study
on ex situ biogas upgrading, and is indicative of a remarkable resi-
lience to the operating conditions suggesting a possible syntrophic
relation with hydrogenotrophic methanogens (Kougias et al.,
2017).
Further details on microbial community composition are pro-
vided in SI. However, among most interesting microorganisms,
10 OTUs were assigned to order Thermoanaerobacterales, mostly
accounting for >1% of the community and increasing at the end
of the experiment (Dataset S1, Figs. 3 and 4). Among them, two
OTUs were identified as genus Thermacetogenium and species

Fig. 3. Heat maps of relative abundance (>0.5%; left part of the panel) and folds change (log2; right part of the panel) of the most interesting microorganisms populating
initial inoculum and reactors at the end of the experiment. Correspondence between colors and relative abundance or folds change is reported in the scale at the top of each
panel. Folds change is represented in red and green for increased and decreased OTUs, respectively.
318 I. Bassani et al. / Bioresource Technology 234 (2017) 310–319

Fig. 4. Comparison between initial inoculum and samples obtained from each reactor at the end of the experiment (R1–R4 are plotted against the initial inoculum and shown
in panel (a) to (d)). The left part of each panel represents the relative abundance (>0.5%), while the right part shows the folds change of OTUs significantly changing in
abundance.

Tepidanaerobacter syntrophicus (96% identity). As shown by COG
and KEGG analysis, genes involved in Wood-Ljungdahl pathway
were highly represented confirming the role of these microorgan-
isms in SAO pathway (Treu et al., 2016a; Westerholm et al., 2011)
(Dataset S2). Moreover, the predictive functional metagenomics
suggested the presence in their genomes of genes for glucose and
pentose metabolism and for amino acids and proteins degradation
indicating that they would be able to utilize different substrates
(Dataset S2). Additionally, the significant increase in relative abun-
dance of 2 OTUs assigned to family Syntrophomonadaceae (30
folds; p < 0.05) is likely related to their syntrophic relation with
hydrogenotrophic methanogens (Dataset S1, Figs. 3 and 4) (Treu
et al., 2016a,b). It has been previously reported that members of
Syntrophomonas genus encode genes involved in menaquinone
biosynthesis (Kougias et al., 2016b), and in turn menaquinone
functions as electron carrier during interspecies electron transfer
in syntrophic communities (Stams and Plugge, 2009).
Regarding Archaea domain, phylum Euryarchaeota accounted for
<4% of the community. Among most abundant OTUs, 3 OTUs were
assigned to Methanothermobacter thermautotrophicus, Methanocul-
leus thermophilus and Methanocorpusculum aggregans (>99% iden-
tity). Interestingly, these microorganisms significantly increased
between 15 and 120-folds (p < 0.05), with M. thermautotrophicus
being the most abundant methanogen (2.6% at the end of the exper-
iment) (Dataset S1, Figs. 3 and 4). Notably, BLASTn search against
NCBI database revealed 100% similarity of M. thermautotrophicus
with several microbial species, such as Methanobacterium thermag-
gregans, M. wolfeii, M. thermophilus, M. thermoflexus, and M. defluvii,
indicating that the most abundant methanogen populating the
community was represented by a new species.
The presence of these hydrogenotrophs is in accordance with
previous studies showing prevalence and increment of these spe-
cies in biogas upgrading reactors (Bassani et al., 2015; Kougias
et al., 2017; Treu et al., 2016b). Interestingly, several hydrogeno-
trophs were maintained in the reactor presumable performing
the same function, without showing competition. This could have
resulted in a more robust hydrogenotrophic methanogenic process,
which would be able to tolerate disturbances, as the different
methanogens might have different tolerance levels. Notably, M.
thermautotrophicus was found with the highest relative abundance
in R2, the reactor presenting the best Y CH4 and output-gas quality
(96% CH4 content). Additionally, R4, which reached remarkable
H2 and CO2 conversion efficiencies, showed the highest relative
abundance of M. thermophilus (1.34%) suggesting a potential
 I. Bassani et al. / Bioresource Technology 234 (2017) 310–319
synergistic function between the two methanogens. In conclusion,
the proliferation of hydrogenotrophic methanogens and the con-
comitant increment of syntrophic bacteria are indicative of the
effect of the H2 on the microbial consortium selectively stimulating
hydrogenotrophic methanogenesis.
4. Conclusions
The configurations containing larger pore size diffusion devices
showed the best kinetics and output-gas quality due to the
achievement of better reactor mixing. The increment of gas recir-
culation flow rate led to improved output gas quality, generating
biogas with 96% CH4 content that can be used as transportation
fuel. Finally, microbial analysis revealed a significant increase in
relative abundance of hydrogenotrophic archaea, with Methanoth-
ermobacter thermautotrophicus being the most abundant methano-
gen. The concomitant proliferation of syntrophic bacteria, such as
Anaerobaculum mobile, or members of Thermoanaerobacterales
and Syntrophomonadaceae confirmed the selection-effect of H2
shaping the microbial community and enhancing hydrogeno-
trophic pathway.
Acknowledgements
We thank Hector Garcia and Hector Diaz for technical assis-
tance. Illumina sequencing was performed at the Ramaciotti Centre
for Genomics (Sydney, Australia). This work was supported by the
Danish Council for Strategic Research under the project ‘‘SYMBIO 
Integration of biomass and wind power for biogas enhancement
and upgrading via hydrogen assisted anaerobic digestion”, contract
12-132654.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2017.03.
055.
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