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Towards biohythane production from biomass:

Influence of operational stage on anaerobic
fermentation and microbial community

Buchun Si a, Zhidan Liu a,*, Yuanhui Zhang b, Jiaming Li a, Ruixia Shen a,

Zhangbing Zhu a, Xinhui Xing c
a
Laboratory of Environment-Enhancing Energy (E2E), College of Water Resources and Civil Engineering, China
Agricultural University, Beijing, 100083, China
b
Department of Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign, Urbana,
61801, USA
c
Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China

article info abstract

Article history: Biohythane consisting of biohydrogen and biomethane via two-stage fermentation is a
Received 30 April 2015 promising energy carrier for vehicle use. In this study, one-stage biomethane system using
Received in revised form upflow anaerobic sludge blanket (UASB) and packed bed reactor (PBR) was shifted to the
5 June 2015 two-stage biohythane system to study the influence of operational stage. Compared with
Accepted 10 June 2015 biomethane system, the biohythane process achieved higher COD removal and energy
Available online 3 July 2015 recovery. Particularly, the total COD removal in the PBR system rose significantly from 74.0
to 97.3%, corresponding to an increased energy recovery from 54.2 to 67.1%. The first-stage
Keywords: hydrogen fermentation had a positive effect on subsequent biomethane production in
Biohythane biohythane system. The analysis of microbial diversity using Illumina MiSeq sequencing
Operational stage showed significant changes of microorganisms in biomethane reactor, which revealed the
Biohydrogen variation of biochemical pathways. Compared to biomethane system, the relative abun-
Two-stage dance of acidogenesis bacteria was reduced in biohythane system, such as family Clos-
Microbial diversity tridiaceae. By contrast, the amount of acetogens (Syntrophaceae, Syntrophomonadaceae and
Desulfovibrionaceae) and acetate-oxidizing bacteria (Spirochaetes) was increased. The archaea
community remained stable, and mainly consisted of acetoclastic methanogens from
family Methanosaetaceae. These results indicated the biomethane reactors in biohythane
system had more efficient acidogenesis and acetate-utilizing microbial community.
Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.

environmentally friendly process and fast speed [1e3]. The

Introduction biohydrogen could be obtained from various industrial wastes
and non-food feedstock, such as lignocellulose [4,5]. However,
Biohydrogen production through dark fermentation is a biohydrogen production alone does not significantly reduce
promising method for hydrogen production due to its

* Corresponding author. Tel./fax: þ86 10 6273 7329.

E-mail address: zdliu@cau.edu.cn (Z. Liu).
http://dx.doi.org/10.1016/j.ijhydene.2015.06.045
0360-3199/Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
4430 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 4 4 2 9 e4 4 3 8
the organic content of feedstock [6]. The chemical oxygen
demand (COD) removal was below 20% during the process [7],
representing low energy recovery [8]. The residue after
hydrogen fermentation is mainly volatile fatty acids (VFAs)
and alcohols, needing further treatment.
Biohythane consisting of biohydrogen and biomethane via
two-stage fermentation is a promising energy carrier [9].
Compared to one-stage biogas production, the two-stage
fermentation has its advantages of enhancing the energy re-
covery and COD removal [6,10], and working at a higher
organic loading rate (OLR) [11]. A number of studies have been
investigated to compare the performance of one-stage bio-
methane and two-stage biohythane system [11,12]. In a two-
stage system, the first-stage hydrogen fermentation may
provide a couple of favorable conditions for subsequent bio-
methane production, and lead to the change of microbial
community in biomethane reactor. The microbial community
in biomethane reactor should be further studied to under-
stand the process. A number of studies reported the charac-
terization of microbial community for biohydrogen and
biomethane production in biohythane system [7,13e17]. Some
species belonging to class Clostridia, such as Clostridium
butyricum, Clostridium tyrobutyricum and Clostridium acetobuty-
licum were reported as the main biohydrogen producers
[15,18e20]. The methane-producers in biohythane system
were found mainly composed of acetoclastic methanogens
Methanosarcina [16,17]. However, limited information is avail-
able on the dynamic changes of anaerobic fermentation and
microbial community with the shift of operational stage.
The purposes of this study were 1) to investigate the effect
of operational stage on the anaerobic fermentation towards
biohythane production using the upflow anaerobic sludge
blanket (UASB) reactor and packed bed reactor (PBR); 2) to
study the dynamic changes of microbial community using
Illumina MiSeq sequencing.
Materials and methods
Inoculum and feedstock
The seed sludge was taken from the anaerobic digester of
Xiaohongmen Municipal Sewage Treatment Plant (Beijing,
China) and used as inoculum. As for hydrogen fermentation,
the sludge was heat treated at 100  C for 15 min to inactivate
methanogenesis and harvest anaerobic spore-forming bacte-
ria [21]. Biohydrogen and biomethane production were per-
formed in a volume inoculation ratio of 20% (v/v) and 60% (v/
v), respectively. The reactors were separately operated using
synthetic wastewater to enrich microbial consortium. Glucose
was used as the carbon source, whereas NH4Cl served as the
nitrogen source. The carbon/nitrogen ratios of substrate for
hydrogen and methane production were adjusted as 47 and
25, respectively [22,23]. The concentration of glucose and
NH4Cl was changed to obtain the desired OLRs. In addition,
the substrate contained (mg/L): K2HPO4 250, KH2PO4 250,
MgCl2 300, CoCl2 25, ZnCl2 11.5, CuCl2 10.5, CaCl2 5, MnCl2 15,
NiSO4 16, FeCl3 25 [24]. The alkalinity was supplemented by
sodium bicarbonate as 0.5 g/g COD for hydrogen reactor and
1 g/g COD for methane reactor. The pH of substrate was
adjusted to 5.7 ± 0.1 for biohydrogen production prior to use,
whereas it was not controlled for biomethane production.
Experimental setup
UASB and PBR were made from transparent acrylic with the
working volume of 2.5 L, and a height-to-diameter ratio of 6:1.
The carbon nanotubes (CNTs) were added into UASB at
100 mg/L to accelerate the granules formation [25], and poly-
ethylene rings were packed in PBR [24]. The reactors were
maintained at 37  C using the water jacket. The methane and
hydrogen reactors were separately operated over 200 days to
enrich the microbes. The hydrogen-producing reactors were
operated as previous reported [24]. The OLRs of methane-
producing reactors were stepwise increased from 0.5 to 16 g
COD/L/d. The hydrogen and methane reactor were sequen-
tially connected to establish a biohythane system when bio-
hydrogen and biomethane were stable in its respective
reactor. There were two series of biohythane systems (Table
1). One consisted of two PBRs for biohydrogen and bio-
methane production in sequence, and the other was
composed of two UASB reactors. The effluent from first-stage
reactors was firstly collected and its alkalinity was then
adjusted using the sodium bicarbonate (0.5 g/L). The effluent
was finally fed to biomethane reactors.
Analytical methods
Gas volumes were measured daily using gas meters at room
temperature (25 ± 3  C) and corrected under standard condi-
tion (273.15 K, 101.325 kPa). Gas composition was determined
daily, including hydrogen, methane and carbon dioxide,
through a gas chromatography (GC1490, Agilent Technologies,
USA) as previous described [24]. The produced VFAs were
analyzed by a high performance liquid chromatography (Shi-
madzu 10A, Japan) equipped with an ultraviolet detector and a
synergi 4u Hydro-RP (Phenomenex) column. 5 mM H2SO4 was
used as mobile phase at a flowrate of 1 mL/min and the oven
temperature was 40  C. Samples were first centrifuged at
4000 rpm for 10 min and then filtered using a 0.45 um mem-
brane filter before COD and VFA analysis. COD was measured
according to standard methods [26]. The microbial samples
were collected and stored at 20  C for morphology observa-
tion and phylogenetic diversity analysis. The microbial
morphology was observed by scanning electron microscopy
(SEM) (Quanta 200, FEI, USA) as previously described [25].
The phylogenetic diversity of the microbial consortium
was analyzed via Illumina MiSeq sequencing. Primers 515F (50 -
barcode-GTGCCAGCMGCCGCGG-30 ) and 907R (50 -CCG
0
TCAATTCMTTTRAGTTT-3 ) for bacteria were used [24].
Primers Arch344F (50 -ACGGGGYGCAGCAGGCGCGA-30 ) and
Arch915R (50 -GTGCTCCCCCGCCAATTCCT-30 ) for archaea were
used [27]. The PCR process was conducted as previously
described [24]. Amplicons were extracted from 2% agarose gels
and purified using the AxyPrep DNA gel extraction kit (Axygen
Biosciences, USA) and quantified using QuantiFluor ST
(Promega, USA). Purified amplicons were pooled in equimolar
and paired-end sequenced on an Illumina MiSeq platform.
The raw reads were deposited into the National Center for
Biotechnology Information (NCBI) Sequence Read Archive
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 4 4 2 9 e4 4 3 8 4431

Table 1 e Setup of one-stage biomethane system and two-stage biohythane system.

One-stage biomethane system Two-stage biohythane system
UASBM1 PBRM1 UASBH UASBM2 PBRH PBRM2
HRT (h) 12 12 12 12 12 12
Working volume (L) 2.5 2.5 2.5 2.5 2.5 2.5
Initial pH 7.8e8.0 7.8e8.0 5.7 ± 0.1 5.8 ± 0.1 5.7 ± 0.1 5.8 ± 0.1

(SRA) database. Raw fastq files were demultiplexed and
quality-filtered using Quantitative Insights into Microbial
Ecology (QIIME). The phylogenetic affiliation of each 16S rRNA
gene sequence was analyzed by an RDP Classifier (http://rdp.
cme.msu.edu/) against the silva (SSU115) 16S rRNA database
using a confidence threshold of 70%.
effluent of hydrogen production mainly consisted of acetate,
lactate, and butyrate (Table 2) which were common metabolic
products during the biohydrogen fermentation as previous
reported [28]. The effluent was collected and used for the
substrate of methane production.

Calculations Biomethane production

The energy production rate of biohydrogen (EH2, kJ/L/d) was The biomethane production was stable for the one-stage
calculated as: system (Fig. 2), with a methane production rate (MPR) of
3.89 ± 0.20 and 4.68 ± 0.22 L/L/d in PBR and UASB, respectively.
EH2 ¼ HPR=22:4  HVH2 (1) The stable biomethane production was also observed in two-
stage PBR (3.89 ± 0.22 L/L/d) and UASB (3.90 ± 0.15 L/L/d). The
where HPR was the hydrogen production rate (L/L/d), and
first-stage hydrogen fermentation seemed to improve the
HVH2 was the heating value of hydrogen (242 kJ/mol) [8].
subsequent biomethane production (Fig. 2). COD removal
The energy production rate of biomethane (ECH4, kJ/L/d)
increased from 74.0 ± 3.5 to 97.1 ± 1.6% in PBR and from
was calculated as:
95.4 ± 0.6 to 96.5 ± 0.1% in UASB. This was consistent with the
ECH4 ¼ MPR=22:4  HVCH4 (2) distribution of organic compounds in effluents of methane
reactors. VFAs were not detected after two-stage biohythane
where MPR was the methane production rate (L/L/d), and
production. In comparison, acetate and lactate were detected
HVCH4 was the heating value of methane (801 kJ/mol) [8].
after one-stage biomethane production (Table 2). Specifically,
The energy recovery (h, %) was proposed to represent the
a high concentration of lactate (913 ± 105 mg/L) was observed
ratio of produced energy and chemical energy of feedstock.
The produced energy included the energy from biohydrogen
production (EH2) and biomethane production (ECH4). The en-
ergy recovery can be calculated as:

EH2 þ ECH4
h¼  100% (3)
m=180  HVg

where m was of the glucose loading rate (g/L/d), 180 was the
molar mass of glucose (g/mol), and HVg was the heating value
of glucose (2888 kJ/mol) [8].

Results and discussion

Biohydrogen production

Both the UASB and PBR had stable performance of bio-

hydrogen production (Fig. 1). UASB had an HPR of 2.69 ± 0.13 L/
L/d with hydrogen content of 50.2 ± 1.6%, whereas PBR ach-
ieved a lower HPR (1.05 ± 0.15 L/L/d) and hydrogen content
(25.8 ± 2.5%). The lower hydrogen production rate in PBR
partly resulted from the significant hydrogenotrophic meth-
anogenesis, which would consume the produced hydrogen
[24]. As a result, the methane content in PBR reached
14.8 ± 4.8%. The final pH in all biohydrogen reactors was
decreased to about 4 due to the formation of VFAs (Table 2).
PBR and UASB had COD removal rates of 23.6 ± 4.1 and
23.4 ± 2.4% after hydrogen fermentation, respectively. The Fig. 1 e Biohydrogen production in UASB and PBR.
4432 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 4 4 2 9 e4 4 3 8

Table 2 e Summary of reactors performances at steady-states.

One-stage biomethane system Two-stage biohythane system
PBRM1 UASBM1 PBRH PBRM2 UASBH UASBM2
a
Formate (mg/L) N.D. N.D. N.D. N.D. N.D. N.D.
Acetate (mg/L) 191 ± 76 39 ± 12 1164 ± 30 N.D. 814 ± 120 N.D.
Lactate (mg/L) 913 ± 105 98 ± 16 18 ± 16 N.D. 289 ± 31 N.D.
Butyrate (mg/L) N.D. N.D. 2151 ± 77 N.D. 2114 ± 214 N.D.
Propionate (mg/L) 88 ± 12 36 ± 15 N.D. N.D. N.D. N.D.
Succinate (mg/L) 106 ± 14 53 ± 16 16 ± 7 N.D. 37 ± 15 N.D.
COD removal rate (%) 74.0 ± 3.5 95.4 ± 0.6 23.6 ± 4.1 97.1 ± 1.6 23.4 ± 2.4 96.5 ± 0.1
Total COD removal (%) 74.0 ± 3.5 95.4 ± 0.6 97.3 ± 0.8 97.8 ± 1.2
Hydrogen content (% v/v) 0.5 ± 0.4 0.8 ± 0.2 25.8 ± 2.5 N.D. 50.2 ± 1.6 N.D.
Methane content (% v/v) 49.2 ± 1.3 53.3 ± 1.3 14.8 ± 4.8 74.1 ± 1.2 1.7 ± 0.8 73.2 ± 1.3
HPR (L/L/d) 0.04 ± 0.04 0.08 ± 0.01 1.05 ± 0.15 N.D. 2.69 ± 0.13 N.D.
MPR (L/L/d) 3.89 ± 0.20 4.68 ± 0.22 0.61 ± 0.06 3.89 ± 0.22 0.09 ± 0.05 3.90 ± 0.15
EH2 (kJ/L/d) 0.4 0.8 11.4 N.D. 29.0 N.D.
ECH4(kJ/L/d) 138.9 167.3 21.76 139.3 3.3 139.6
ET (kJ/L/d)b 139.3 168.1 172.2 171.9
a
N.D. ¼ not detected.
b
ET ¼ EH2 þ ECH4.

in one-stage PBR system. In addition, methane content was
dramatically increased in both UASB (73.2 ± 1.3%) and PBR
(74.1 ± 1.2%) with the shift of operational stage (Table 2).
Similar high methane content (73.0e74.6%) was observed by
Kyazze et al. [29] using a two-stage system fed with sucrose.
Energy efficiency of two-stage process
The two-stage PBR and UASB system achieved total energy
recovery of 67.1% and 67.0%, respectively, which was higher
than respective one-stage system (Table 3). The biohydrogen
production only contributed to 11.3% of total energy recovery
in two-stage UASB system and much a lower value (4.4%) in
PBR system due to the significant hydrogenotrophic meth-
anogenesis. A highest energy recovery (82.1%) was reported
using a glucose-fed batch two-stage system [8]. However, such
high energy recovery was achieved at the cost of long batch
fermentation time (over 15 d). Other studies showed much
less energy recoveries in a range of 6.2e23.8% using contin-
uous glucose-fed two-stage system [30,31]. In comparison, a
higher energy recovery rate (67%) was achieved at continuous
operation in this study (Table 3), probably due to higher
biomass concentration in UASB and PBR. Note that the ratio of
H2/(H2 þ CH4) in the UASB and PBR two-stage system were 0.40
and 0.19 in this study, respectively. The hydrogen content in
PBR well matched the suggested range (0.1e0.25) for the pro-
duction of clean vehicle fuel hythane [9].

The changes of microbial diversity in biomethane reactors

The shift of operational stage remarkably affected the distri-

Fig. 2 e Biomethane production in UASB and PBR.
bution of microbial diversity in the biomethane reactors.
Table 4 showed the comparison of microbial diversity (bac-
teria and archaea community) using Shannon, Simpson,
Chao, ACE and operational taxonomic units (OTUs).
Compared with one-stage systems, the two-stage systems
had higher Shannon and lower Simpson indexes for bacteria
community, indicating higher diversity of bacterial species.
Moreover, the two-stage systems had higher OTUs, ACE and
Chao indexes, suggesting higher bacteria richness. The higher
diversity and richness of bacterial species might benefit the
conversion of intermediates into acetate, which was the main
substrate of methanogens. As for the archaea community, the
UASB and PBR system showed different changes with the shift
of operational stage (Table 4). Compared with UASBM1,
UASBM2 showed a higher diversity of archaea species and
richness. However, the PBR system exhibited a converse
change (Table 4).
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Table 3 e Comparison of biohythane production in this study with literature.

Substrate H2 reactor H2 yield mol/mol CH4 reactor CH4 yield H2/H2 þ CH4 h (%) Reference
mol/mol (v/v)
Operation HRT (h) Operation HRT (h)
Glucose CSTR,Ba e 2.75 CSTR,B e 2.13 0.56 82.1 [8]
Glucose CSTR,B e 2.33 CSTR,B e 0.8 0.75 41.7 [40]
Glucose CSTR,Cb 9.6 0.34 CSTR,C 72 0.12 0.74 6.2 [31]
Glucose UASB,C 5 0.73 UASB,C 13 0.64 0.53 23.8 [30]
Glucose CSTR,B e 1.39 CSTR,B e 2.00 0.41 67.1 [41]
Glucose UASB,C 12 1.35 UASB,C 12 2.01 0.40 67.0 This study
Glucose PBR,C 12 0.53 PBR,C 12 2.26 0.19 67.1 This study
a
B ¼ batch experiments.
b
C ¼ continuous experiments.

Remarkable changes of bacteria community in the bio-
methane reactors were observed using Illumina MiSeq
sequencing with the shift of operational stage. Functions of
dominant families were classified according to the literature,
although many functions were still unknown (Table 5). The
bacteria community in the two-stage biohythane systems
showed a lower population of families of Bacteroidaceae, Clos-
tridiaceae, Erysipelotrichaceae, Veillonellaceae than one-stage
systems (Fig. 3A). All these families could be classified to the
acidogenesis bacteria (Table 5). For instance, the family Clos-
tridiaceae was dominant in biohydrogen reactor for hydrogen
and VFAs production [24], and its relative abundance was
decreased from 4.4% in UASBM1 to zero in UASBM2. Similar
reduction was found in PBR from 13.6% to zero. On the con-
trary, the abundance of other acidogenesis families (Synergis-
taceae, Thermotogaceae, Porphyromonadaceae, Rikenellaceae,
Ruminococcaceae, Christensenellaceae, Sphingobacteriaceae) was
increased with the shift of operational stage. These results
suggested that the distribution of acidogenesis bacteria was
significantly changed with the shift of operational stage, and
its total relative abundance was decreased (Fig. 3B). This
benefited the physical separation of hydrogen and methane
production in the biohythane system.
Acetogens are another important bacteria community
during biomethane production, which often co-culture with
hydrogenotrophic methanogens and responsible to convert
butyrate, lactate and propionate into acetate [32]. The relative
abundance of families Syntrophaceae, Syntrophomonadaceae and
Desulfovibrionaceae rose significantly (Table 5). Specifically, the
amount of acetogens was increased from 3.60 to 10.41% in
UASB, and 1.47e3.69% in PBRM2, respectively (Fig. 3B). The
higher distribution of syntrophic acetogens would benefit the
conversion of VFAs produced from hydrogen fermentation. As
the degradation of fatty acids is often regarded as the rate-
limiting step for biomethane production, it is essential that
bioreactors be operated under suitable conditions for the
bacteria capable of syntrophic metabolism [33]. Hence, the
increased amount of acetogens would enhance methane
production and COD removal.
Another interesting finding was that family Spirochaetaceae,
which belongs to class Spirochaetes, was observed in UASBM1
and PBRM1, with a relative abundance of 13.1 and 4.8%,
respectively. These values were drastically increased to 21.05
and 14.4% in UASBM2 and PBRM2, respectively. The class Spi-
rochaetes are frequently detected in anaerobic digesters and
classified as syntrophic acetate-oxidizing bacteria [34,35],
which could catalyze the following acetate-oxidizing reaction
(Eq. (4)):
0

CH3 COO þ 4H2 O/4H2 þ 2HCO 3 þH
þ
DG0 ¼ þ105 kJ (4)
This reaction is difficult to take place at the standard
conditions and pH 7 due to positive changes of Gibbs free
energy (DG0 ¼ þ105 kJ). However, the high concentration of
acetate after hydrogen fermentation and syntrophic interac-
tion between Spirochaetes and hydrogenotrophic methanogens
made the reaction possible [35]. This deduction was supported
by Fig. 3C, where the hydrogen-consuming families of Meth-
anobacteriaceae and Methanospirillaceae were not significantly
decreased in the two-stage biohythane systems. Shimada
et al. [36] also found high amounts of hydrogenotrophic
methanogens and acetate-oxidizing bacteria in two-stage
anaerobic digestion. The acetate-oxidizing bacteria and ace-
toclastic methanogens would build an efficient acetate-
utilizing system enhancing acetogenesis.
Distinguished from significant changes of bacterial com-
munity, the archaea community seemed to remain consistent
regardless of operational stages. This was also supported by
Kim et al. [37]. The dominant archaea in all biomethane

Table 4 e Diversity analysis of microbial community for clustering at 97% identity.

Sample Bacteria community Archaea community
Chao Shannon Simpson ACE OTUs Chao Shannon Simpson ACE OTUs
PBRM1 269 3.01 0.0988 264 207 36 0.36 0.8580 52 28
PBRM2 393 3.8 0.0466 397 343 12 0.24 0.9195 12 12
UASBM1 221 3.26 0.0655 223 190 23 0.39 0.8166 26 21
UASBM2 365 3.8 0.0598 370 321 53 0.72 0.7166 53 51
 4434
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 4 4 2 9 e4 4 3 8
Table 5 e Bacteria and archaea families in biomethane reactors.
Families Function Taxonomy (phylum, class) Metabolic features Reference
Erysipelotrichaceae Acidogenesis Firmicutes, Erysipelotrichia Some species ferment glucose, major metabolic end products are butyrate, lactate and formate [42]
Clostridiaceae Acidogenesis Firmicutes, Clostridia Ferment glucose; metabolic end products are hydrogen, butyrate, acetate and lactate [20,43]
Bacteroidaceae Acidogenesis Bacteroidetes, Bacteroidia Some species ferment glucose, metabolic end products are acetate, CO2 and hydrogen [44]
Veillonellaceae Acidogenesis Firmicutes, Negativicutes Some species ferment glucose, metabolic end products are acetate, propionate, isobutyrate, butyrate [45]
and isovalerate
Synergistaceae Acidogenesis Synergistetes, Synergistia Some species ferment glucose and organic acids, metabolic end products are acetate, CO2 and [46,47]
hydrogen, co-culture with the hydrogenotrophic methanogens
Syntrophaceae Acetogenesis Proteobacteria, Deltaproteobacteria Propionate and butyrate-utilizing bacteria, syntrophic bacteria by co-culture with hydrogenotrophic [48,49]
methanogens
Syntrophomonadaceae Acetogenesis Firmicutes, Clostridia Some species utilize butyrate; syntrophic association with hydrogenotrophic methanogens [50,51]
Thermotogaceae Acidogenesis Thermotogae, Thermotogae Able to ferment carbohydrates and peptides [52]
Porphyromonadaceae Acidogenesis Bacteroidetes Bacteroidia Some species involved in hydrolysis and acidogenesis [53]
Rikenellaceae Acidogenesis Bacteroidetes, Bacteroidia Carbohydrate-fermenting, hydrogen-producing bacterium [54]
Ruminococcaceae Acidogenesis Firmicutes, Clostridia Ferment glucose; metabolic end products are hydrogen and VFAs [55]
Desulfovibrionaceae Acetogenesis Proteobacteria, Deltaproteobacteria Some species utilize lactate and pyruvate; major metabolic end products are acetate, hydrogen and [56]
CO2;
Christensenellaceae Acidogenesis Firmicutes, Clostridia Some species ferment glucose, major metabolic end products are acetate and butyrate [57]
Sphingobacteriaceae Acidogenesis Bacteroidetes, Sphingobacteriia Some species ferment glucose, major metabolic end products are VFAs [58]
Spirochaetaceae Acetate-oxidizing Spirochaetae, Spirochaetes Major metabolic end products are hydrogen and CO2, [34]
Anaerolineaceae Acidogenesis Chloroflexi, Anaerolineae Some species ferment glucose, major metabolic end products are VFAs and hydrogen [59]
Methanosaetaceae Methanogenic Euryarchaeota, Methanomicrobia Acetoclastic methanogens [60]
Methanobacteriaceae Methanogenic Euryarchaeota, Methanobacteria Hydrogenotrophic methanogens [60]
Methanospirillaceae Methanogenic Euryarchaeota, Methanomicrobia Hydrogenotrophic methanogens [60]
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 4 4 2 9 e4 4 3 8 4435

Fig. 3 e Taxonomic classification bacterial (A, B) and archaea community (C, D) of biomethane PBRM1, UASBM1 in one-stage
system and PBRM2 and UASBM2 in two-stage system at the families levels through Illumina Miseq sequencing. “Others”
included families which were unclassified and less than 1% of total composition.

reactors came from family Methanosaetaceae with a distribu-
tion of 92.5% (PBRM1), 96.0% (PBRM2), 90.0% (UASBM1) and 84.2%
(UASBM2). Families Methanosaetaceae and Methanosarcinaceae
are two main acetoclastic methanogens. Methanosarcina out-
competes Methanosaeta at higher acetate concentration
(>1.9 mM) [38]. The initial acetate concentrations for second-
stage biomethane production in this study were as high as
19.4 mM (UASBM2) and 13.6 mM (PBRM2), which compelled
family Methanosaetaceae to locate in the inner part of granules
or biofilms with relatively lower concentration of acetate [39].
In addition, hydrogenotrophic methanogens including Meth-
anospirillaceae and Methanobacteriaceae were also observed
during biomethane production (Fig. 3C). The larger amount of
hydrogenotrophic methanogens in UASB than PBR was prob-
ably due to the higher abundance of syntrophic acetogens and
acetate-oxidizing bacteria as above discussed.
Though the same inoculum was used in all reactors, an
efficient two-stage biofuels system was built up based on the
separated and enriched microbes. This was due to the proper
operation of these reactors.
The SEM images (Fig. 4) showed microbes morphology in
the inner part of granules and biofilms. The coccus-shaped
and bamboo-like microorganisms were observed in all of
them. The bamboo-like microorganisms were dominant both
in granules (Fig. 4A and B) and biofilms (Fig. 4C and D),
exhibiting the typical characteristic of family Meth-
anosaetaceae. Obviously, there were much more bamboo-like
microorganisms in biofilms than granules, which further
supported the analysis of microbial diversity using Illumina
Miseq sequencing.
Conclusion
In this study, the influence of operational stage on anaerobic
fermentation and microbial community was investigated
through two series of UASB and PBR. Compared with one-
stage system, the two-stage biohythane system achieved
higher COD removal rate and energy recovery rate. The first-
stage hydrogen fermentation benefited subsequent bio-
methane production. The analysis of microbial diversity using
Illumina Miseq sequencing revealed significant changes of
distribution of microbial community with the shift of opera-
tional stage. Compared with biomethane production in one-
stage system, the two-stage system had a lower abundance
of acidogenesis bacteria. The amount of both acetogens and
acetate-oxidizing bacteria was increased. This may suggest an
efficient biochemical pathway for biomethane production in
two-stage system. Distinguished from bacterial community,
the archaea seemed to remain consistent and mainly
4436 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 1 ( 2 0 1 6 ) 4 4 2 9 e4 4 3 8
Fig. 4 e SEM images of biomethane microbial community of UASBM1 (A) and PBRM1 (C) in one-stage system, and UASBM2 (B)
and PBRM2 (D) in two-stage system.
consisted of family Methanosaetaceae regardless of operational
stages.
Acknowledgment
This work was financially supported by the National Key
Technology Support Program of China (2014BAD02B03), Na-
tional Natural Science Foundation of China (21106080) and the
Chinese Universities Scientific Fund (2012RC030).
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