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Comprehensive microbial analysis of combined

mesophilic anaerobicethermophilic aerobic
process treating high-strength food wastewater

Hyun Min Jang a, Jeong Hyub Ha a,b,*, Jong Moon Park a,b,c,**,
Mi-Sun Kim d, Sven G. Sommer e
a
School of Environmental Science and Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro,
Pohang 790-784, Republic of Korea
b
Department of Chemical Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro, Pohang 790-
784, Republic of Korea
c
Division of Advanced Nuclear Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro,
Pohang 790-784, Republic of Korea
d
Biomass and Waste Energy Laboratory, Korea Institute of Energy Research, 152 Gajeong-ro, Yuseong-gu, Daejeon
305-343, Republic of Korea
e
Institute of Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, University
of Southern Denmark, Niels Bohrs All
e 1, DK-5230 Odense M, Denmark

article info abstract

Article history:
Received 1 October 2014
Received in revised form
27 January 2015
Accepted 28 January 2015
Available online 7 February 2015
Keywords:
High-strength food wastewater
Combined biological process
Methane production
Pyrosequencing
Quantitative real-time PCR (qPCR)
A combined mesophilic anaerobicethermophilic aerobic process was used to treat high-
strength food wastewater in this study. During the experimental period, most of solid
residue from the mesophilic anaerobic reactor (R1) was separated by centrifugation and
introduced into the thermophilic aerobic reactor (R2) for further digestion. Then, ther-
mophilic aerobically-digested sludge was reintroduced into R1 to enhance reactor perfor-
mance. The combined process was operated with two different Runs: Run I with hydraulic
retention time (HRT) ¼ 40 d (corresponding OLR ¼ 3.5 kg COD/m3 d) and Run II with
HRT ¼ 20 d (corresponding OLR ¼ 7 kg COD/m3). For a comparison, a single-stage meso-
philic anaerobic reactor (R3) was operated concurrently with same OLRs and HRTs as the
combined process. During the overall digestion, all reactors showed high stability without
pH control. The combined process demonstrated significantly higher organic matter
removal efficiencies (over 90%) of TS, VS and COD and methane production than did R3.
Quantitative real-time PCR (qPCR) results indicated that higher populations of both bacteria
and archaea were maintained in R1 than in R3. Pyrosequencing analysis revealed relatively
high abundance of phylum Actinobacteria in both R1 and R2, and a predominance of phyla
Synergistetes and Firmicutes in R3 during Run II. Furthermore, R1 and R2 shared genera
(Prevotella, Aminobacterium, Geobacillus and Unclassified Actinobacteria), which suggests
synergy between mesophilic anaerobic digestion and thermophilic aerobic digestion. For

* Corresponding author. Department of Chemical Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro, Pohang,
790-784, Republic of Korea. Tel.: þ82 54 279 8315; fax: þ82 54 279 8659.
** Corresponding author. Department of Chemical Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro, Pohang,
790-784, Republic of Korea. Tel.: þ82 54 279 2275; fax: þ82 54 279 8659.
E-mail addresses: jeonghha@postech.ac.kr (J.H. Ha), jmpark@postech.ac.kr (J.M. Park).
http://dx.doi.org/10.1016/j.watres.2015.01.038
0043-1354/© 2015 Elsevier Ltd. All rights reserved.
296 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3
conducted to clarify the relationship between TVFA/TA ratio
and reactor performance during AD. Furthermore, alkalinity
addition may be required to alleviate the possible acidification
and to maintain appropriate pH condition for methanogenesis
when the proposed combined process is operated with OLR
>7.5 kg COD/m3 d.
The biogas production and methane content in both
combined and control processes were monitored throughout
digestion (Table 2). In Run I at steady state, the combined
process achieved 1.54 ± 0.03 L/L/d biogas production rate with
68.40 ± 0.31% methane content and the control process ach-
ieved 1.00 ± 0.02 L/L/d biogas production rate with
63.10 ± 0.58% methane content. In Run II at steady state, the
combined process achieved 2.67 ± 0.04 L/L/d biogas produc-
tion rate with 66.20 ± 0.48% methane content and the control
process achieved 2.00 ± 0.01 L/L/d biogas production rate with
59.42 ± 0.44%; in both cases, the biogas production was
higher, but the methane content lower, than in Run I at
steady state.
Methane production rate (MPR) and methane yield (MY)
were calculated from the data. The combined process ach-
ieved MPR ¼ 1.05 ± 0.01 (Run I at steady state) and 1.77 ± 0.02 L/
L/d (Run II at steady state) with stable MY ¼ 0.32 ± 0.01 m3 CH4/
kg CODremoved; these values were ~1.5 times higher than those
of the control process (MPR ¼ 0.63 ± 0.01 (Run I at steady state)
and 1.19 ± 0.01 L/L/d (Run II at steady state), MY ¼ 0.22e0.25 m3
CH4/kg CODremoved). These findings indicate that combined
process have potential advantages in terms of methane re-
covery as compared to control process. Therefore, it can be
inferred that the additional digestion in R2 of highly-
concentrated solid residue from R1 resulted in remarkable
degradation of organic matter and increased methane
production.
3.2. Quantification of total bacteria and archaea
Typically, microbial population (expressed as 16S rRNA gene
concentration) in biological reactor is strongly related to the
respond to environmental stress (Klappenbach et al., 2000).
Fig. 2 e Changes in 16S rRNA concentration of total bacteria, total archaea and ratio of archaea to total microbes (the sum of
bacteria and archaea) in different Runs I and II.
Therefore, measurement of 16S rRNA gene concentration is
useful during treatment of high-strength wastewater. qPCR
method was used to measure the concentration of 16S rRNA
gene copies of total bacteria and archaea (Fig. 2). In addition, it
is noteworthy to mention that PCR amplicons was not detec-
ted in R2 with archaea specific primer. This implies that
archaea within the solid residue from R1 completely lost their
activity and were rapidly degraded due to high microbial ac-
tivity under thermophilic aerobic conditions.
In spite of the relatively short HRT (5 d), bacterial 16s rRNA
gene concentration in R2 increased from 8.24 (±0.09)  1010 to
1.02 (±0.07)  1012 copies/mL (~12-fold increase) in Run I and
maintained higher population (~1012 copies/mL) throughout
digestion than in both R1 and R3. This difference in bacterial
populations might be due to the different growth character-
istics between aerobic and anaerobic bacteria. This result
corresponds to previous findings that stable and high bacterial
populations were maintained under thermophilic aerobic
conditions (Jang et al., 2014, 2013). Interestingly, R1 and R3
showed similar variation patterns of bacterial population, but
R1 maintained higher bacteria population
(2.21 (±0.07)  1011 copies/mL in Run I and
6.25 (±0.15)  1011 copies/mL in Run II) than did R3
(6.29 (±0.14)  1010 copies/mL in Run I and
2.15 (±0.11)  1011 copies/mL in Run II) throughout the
experiment (Fig. 2).
Similar to bacteria, archaea population in R1 and R3
increased as OLR increased and R1 showed higher archaea
population (2.79 (±0.25)  1010 copies/mL in Run I and
7.69 (±0.33)  1010 copies/mL at Run II) than did R3
(6.90 (±0.06)  109 copies/mL in Run I and
2.35 (±0.07)  1010 copies/mL Run II) throughout digestion.
Additionally, the percentage of archaea to total microbes was
slightly higher in R1 (11 ± 0.58%) than in R3 (9.8 ± 0.12%).
Collectively, the qPCR results demonstrated that the com-
bined process provided more suitable environment for growth
of both bacteria and archaea, thereby resulting in higher MPR
and methane yield as well as organic removal than did the
control process (Table 2).
 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3
3.3. Pyrosequencing data
For in-depth understanding of microbial communities in the
reactors pyrosequencing was used with 16s rRNA-specific
oligonucleotide primers, which usually cover all bacteria and
archaea. Totals of 38002 raw bacterial sequences and 23677
raw archaeal sequences were obtained (Table S3). After
filtering, totals of 36867 (97% of raw sequences) clean bacterial
sequences and 23558 (99% of raw sequences) clean archaeal
sequences were obtained. The average lengths were
473 ± 4.72 bp for bacteria and 514 ± 2.37 bp for archaea; these
lengths are well-matched with those of the hypervariable re-
gions that were targeted. Based on clean sequences, phylo-
types and diversity indices of bacterial and archaeal
communities were calculated (Table S3).
None of rarefaction curves reached saturation phase; this
means that the sequences collected do not fully represent the
microbial community diversity. This phenomenon is
commonly observed during pyrosequencing, probably
because environmental samples are complex and contain rare
species with low abundance (Ashby et al., 2007; Claesson et al.,
2009). Meanwhile, Good's coverage values (estimator of sam-
pling completeness) of all samples represented > 95% of
coverage (Table S3); therefore the diversity and richness in
this study are sufficient to characterize the major phylotypes
in each sample.
3.4. Composition and possible functions of major
bacterial species
Taxonomic classification of clean sequences obtained from
pyrosequencing provides comprehensive insight into the mi-
crobial communities and their possible functions assuming
that closely-affiliated taxa share similar metabolic pathways
and capabilities (Rivie re et al., 2009; Zhang et al., 2010). In this
study, the relative composition of the unclassified sequences
at all taxonomic levels was within typical ranges reported
previously (Lee et al., 2008; Lu et al., 2012; Sundberg et al.,
2013). According to taxonomic classification and calculated
Shannon-Weaver Indices (Table S3), all bacterial samples
showed higher diversity and taxonomic distribution than
those of archaeal samples; this finding is in agreement with
the results of earlier studies of microbial communities in the
full-scale biogas digesters (Regueiro et al., 2012; Sundberg
et al., 2013).
The taxonomic distributions of each bacterial sample were
determined at the phylum and class level (Fig. 3). Unlike
Anaerobic seed, Bacteroidetes was most abundant phylum in
both anaerobic reactors and was detected in all reactors dur-
ing the steady-state condition in Runs I and II. In contrast, the
relative abundances of phyla Chloroflexi (most abundant
phylum in Anaerobic seed) and Proteobacteria (second major
phylum) in both anaerobic reactors decreased significantly in
Runs I and II. The decline of those two phyla in this study may
be due to the difference in characteristics of the substrates, as
recently reported by Sundberg et al. (2013). Also, several
studies mentioned potential function of microorganisms
assigned into the phylum in AD mainly related to hydrolysis of
organic matter and production of acetic acid (Kragelund et al.,
2008; Riviere et al., 2009). Thus, the dominance of phylum
297
Bacteroidetes in both anaerobic reactors indicates that bacteria
of this phylum may have important functions in hydrolysis
and acetogenesis.
Interestingly, phyla distributions were markedly different
between R1 and R3 at Run II. PCoA also demonstrated clear
regional separation (Fig. S2a). In R1, phyla Synergistetes (main
class Synergistia) decreased from 17.82% in Run I to 4.07% in
Run II, Firmicutes (main classes Clostridia and Bacilli) decreased
from 10.71% in Run I to 8.91% in Run II, whereas the phylum
Actinobacteria (main class Actinobacteria) increased from 6.05%
in Run I to 17.55% in Run II. The large proportion of members
of the phylum Actinobacteria is indicative of their contribution
to efficient degradation of complex organic materials and
production of organic acids in R1 (Ventura et al., 2007). In R3,
members of the phyla Synergistetes increased from 18.25% in
Run I to 28.12% in Run II, and those of Firmicutes (main class
Clostridia) increased from 11.29% in Run I to 14.24% in Run II;
whereas the relative abundance of phylum Actinobacteria was
~3.3% in both Runs I and II. Members of the phyla Synergistetes
and Firmicutes commonly appear in AD; they can degrade
proteins, lipids and carbohydrate with hydrolytic enzymes
(Guo et al., 2014; Sundberg et al., 2013). In addition, some of
them can obtain energy by oxidizing organic acids (syntrophic
reaction), especially by fermenting VFAs to H2 and CO2 (Ito
re et al., 2009). Therefore, the predominance
et al., 2011; Rivie
of the phyla Synergistetes and Firmicutes strongly suggests that
syntrophic metabolism is highly activated in R3, especially
during Run II.
It is noteworthy that R2 maintained higher bacterial di-
versity and taxonomic distribution than did both R1 and R3
throughout digestion (Table S3). Also, the bacterial commu-
nity composition of thermophilic aerobic seed differed
greatly from those of Runs I and II, probably due to the dif-
ference in characteristics of substrate and operating param-
eters (e.g., reactor configuration and aeration efficiency), as
recently reported by Piterina et al. (2012). In Run I, abundance
of the phylum Firmicutes (most abundant phylum in ther-
mophilic aerobic seed) decreased to ~17%, then remained
steady during rest of the operating period. It should addi-
tionally be noted that most species of class Clostridia (~5% in
Runs I and II) are obligate anaerobes. The presence of a sig-
nificant proportion of such anaerobic bacteria in TAD has
been reported by several authors (Juteau et al., 2004b; LaPara
et al., 2002) and is probably due to the low oxygen solubility
under thermophilic conditions. The variation patterns of ORP
in R2 (Table 2) are consistent with the existence of an
anaerobic period caused by the step-feeding system, whether
they have metabolic activities in R2 remains unclear. Unlike
phylum Firmicutes, phylum Actinobacteria (mainly class Acti-
nobacteria) was not a major phylum in Seed (3.33%), but
significantly increased to 22.98% in Run I and 24.55% in Run II,
and became the most abundant phylum in R2. Although this
phylum is frequently detected in the ATAD process (never as
the dominant phylum) (Hayes et al., 2011; Juteau et al., 2004b),
this is the first observation that members of phylum Actino-
bacteria emerge as the dominant phylum in TAD. Their pre-
dominance in this study suggests that they were selectively
enriched from the thermophilic aerobic seed and had an
important function related to organic matter removal
throughout digestion.
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 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3

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

Comprehensive microbial analysis of combined

mesophilic anaerobicethermophilic aerobic
process treating high-strength food wastewater

Hyun Min Jang a, Jeong Hyub Ha a,b,*, Jong Moon Park a,b,c,**,
Mi-Sun Kim d, Sven G. Sommer e
a
School of Environmental Science and Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro,
Pohang 790-784, Republic of Korea
b
Department of Chemical Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro, Pohang 790-
784, Republic of Korea
c
Division of Advanced Nuclear Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro,
Pohang 790-784, Republic of Korea
d
Biomass and Waste Energy Laboratory, Korea Institute of Energy Research, 152 Gajeong-ro, Yuseong-gu, Daejeon
305-343, Republic of Korea
e
Institute of Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, University
of Southern Denmark, Niels Bohrs All
e 1, DK-5230 Odense M, Denmark

article info abstract

Article history:
Received 1 October 2014
Received in revised form
27 January 2015
Accepted 28 January 2015
Available online 7 February 2015
Keywords:
High-strength food wastewater
Combined biological process
Methane production
Pyrosequencing
Quantitative real-time PCR (qPCR)
A combined mesophilic anaerobicethermophilic aerobic process was used to treat high-
strength food wastewater in this study. During the experimental period, most of solid
residue from the mesophilic anaerobic reactor (R1) was separated by centrifugation and
introduced into the thermophilic aerobic reactor (R2) for further digestion. Then, ther-
mophilic aerobically-digested sludge was reintroduced into R1 to enhance reactor perfor-
mance. The combined process was operated with two different Runs: Run I with hydraulic
retention time (HRT) ¼ 40 d (corresponding OLR ¼ 3.5 kg COD/m3 d) and Run II with
HRT ¼ 20 d (corresponding OLR ¼ 7 kg COD/m3). For a comparison, a single-stage meso-
philic anaerobic reactor (R3) was operated concurrently with same OLRs and HRTs as the
combined process. During the overall digestion, all reactors showed high stability without
pH control. The combined process demonstrated significantly higher organic matter
removal efficiencies (over 90%) of TS, VS and COD and methane production than did R3.
Quantitative real-time PCR (qPCR) results indicated that higher populations of both bacteria
and archaea were maintained in R1 than in R3. Pyrosequencing analysis revealed relatively
high abundance of phylum Actinobacteria in both R1 and R2, and a predominance of phyla
Synergistetes and Firmicutes in R3 during Run II. Furthermore, R1 and R2 shared genera
(Prevotella, Aminobacterium, Geobacillus and Unclassified Actinobacteria), which suggests
synergy between mesophilic anaerobic digestion and thermophilic aerobic digestion. For

* Corresponding author. Department of Chemical Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro, Pohang,
790-784, Republic of Korea. Tel.: þ82 54 279 8315; fax: þ82 54 279 8659.
** Corresponding author. Department of Chemical Engineering, Pohang University of Science and Technology, 77, Cheongam-Ro, Pohang,
790-784, Republic of Korea. Tel.: þ82 54 279 2275; fax: þ82 54 279 8659.
E-mail addresses: jeonghha@postech.ac.kr (J.H. Ha), jmpark@postech.ac.kr (J.M. Park).
http://dx.doi.org/10.1016/j.watres.2015.01.038
0043-1354/© 2015 Elsevier Ltd. All rights reserved.
 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3 295

Table 2 e Summary of the reactor performance of combined (R1 and R2) and control (R3) processes at the two different
conditions.
Parameters Run I (HRT: 40 d) Run II (HRT: 20d)
Combined process R3 (control) Combined process R3 (control)
R1 (MAD) R2 (TAD) Final effluent R1 (MAD) R2 (TAD) Final effluent
pH 7.43 ± 0.11 8.25 ± 0.04 7.43 ± 0.11 7.33 ± 0.09 7.37 ± 0.01 8.21 ± 0.02 7.37 ± 0.01 6.75 ± 0.04
TA (g CaCO3/L) 3.22 ± 0.07 1.45 ± 0.08 3.22 ± 0.07 3.29 ± 0.02 3.22 ± 0.02 1.43 ± 0.02 3.22 ± 0.02 2.23 ± 0.03
ORP(mV) 405 ±8 38a ~ 67b e 402 ± 4 403 ± 6 37a ~ 67b e 402 ±3
TS (g/L) 40.06 ± 1.99 56.68 ± 0.49 8.88 ± 0.21 37.74 ± 0.28 51.08 ± 0.82 72.12 ± 0.28 8.89 ± 0.19 43.68 ± 0.12
VS (g/L) 20.47 ± 1.01 29.80 ± 0.26 3.94 ± 0.18 20.63 ± 0.31 26.10 ± 0.42 37.92 ± 0.15 3.98 ± 0.22 23.88 ± 0.24
TCOD (g/L) 30.92 ± 0.92 47.29 ± 0.26 9.49 ± 0.58 29.98 ± 0.58 43.49 ± 0.39 61.42 ± 0.42 9.07 ± 0.06 40.32 ± 0.42
SCOD (g/L) 3.43 ± 0.03 3.29 ± 0.11 3.43 ± 0.03 2.11 ± 0.08 4.81 ± 0.11 4.06 ± 0.04 4.81 ± 0.11 3.20 ± 0.01
TOA (g COD/L) 2.86 ± 0.07 e 2.86 ± 0.07 0.89 ± 0.01 3.28 ± 0.01 e 3.28 ± 0.01 1.99 ± 0.05
Acetic acid (g COD/L) 1.52 ± 0.01 e 1.52 ± 0.01 0.89 ± 0.01 1.73 ± 0.01 e 1.73 ± 0.01 1.24 ± 0.03
Propionic acid (g COD/L) 1.43 ± 0.03 e 1.43 ± 0.03 e 1.55 ± 0.01 e 1.55 ± 0.01 0.75 ± 0.01
TVFA (g acetic acid/L) 2.67 ± 0.05 e 2.67 ± 0.05 0.83 ± 0.01 3.07 ± 0.01 e 3.07 ± 0.01 1.86 ± 0.05
TVFA/TA (g acetic acid/g 0.83 ± 0.09 e 0.83 ± 0.09 0.25 ± 0.02 0.95 ± 0.01 e 0.95 ± 0.01 0.83 ± 0.06
CaCO3)
TS removal efficiency 92.52 ± 0.35 68.15 ± 0.75 92.54 ± 0.42 63.13 ± 0.39
(% of feed)
VS removal efficiency 96.31 ± 0.41 80.63 ± 0.48 96.30 ± 0.28 77.58 ± 0.27
(% of feed)
COD removal efficiency 93.20 ± 0.28 78.52 ± 0.24 93.51 ± 0.11 71.11 ± 0.65
(% of feed)
Biogas production rate 1.54 ± 0.03 1.00 ± 0.02 2.67 ± 0.04 2.00 ± 0.01
(L/L/d)
Methane content (%) 68.40 ± 0.31 63.10 ± 0.58 66.20 ± 0.48 59.42 ± 0.44
Methane production rate 1.05 ± 0.01 0.63 ± 0.01 1.77 ± 0.02 1.19 ± 0.01
(L CH4/L/d)
Methane yield (m3 CH4/kg 0.32 ± 0.01 0.23 ± 0.01 0.32 ± 0.01 0.24 ± 0.01
COD removed)
a
After the feeding.
b
About 3 h after the feeding.

A considerable amount of organic matter was degraded in
both combined and control processes. In Run I at steady state,
the control process achieved removal (%) of 68.15 ± 0.75 for TS,
80.63 ± 0.48 for VS and 78.52 ± 0.24 for COD; the combined
process achieved 92.52 ± 0.35 for TS, 96.31 ± 0.41 for VS and
93.20 ± 0.28 for COD (Table 2). In Run II at steady state, the
increase in OLR from 3.5 to 7 kg COD/m3 d caused higher
concentrations of TS, VS and COD in all reactors. After the
initial period of Run II, the control process showed slightly
decreased removal (%) (63.13 ± 0.39 for TS, 77.58 ± 0.27 for VS
and 71.11 ± 0.65 for COD) compared to Run I at steady state.
But the removal (%) in the combined process (92.54 ± 0.42 for
TS, 96.30 ± 0.28 for VS and 93.51 ± 0.11% for COD) in Run II at
steady state did not differ from those in Run I at steady state.
These results indicate that efficient degradation of separated
(concentrated) anaerobic sludge in R2 significantly increased
the removal efficiency in terms of TS, VS and COD. In addition,
the collaboration between R1 and R2 might provide remark-
able differences in organic removal between combined and
control processes (Miah et al., 2005).
To better understand the reactor performance, organic
acids that are known to be major intermediates were ana-
lysed. Only two organic acids (acetic and propionic acid) were
detected in both R1 and R3; no organic acids were detected in
R2 (Table 2). In Run I at steady state, TOA concentration was
2.86 ± 0.07 g COD/L in R1 (final effluent of combined process)
and 0.89 ± 0.01 g COD/L in R3. With increased OLR, increased
TOA concentration was observed in R1 (3.28 ± 0.01 g COD/L)
and R3 (1.99 ± 0.05 g COD/L). This suggests that HRT of R1 and
R2 were insufficient to allow consumption of all organic acids
in feedstock and produced in the reactors. The TOA concen-
tration was higher in R1 than R3, possibly because HRT was
shorter in R1 than in R3 during the overall digestion (Table S1).
Degradable organic matter recirculated from R2 might also
contribute to the relatively high concentration of TOA in R1.
Based on the organic acid analysis in the reactor, the TVFA/
TA ratio was calculated; this ratio has been used as an
acidification-risk parameter in AD (Kafle and Kim, 2013;
Schievano et al., 2012). In general, TVFA/TA < 0.4 is consid-
ered to be favourable for stable methanogenesis without in-
hibition by acidification (Rinco
n et al., 2008). In this study,
TVFA/TA was >0.8 in R1 in both Runs at steady state, and in R3
during Run II at steady state. This high TVFA/TA ratio is
mainly due to: (1) high concentration of easily-biodegradable
components (i.e., VS and SCOD) and >69 g COD/L of organic
acids in the feedstock (Table 1), and (2) relatively insufficient
retention time (especially R1). Although R1 and R3 showed
high TVFA/TA ratio, both R1 and R3 operated stably
throughout digestion. This observation is consistent with a
recent report that showed stable reactor operation with TVFA/
TA > 0.9 during the co-digestion of apple waste and swine
manure (Kafle and Kim, 2013). Further studies need to be
296 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3
conducted to clarify the relationship between TVFA/TA ratio
and reactor performance during AD. Furthermore, alkalinity
addition may be required to alleviate the possible acidification
and to maintain appropriate pH condition for methanogenesis
when the proposed combined process is operated with OLR
>7.5 kg COD/m3 d.
The biogas production and methane content in both
combined and control processes were monitored throughout
digestion (Table 2). In Run I at steady state, the combined
process achieved 1.54 ± 0.03 L/L/d biogas production rate with
68.40 ± 0.31% methane content and the control process ach-
ieved 1.00 ± 0.02 L/L/d biogas production rate with
63.10 ± 0.58% methane content. In Run II at steady state, the
combined process achieved 2.67 ± 0.04 L/L/d biogas produc-
tion rate with 66.20 ± 0.48% methane content and the control
process achieved 2.00 ± 0.01 L/L/d biogas production rate with
59.42 ± 0.44%; in both cases, the biogas production was
higher, but the methane content lower, than in Run I at
steady state.
Methane production rate (MPR) and methane yield (MY)
were calculated from the data. The combined process ach-
ieved MPR ¼ 1.05 ± 0.01 (Run I at steady state) and 1.77 ± 0.02 L/
L/d (Run II at steady state) with stable MY ¼ 0.32 ± 0.01 m3 CH4/
kg CODremoved; these values were ~1.5 times higher than those
of the control process (MPR ¼ 0.63 ± 0.01 (Run I at steady state)
and 1.19 ± 0.01 L/L/d (Run II at steady state), MY ¼ 0.22e0.25 m3
CH4/kg CODremoved). These findings indicate that combined
process have potential advantages in terms of methane re-
covery as compared to control process. Therefore, it can be
inferred that the additional digestion in R2 of highly-
concentrated solid residue from R1 resulted in remarkable
degradation of organic matter and increased methane
production.
3.2. Quantification of total bacteria and archaea
Typically, microbial population (expressed as 16S rRNA gene
concentration) in biological reactor is strongly related to the
respond to environmental stress (Klappenbach et al., 2000).
Fig. 2 e Changes in 16S rRNA concentration of total bacteria, total archaea and ratio of archaea to total microbes (the sum of
bacteria and archaea) in different Runs I and II.
Therefore, measurement of 16S rRNA gene concentration is
useful during treatment of high-strength wastewater. qPCR
method was used to measure the concentration of 16S rRNA
gene copies of total bacteria and archaea (Fig. 2). In addition, it
is noteworthy to mention that PCR amplicons was not detec-
ted in R2 with archaea specific primer. This implies that
archaea within the solid residue from R1 completely lost their
activity and were rapidly degraded due to high microbial ac-
tivity under thermophilic aerobic conditions.
In spite of the relatively short HRT (5 d), bacterial 16s rRNA
gene concentration in R2 increased from 8.24 (±0.09)  1010 to
1.02 (±0.07)  1012 copies/mL (~12-fold increase) in Run I and
maintained higher population (~1012 copies/mL) throughout
digestion than in both R1 and R3. This difference in bacterial
populations might be due to the different growth character-
istics between aerobic and anaerobic bacteria. This result
corresponds to previous findings that stable and high bacterial
populations were maintained under thermophilic aerobic
conditions (Jang et al., 2014, 2013). Interestingly, R1 and R3
showed similar variation patterns of bacterial population, but
R1 maintained higher bacteria population
(2.21 (±0.07)  1011 copies/mL in Run I and
6.25 (±0.15)  1011 copies/mL in Run II) than did R3
(6.29 (±0.14)  1010 copies/mL in Run I and
2.15 (±0.11)  1011 copies/mL in Run II) throughout the
experiment (Fig. 2).
Similar to bacteria, archaea population in R1 and R3
increased as OLR increased and R1 showed higher archaea
population (2.79 (±0.25)  1010 copies/mL in Run I and
7.69 (±0.33)  1010 copies/mL at Run II) than did R3
(6.90 (±0.06)  109 copies/mL in Run I and
2.35 (±0.07)  1010 copies/mL Run II) throughout digestion.
Additionally, the percentage of archaea to total microbes was
slightly higher in R1 (11 ± 0.58%) than in R3 (9.8 ± 0.12%).
Collectively, the qPCR results demonstrated that the com-
bined process provided more suitable environment for growth
of both bacteria and archaea, thereby resulting in higher MPR
and methane yield as well as organic removal than did the
control process (Table 2).
 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3
3.3. Pyrosequencing data
For in-depth understanding of microbial communities in the
reactors pyrosequencing was used with 16s rRNA-specific
oligonucleotide primers, which usually cover all bacteria and
archaea. Totals of 38002 raw bacterial sequences and 23677
raw archaeal sequences were obtained (Table S3). After
filtering, totals of 36867 (97% of raw sequences) clean bacterial
sequences and 23558 (99% of raw sequences) clean archaeal
sequences were obtained. The average lengths were
473 ± 4.72 bp for bacteria and 514 ± 2.37 bp for archaea; these
lengths are well-matched with those of the hypervariable re-
gions that were targeted. Based on clean sequences, phylo-
types and diversity indices of bacterial and archaeal
communities were calculated (Table S3).
None of rarefaction curves reached saturation phase; this
means that the sequences collected do not fully represent the
microbial community diversity. This phenomenon is
commonly observed during pyrosequencing, probably
because environmental samples are complex and contain rare
species with low abundance (Ashby et al., 2007; Claesson et al.,
2009). Meanwhile, Good's coverage values (estimator of sam-
pling completeness) of all samples represented > 95% of
coverage (Table S3); therefore the diversity and richness in
this study are sufficient to characterize the major phylotypes
in each sample.
3.4. Composition and possible functions of major
bacterial species
Taxonomic classification of clean sequences obtained from
pyrosequencing provides comprehensive insight into the mi-
crobial communities and their possible functions assuming
that closely-affiliated taxa share similar metabolic pathways
and capabilities (Rivie re et al., 2009; Zhang et al., 2010). In this
study, the relative composition of the unclassified sequences
at all taxonomic levels was within typical ranges reported
previously (Lee et al., 2008; Lu et al., 2012; Sundberg et al.,
2013). According to taxonomic classification and calculated
Shannon-Weaver Indices (Table S3), all bacterial samples
showed higher diversity and taxonomic distribution than
those of archaeal samples; this finding is in agreement with
the results of earlier studies of microbial communities in the
full-scale biogas digesters (Regueiro et al., 2012; Sundberg
et al., 2013).
The taxonomic distributions of each bacterial sample were
determined at the phylum and class level (Fig. 3). Unlike
Anaerobic seed, Bacteroidetes was most abundant phylum in
both anaerobic reactors and was detected in all reactors dur-
ing the steady-state condition in Runs I and II. In contrast, the
relative abundances of phyla Chloroflexi (most abundant
phylum in Anaerobic seed) and Proteobacteria (second major
phylum) in both anaerobic reactors decreased significantly in
Runs I and II. The decline of those two phyla in this study may
be due to the difference in characteristics of the substrates, as
recently reported by Sundberg et al. (2013). Also, several
studies mentioned potential function of microorganisms
assigned into the phylum in AD mainly related to hydrolysis of
organic matter and production of acetic acid (Kragelund et al.,
2008; Riviere et al., 2009). Thus, the dominance of phylum
297
Bacteroidetes in both anaerobic reactors indicates that bacteria
of this phylum may have important functions in hydrolysis
and acetogenesis.
Interestingly, phyla distributions were markedly different
between R1 and R3 at Run II. PCoA also demonstrated clear
regional separation (Fig. S2a). In R1, phyla Synergistetes (main
class Synergistia) decreased from 17.82% in Run I to 4.07% in
Run II, Firmicutes (main classes Clostridia and Bacilli) decreased
from 10.71% in Run I to 8.91% in Run II, whereas the phylum
Actinobacteria (main class Actinobacteria) increased from 6.05%
in Run I to 17.55% in Run II. The large proportion of members
of the phylum Actinobacteria is indicative of their contribution
to efficient degradation of complex organic materials and
production of organic acids in R1 (Ventura et al., 2007). In R3,
members of the phyla Synergistetes increased from 18.25% in
Run I to 28.12% in Run II, and those of Firmicutes (main class
Clostridia) increased from 11.29% in Run I to 14.24% in Run II;
whereas the relative abundance of phylum Actinobacteria was
~3.3% in both Runs I and II. Members of the phyla Synergistetes
and Firmicutes commonly appear in AD; they can degrade
proteins, lipids and carbohydrate with hydrolytic enzymes
(Guo et al., 2014; Sundberg et al., 2013). In addition, some of
them can obtain energy by oxidizing organic acids (syntrophic
reaction), especially by fermenting VFAs to H2 and CO2 (Ito
re et al., 2009). Therefore, the predominance
et al., 2011; Rivie
of the phyla Synergistetes and Firmicutes strongly suggests that
syntrophic metabolism is highly activated in R3, especially
during Run II.
It is noteworthy that R2 maintained higher bacterial di-
versity and taxonomic distribution than did both R1 and R3
throughout digestion (Table S3). Also, the bacterial commu-
nity composition of thermophilic aerobic seed differed
greatly from those of Runs I and II, probably due to the dif-
ference in characteristics of substrate and operating param-
eters (e.g., reactor configuration and aeration efficiency), as
recently reported by Piterina et al. (2012). In Run I, abundance
of the phylum Firmicutes (most abundant phylum in ther-
mophilic aerobic seed) decreased to ~17%, then remained
steady during rest of the operating period. It should addi-
tionally be noted that most species of class Clostridia (~5% in
Runs I and II) are obligate anaerobes. The presence of a sig-
nificant proportion of such anaerobic bacteria in TAD has
been reported by several authors (Juteau et al., 2004b; LaPara
et al., 2002) and is probably due to the low oxygen solubility
under thermophilic conditions. The variation patterns of ORP
in R2 (Table 2) are consistent with the existence of an
anaerobic period caused by the step-feeding system, whether
they have metabolic activities in R2 remains unclear. Unlike
phylum Firmicutes, phylum Actinobacteria (mainly class Acti-
nobacteria) was not a major phylum in Seed (3.33%), but
significantly increased to 22.98% in Run I and 24.55% in Run II,
and became the most abundant phylum in R2. Although this
phylum is frequently detected in the ATAD process (never as
the dominant phylum) (Hayes et al., 2011; Juteau et al., 2004b),
this is the first observation that members of phylum Actino-
bacteria emerge as the dominant phylum in TAD. Their pre-
dominance in this study suggests that they were selectively
enriched from the thermophilic aerobic seed and had an
important function related to organic matter removal
throughout digestion.
298 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3

Fig. 3 e Distribution and relative abundance of taxa in bacterial samples based on sequences derived from pyrosequencing.
Inner circle: composition at phylum level; outer circle: composition at class level. Sequences showing with percentage of
reads >1.0% in all samples were grouped into ‘Others’.

Genus-level classification was conducted to further deter-
mine the phylogenetic discrimination and possible functions
of the bacterial members in the reactors (Fig. 5a). Throughout
digestion, both R1 and R3 also showed discernible distinctions
in distribution of sequences at the genus level within phylum
Firmicutes (Fig. 5a). The members assigned into genus Clos-
tridium (belong to class Clostridia), which are syntrophic bac-
teria (Schnürer et al., 1996), were predominant (~8%) in R3, but
relatively uncommon (~1%) in R1. Interestingly, the genus
Geobacillus (belong to class Bacilli) was observed at ~6% of
abundances in R1, and was predominant (~11%) in R2; the
presence of this genus in thermophilic aerobic condition
(mainly produce lytic enzyme) has been reported previously,
but it is uncommon in anaerobic conditions. Other genera
such as Prevotella and Aminobacterium, which are mostly
anaerobic bacteria, were also detected in both R1 and R2. In
particular, R1 and R2 shared a significant proportion (4.09%
and 14.24% in R1; 19.28% and 17.64% in R2 at Runs I and II,
respectively) of one uncharacterized genus Unclassified Acti-
nobacteria throughout digestion. This may in fact be signifi-
cantly important in maintenances of bacterial population and
reactor performances. This was also highly in agreement with
 w a t e r r e s e a r c h 7 3 ( 2 0 1 5 ) 2 9 1 e3 0 3
(belong to order Methanosarcinales) were the two main genera
in R1. Interestingly, during Run II, the relative abundance of
genus Methanosarcina reached up to 95.02%, whereas members
of the genus Methanosaeta were completely inhibited or
washed out in R1. In contrast, in R3, >50% of sequences was
assigned to genus Methanosaeta, whereas members of the
genus Methanosarcina were never detected. Also, Meth-
anobacterium (belong to order Methanobacteriales; 17.58% in Run
I and 16.64% in Run II) and Methanoculleus (belong to order
Methanomicrobiales; 17.11% in Run I and 24.64% in Run II) were
the two main genera in R3. These genera are related to
hydrogenotrophic methanogenesis and are predominant
during AD of food-related wastes (Kim et al., 2014).
Typically, members of genus Methanosarcina can produce
methane by both aceticlastic and hydrogenotrophic meth-
anogenesis pathways (Garcia et al., 2000). Furthermore, in AD
the predominant methanogenic genera change from Meth-
anosaeta to Methanosarcina, as a result of changes in operating
parameters, particularly by increase in VFAs (especially acetic
acid) and decrease in HRT (De Vrieze et al., 2012; McMahon
et al., 2004). The change occurs mainly because these genera
have different growth characteristics. Members of genus
Methanosarcina can achieve stable growth at higher concen-
tration of VFAs and have higher specific growth rates than
genus Methanosaeta (Conklin et al., 2006). Thus, the relatively
high concentration of TVFA (3.07 ± 0.01 g acetic acid/L) (Table
2) and short HRT (13.75 day) (Table S1) during Run II probably
contributed to the predominance of genus Methanosarcina in
R1. Given this information, the predominance of Meth-
anosarcina in the methanogenic community in R1 strongly
indicates that members of the genus probably have important
functions in methanogenesis and may lead to a better per-
formance with stable conditions at low HRT, compared to R3.
4. Conclusions
In this study, a combined MAD (R1)-TAD (R2) process was used
to treat high-strength food wastewater. The specific conclu-
sions can be written as follows:
 The proposed combined MAD (R1)-TAD (R2) process
showed efficient treatment of organic matter removal (i.e.,
COD, TS and VS), and higher methane production than did
the control process (R3).
 Higher populations (based on the concentration of 16s
rRNA gene copy number) of both bacteria and archaea were
maintained in R1 than in R3 during the entire digestion.
 R1 and R2 shared a significant proportion of bacterial
genera (Prevotella, Aminobacterium, Geobacillus and Unclas-
sified Actinobacteria) and the predominant genus of meth-
anogenic archaea shifted from Methanosaeta to
Methanosarcina in R1.
 The qPCR and high-throughput pyrosequencing results
demonstrated that the synergisms between MAD and TAD
improved reactor performance by establishing substantial
microbial communities.
 To clarify the mechanism of collaboration between MAD
and TAD, further studies of the function of shared micro-
organisms should be conducted using other molecular
301
biology technologies (e.g., metagenomics, microarrays,
single cell genomics).
Acknowledgements
This work was conducted under the framework of Research
and Development Program of the Korea Institute of Energy
Research (KIER) (B4-2474-02) and supported by the Advanced
Biomass R&D Center (ABC) of Korea Grant funded by the
Ministry of Education, Science, and Technology (ABC-
2013059453). The research was partially supported by
BK21 þ program through the National Research Foundation of
Korea funded by the Ministry of Education, Science and
Technology, the Manpower Development Program for Marine
Energy funded by Ministry of Land, Transportation and Mari-
time Affairs (MLTM) of Korean government. This research was
also supported by POSCO and the Korea Institute of Energy
Technology Evaluation and Planning (KETEP) grant funded by
the Korea Government Ministry of Knowledge Economy (No.
2012K130y). This research was a part of the project titled
‘Technology Development of Marine Industrial Biomaterials’,
funded by the Ministry of Oceans and Fisheries, Korea.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.watres.2015.01.038.
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