Bioresource Technology 222 (2016) 470–477

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

Performance, kinetics behaviors and microbial community of internal

circulation anaerobic reactor treating wastewater with high organic
loading rate: Role of external hydraulic circulation
Gan Luo, Jun Li, Yan Li ⇑, Zhu Wang, Wen-Tao Li, Ai-Min Li
State Key Laboratory of Pollution Control and Resources Reuse, School of the Environment, Nanjing University, Nanjing 210023, China

h i g h l i g h t s

3

The critical OLR for IC reactor effectively operation was close to 19 kgCOD/m /d.

Withdrawal of external hydraulic circulation led to deterioration of IC reactor.

Substrate metabolic pathway shifted from propionate to hybrid fermentation.

Kinetics of substrate degradation and methane production was studied.

High-throughput sequencing showed the shift of bacterial and archaea community.

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

Article history: Performance of internal circulation anaerobic reactor (IC) treating wastewater at high organic loading
Received 28 July 2016 rate (OLR) and role of external hydraulic circulation were evaluated. When the OLR was increased from
Received in revised form 5 October 2016 2.50 to 18.94 kgCOD/m3/d, COD removal decreased to 85% slightly and methane production increased to
Accepted 6 October 2016
4.49 L/L/d with the upflow velocity of 1.0 m/h resulted from the additional hydraulic circulation.
Available online 13 October 2016
Withdrawal of external hydraulic circulation led to decrease of COD removal to lower than 60% drasti-
cally and methane production by 81%. Accumulation of volatile fatty acids caused decline of pH to below
Keywords:
6.0 and the shift of substrate metabolic pathway to the hybrid fermentation. In addition, both maximum
Internal circulation anaerobic (IC) reactor
Volatile fatty acids (VFA)
methane production rate and maximum substrate degradation rate obtained from mathematical models
Kinetics behavior decreased significantly. Hydrogenotrophic methanogens including Methanobacterium and
Microbial community Methanocorpusculum predominated in the anaerobic sludge and the shift of microbial community was
High-throughput sequencing also observed.
Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction circulation system, IC reactor possesses many traits such as large

Anaerobic digestion has been widely applied for the treatment
of wastewater which characterized by high concentration of
organic matters from industries such as paper-making, food pro-
cessing and pharmacy due to its low cost, little sludge production
and recovery of energy in the form of methane (CH4). During the
past decades, many types of anaerobic reactor have been devel-
oped, including up-flow anaerobic sludge blanket (UASB),
expanded granule sludge bed (EGSB), internal circulation (IC) reac-
tor and so on. IC reactor, which integrates two UASB vertically, is
an excellent representative of the third-generation anaerobic reac-
tors. Due to its unique bilayer structure and gas-driven internal
⇑ Corresponding author.
E-mail address: liyan_0921@126.com (Y. Li).
height-diameter ratio, effective biomass retention and improved
mixing intensity (Cui et al., 2011). Therefore, IC reactor offers a
promising alternative for the industrial wastewater treatment with
high organic loading rate (OLR). Generally, the OLR applied for IC
reactor could be 3–5 times higher than that for UASB reactor due
to the better mass transfer resulted from the internal circulation
of intermixture (Jiang et al., 2014).
Anaerobic digestion consists of a series of microbial processes
including acidogenesis, acetogenesis and methanogenesis which
are operated by acidogenic bacteria, acetogenic bacteria and
methanogenic archaea, respectively. These microorganisms differ
widely in terms of physiology, nutritional needs, growth kinetics,
and sensitivity to environmental conditions. They are interdepen-
dent and interactional in anaerobic digestion and the balance of
these microorganisms makes the whole process of anaerobic

http://dx.doi.org/10.1016/j.biortech.2016.10.023
0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
 G. Luo et al. / Bioresource Technology 222 (2016) 470–477 471

digestion a dynamic equilibrium process. Relatively high methano- Table 1

genic activity of sludge is a precondition for efficient and stable Operation parameters applied in different stages.

pollutant removal and energy recovery, since methanogens that Stages Time (d) Influent COD OLR HRT Vup
are responsible for utilization of acetate and H2/CO2 for methane (mg/L) (kgCOD/m3/d) (d) (m/h)
formation have higher susceptibility to environmental factors I 1–58 5000 2.50 2.00 1.0
and lower substrate metabolism rate (Chen et al., 2008; II 59–81 5000 4.43 1.13 1.0
Hao et al., 2012). For instance, deterioration even failure often III 82–98 5000 7.58 0.66 1.0
IV 99–116 8000 12.12 0.66 1.0
occurred when anaerobic reactors are encountered with high con- V 117–193 10,000 15.15 0.66 1.0
tent of methanogenic inhibitors (Zhao et al., 2014). In addition, over- VI 193–239 12,500 18.94 0.66 1.0
loaded with organic matter or high organic load rate may also break VII 240–271 12,500 18.94 0.66 0.06
the balance, leading to the failure of anaerobic methanogenesis.
Good mass transfer between sludge and substrate plays a key
role in pollutant removal and methane production in anaerobic and 4.0 g/L NaHCO3 as pH buffer was fed by peristaltic pump into
reactor. Higher liquid up-flow velocity in anaerobic reactor con- the reactor from the bottom. Trace elements were added into the
tributes to thorough liquid–solid mixing (Ding and Wang, 2005; synthetic wastewater as described by our previous study
Wang et al., 2015b; Zhang et al., 2009). Although IC reactor can (Liao et al., 2013). The IC reactor was operated for 271 days contin-
treat wastewater with high OLR (Jiang et al., 2014; Xu et al., uously which covers 7 stages (Stage I–VII) according to the opera-
2013), to address the deterioration related with higher OLR, an tion parameters of hydraulic retention time (HRT), influent COD,
additional external hydraulic circulation could be applied for oper- OLR and Vup (Table 1). In Stage I, low COD content (5000 mg/L)
ation of IC reactor to elevate the up-flow velocity for enhanced and long HRT (2.0 d) was selected in favor of startup of the IC reac-
mass transfer, which could achieve higher COD removal rate and tor. Thereafter, considering the treatment capacity of IC reactor,
methane yield (Wang et al., 2014). However, previous studies the HRT was reduced from 2.0 d of Stage I to 0.66 d of Stage III
mainly focused on the improved performance of IC reactor with to elevate the OLR with a fixed influent COD content (5000 mg/
hydraulic circulation (Wang et al., 2014), lack of comprehensive L). To stress the role of external hydraulic circulation, shorter
evaluation on the effect of external circulation in terms of the HRT should be avoided because high influent flow rate also led
kinetics behavior of sludge and microbial community. Further- to an increased upflow velocity. Therefore, in the following stages
more, especially for a highly organic-loaded IC reactor, the influ- (Stage IV – VI), the influent COD concentration was elevated con-
ence of revocatory external hydraulic circulation on the tinuously from 5000 to 12500 mg/L with the fixed HRT of 0.66 d,
performance, consequent dynamic behavior and microbial com- leading to the increasing OLR from 7.58 to 18.94 kgCOD/m3/d.
munity remains unknown. The Vup was controlled by the flow rate of external hydraulic circu-
Therefore, in this study, a lab-scale IC reactor with an external lation. In the last stage (Stage VII) which had the same OLR and
hydraulic circulation was setup to study its performance in terms HRT with Stage VI, the external hydraulic circulation was with-
of COD removal and methane production under the increased drawn to evaluate its role at high OLR. A water bath was employed
OLR. At its tolerance OLR level, external hydraulic circulation was for a constant temperature of 35 ± 1 °C.
withdrawn to study its role on performance of IC reactor. Besides,
batch experiments and mathematical models were applied to elu-
cidate the kinetics of substrate degradation and methane produc- 2.2. Batch experiments and kinetics models
tion of the anaerobic sludge with and without external hydraulic
circulation. High throughput sequencing was also conducted for To evaluate the role of external hydraulic circulation on the
underlying microbiological mechanism of external hydraulic circu- microbial activity and dynamic behavior, anaerobic sludge was
lation on IC reactor. The study provided comprehensive under- extracted from IC reactor on the 227th day (stage VI, B1) and
standing for the IC reactor treating wastewater with high organic 270th day (stage VII, B2) for batch experiments, respectively. About
loading. 50 mL anaerobic sludge with VSS of 18.90 and 52.96 g/L for B1 and
B2 respectively was added into three identical serum bottles
(250 mL) respectively for parallel test. The anaerobic sludge was
2. Materials and methods then replenished to 200 mL with synthetic nutrient solution con-
taining 5000 mg/L COD (glucose) after washed three times. The ini-
2.1. IC reactor set-up tial pH was adjusted to 7.0 ± 0.1 and 2.0 g/L NaHCO3 was
supplemented to buffer the fluctuation of pH. Then, the bottles
A lab-scale IC reactor was established with a working volume of were placed in a constant temperature shaker setting at 35 °C
24L (Supplementary material, Fig. S1). The IC reactor consists of and 100 rpm after purging the oxygen out by nitrogen gas and seal-
five compartments: influent feed and distribution system, fluidized ing. The methane produced was collected and measured by a liquid
bed area, polishing treatment area, three-phase separation area displacement system of 30 g/L NaOH-NaCl solution. Liquid samples
and recirculation system that consisted of a biogas lifting pipe were collected at predetermined time points for the measurement
and a sludge downer-pipe (Habets and de Boerstraat, 1999). An of substrate and volatile fatty acids (VFA). The results were shown
external hydraulic circulation was applied to provide a proper in average values with the standard deviation between the three
up-flow velocity (Vup). Aerobic activated sludge collected from parallels below 10%.
Jiangxinzhou Wastewater Treatment Plant (Nanjing City, China) Three mathematical models to simulate the biogas production
was used as seed sludge with the initial mixed liquor suspended and substrate degradation were used, including the Modified Gom-
solid (SS) and mixed liquor volatile suspended solid (VSS) of pertz function (GM) (Eq. (1)), the Logistic model (LM) (Eq. (2)) and
5200 mg/L 3200 mg/L respectively according to the literature the Transference model (TM) (Eq. (3)). In these three equations, P is
(Kobayashi et al., 2009). represented for the cumulative methane production (mL) or sub-
The wastewater used in this study is synthetic wastewater and strate degradation amount (mg/L), Pm for the methane production
simulated the composition of actual molasses wastewater which is potential (mL/gVSS) or substrate degradation potential (mg/L/
a kind of typical high organic strength wastewater. Synthetic gVSS), Rm for maximum methane production rate (mL/h/gVSS) or
wastewater containing glucose, NH4Cl, KH2PO4 as basic nutriment maximum substrate degradation rate (mg/L/gVSS/h), k for lag time
472 G. Luo et al. / Bioresource Technology 222 (2016) 470–477
for methane production or substrate degradation (h) and t for reac-
tion time (h). Notably, the Pm and Rm was normalized to VSS after
modeling.
  
eðk  tÞ
P ¼ Pm exp  exp Rm þ1
Pm
Pm
P¼
1 þ exp ð4Rm ðk  tÞ=Pm þ 2Þ
  
Rm ð t  k Þ
P ¼ Pm ð1  exp 
Pm
2.3. DNA extraction, Polymerase chain reaction (PCR) and sequencing
Anaerobic sludge was collected from the IC reactor at 227th day
(stage VI, S1) and 270th day (stage VII, S2). DNA was extracted by
using FastDNAÒ SPIN Kit for Soil (MP Biomedicals, USA) and ampli-
fied with the primers 27F/338R for bacteria (Ravel et al., 2011) and
524F_10ext/Arch958R for archaea (Pires et al., 2012), respectively
(Table S1). The PCR was conducted with a reaction system of
20 lL containing 0.8 lL of each primers, 2 lL 10buffer, 2 lL
dNTPs, 0.2 lL rTaq Polymerase, 0.2 lL BSA, 10 ng template DNA
and ddH2O. The amplification of DNA was performed under the
following procedures: 95 °C for 3 min, 27 cycles for bacteria (35
cycles for archaea) of 95 °C for 30 s, 55 °C for bacteria (52 °C for
archaea) for 30 s, 72 °C for 45 s, and a final extension at 72 °C for
10 min. High-throughput sequencing was conducted at Majorbio
Bio-pharm Technology Co., Ltd (Shanghai, China) on Illumina
Mi-seq platform. Corresponding sequence with specific barcodes
inside each sample was picked out by using Mothur (http://
www.mothur.org/). Then data was denoised as described by
previous study (Guo et al., 2015). After noise reduction, 32243
reads for bacteria and 31587 reads for archaea were drawn ran-
domly from the samples for comparison at the same sequencing
depth. Downstream taxonomic assignment was performed by
Ribosomal Database Project (RDP) (http://rdp.cme.msu.edu/)
Classifier with confidence threshold of 50%. Mothur was applied
for calculation of richness and diversity indices including opera-
tional taxonomic units (OTUs), Chaos and Shannon Index at 3%
cutoff level.
2.4. Analytical methods
SS, VSS and COD were determined by standard methods. VFA
including acetate, propionate, butyrate and pentanoate were
detected with gas chromatography (7890A, Agilent, USA) equipped
with hydrogen flame ionization detector. A wet gas flow meter
(LMF-1, Changchun Automobile Filter, China) was used for the
measurement of biogas production in each operational stage of
the IC reactor. The composition of biogas (CH4, CO2, H2) was eval-
uated by using a gas chromatography (SP7820, Rainbow Chemical
Instrument Co., Ltd, China) with a thermal conductivity detector.
Glucose content in batch experiments was analyzed with the mod-
ified Anthrone method by using glucose as the standard substance.
3. Results and discussion
3.1. Overall performance
Fig. 1 shows the performance of IC reactor in terms of COD
removal at varied OLR. During stage I, about 44 days were required
for the COD removal reaching up to 90%. The maximum COD
removal of 96.4% was observed on the 56th day, indicating setup
of IC reactor. Cultivation stage shorter than 30 days for IC reactor
ð1Þ
ð2Þ
ð3Þ
Fig. 1. Effluent COD content (left axis) and its removal rate (right axis) during the
operation stages.
was reported previously using granule or flocculent sludge as
inoculum (Wang et al., 2014; Xu et al., 2013). Longer startup stage
in this study could be attributed to the unfavorable aerobic seed
sludge. In the following stages, a transient decline of COD removal
efficiency could be noticed after each OLR shock load. Higher OLR
applied led to a prolonged time for recovery of COD removal from
the shock load. The effluent COD increased rapidly to 5452 mg/L
after raising OLR from 15.15 to 18.94 kgCOD/m3/d and then grad-
ually decreased to below 2000 mg/L with the adaption of the
sludge microorganisms. Stable COD removal efficiency over 90%
was obtained for IC reactor at OLR ranging from 2.50 to
12.12 kgCOD/m3/d, while the COD removal decreased to around
85% as the OLR increased to 15.15 and 18.94 kgCOD/m3/d. Besides,
the effluent pH decreased from above 7.0 initially to around
6.5–6.8 at stage VI (Fig. S2). Similar phenomena were reported pre-
viously. Shortening the HRT from 19.2 h to 2.5 h with fixed influent
COD content, which led to OLR increasing from 2.5 to 19.5 kgCOD/
m3/d, the IC reactor treating brewery wastewater could remove
over 80% COD. However, further decrease of HRT to elevate OLR
to 21.5 kgCOD/m3/d led to acidification of IC reactor featured by
the decreased COD removal to 70% and decline of effluent pH to
5.0 (Xu et al., 2013). For a UASB treating a mixture of VFA and alco-
hol, elevating OLR from 12.9 to 15.3 kgCOD/m3/d by reducing HRT
from 0.24 d to 0.20 d led to an obvious decrease of COD removal
from 96% to 73% (Han et al., 2005). Thus, the tolerant OLR level
for IC reactor operation could be identified to be close 19 kgCOD/
m3/d, a value higher than that of UASB.
Fig. 2 shows the average volumetric production rate of biogas
and methane in each stage. During the stages I–III, with the
increasing OLR from 2.50 to 7.58 kgCOD/m3/d by shortening HRT
from 2 d to 0.66 d, the methane production rate (MPR) increased
from 0.44 to 1.88 L/L/d and the biogas production rate (BPR)
increased from 0.83 to 2.58 L/L/d. During stage IV–VI with a fixed
HRT, further increase of MPR to 4.49 L/L/d as well as BPR to
7.58 L/L/d were observed due to the gradually increased influent
COD to 12500 mg/L which led to the elevated OLR to
18.94 kgCOD/m3/d. A positive linearly correlation between MPR
or BPR and OLR could be observed (R2 = 0.98). Furthermore, the
proportion of methane in biogas was ranging from 50% to 60% in
Stage I to VI. Previous study found that the MPR increased from
0.42 to 0.64 L/L/d when the elevated OLR from 10.2 to 26 kgCOD/
m3/d was employed for an up-flow multistage anaerobic reactor
(Sun et al., 2012). Similarly, the BPR also exhibited a linearly
positive correlation with the OLR ranging from 1 to 8 kgCOD/m3/d
applied for a UASB reactor treating starch wastewater (Lu et al.,
 G. Luo et al. / Bioresource Technology 222 (2016) 470–477
Fig. 2. Production rate of methane and biogas in each stage.
2015). Thus, elevation of OLR levels within a specific tolerance
would lead to an enhanced methane and biogas yield.
The concentration of VFA in effluent is depicted in Fig. 3.
Rapid accumulation of acetic, propionic and butyrate acid up to
4100 mg/L in total could be observed in the initial two weeks.
Relatively low methane production activity of aerobic sludge in
anoxic condition might explained the VFA accumulation and worse
performance of the IC reactor in the earlier days (Gray et al., 2002).
At the OLR lower than 12.12 kgCOD/m3/d, except the momentary
increase of acetic and propionic acid after a shock organic load,
total VFA (TVFA) below 400 mg/L was detected in steady phase
of stage I to IV with butyrate and pentanoate lower than 50 mg/L.
Increasing OLR from 15.15 to 18.94 kgCOD/m3/d led to a short-
term accumulation of butyrate and pentanoate up to 477 mg/L
and 572 mg/L respectively, both of which decreased to the level
below 100 mg/L after adapted while the content of acetic and pro-
pionic acid both maintained above 500 mg/L. Gradual accumula-
tion of TVFA up to 1500 mg/L with elevated OLR to 18.94 kgCOD/
m3/d led to a decrease of pH to 6.5–6.8 (Fig. S2), unfavorable pH
to methanogens. Besides, the ratio of propionate to acetate (P/A)
as a critical parameter of anaerobic reactor operation also pre-
sented an uptrend to around 1.0 at stage V and to nearly 1.4 at
stage VI (Fig. S3). A value of P/A below 1.4 was recommended for
stable operation of anaerobic reactors (Ahring et al., 1995). Thus,
a critical OLR level of approximately 19 kgCOD/m3/d for IC reactor
in this study was assured again.
To study its role under high OLR level, external hydraulic
circulation was withdrawn in the stage VII, which led to the Vup
Fig. 3. Effluent content of VFA: orange for TVFA, black for acetate, blue for
propionate, green for butyrate and red for pentanoate. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
473
decreasing from 1.0 m/h to nearly 0.06 m/h resulted from the flow
rate of influent. Consequently, the performance of the IC reactor
declined and a dramatic increase of effluent COD from 1959 mg/L
to 6234 mg/L was observed (Fig. 1). With the adaption of the
microorganism, their activity recovered partly and the effluent
COD gradually decreased and maintained over 5000 mg/L after-
wards. Meanwhile, the MPR dropped to 0.86 L/L/d remarkably
(Fig. 2), accompanied by the drop of methane content in biogas
to 16.2% and the increase of carbon dioxide proportion to 77.4%.
In comparison, the reduction of MPR (3.63 L/L/d) after withdrawal
of external hydraulic circulation was higher than the loss of BPR
(2.15 L/L/d), which could be attributed to that more CO2
(1.59 L/L/d) was produced and entered into the biogas. Further-
more, rapid accumulation of TVFA up to 4538 mg/L, of which pro-
pionic, butyrate and valeric acid increased to 1266, 831 and
1263 mg/L respectively was observed in ten days while acetic acid
maintained at around 1000 mg/L (Fig. 3). Consistent with the accu-
mulation of VFA, the effluent pH decreased from 6.5–6.8 to 5.9
(Fig. S2). Hence it can be concluded that deterioration or acidifica-
tion occurred in the IC reactor at high OLR without external
hydraulic circulation. Reinforced mass transfer in anaerobic system
by hydraulic circulation has been proved previously (Ding and
Wang, 2005; Wang et al., 2015b). It could be speculated that the
utilization of CO2/H2 for methane formation was limited in stage
VII due to the remarkable increase of CO2 proportion in biogas.
High dissolved amount of CO2 in sludge blanket led to the descent
of pH which inhibited the methane formation in turn (Zhang et al.,
2009). Effective elimination of H2 by methanogens plays an essen-
tial role in the conversion of propionic and butyrate acid to acetic
acid (Morris et al., 2013). High hydrogen pressure inhibited the
formation of acetic acid from syntrophy oxidization of other
short-chain fatty acids. Thus, external hydraulic circulation not
only improves the mass transfer but also accelerates the release
even utilization of gaseous products such as CO2 and H2, contribut-
ing to a favorable environment for anaerobic microorganism.
3.2. Substrate degradation and methane production in batch
experiments
Batch experiments were conducted with anaerobic sludge at
stage VI (B1) and stage VII (B2) to study the behavior of substrate
degradation and methane production under high OLR and evaluate
the role of external circulation on the activity of anaerobic sludge.
Fig. 4 shows the methane production, substrate degradation and
VFA content versus reaction time. A linear growth of methane yield
from 0 to 10 h in B1 and 2 to 7 h in B2 was obtained with the
correlation coefficient R2 both higher than 0.98 (Fig. 4). Specific
methanogenic activities (SMA) of anaerobic sludge calculated
based on the slope of linear region were 0.624 and
0.396 g CODCH4 =gVSS=d, corresponding to 270.19 and 171.47 mL
CH4/gVSS/d for B1 and B2 respectively. A similar SMA value of
210 mL CH4/gVSS/d was reported by Xu et al. (2013) at OLR of
19.5 kgCOD/m3/d. In this study, a decrease of SMA by 37.0% was
obtained after withdrawal of external hydraulic circulation. How-
ever, the MPR in stage VII was 80.8% lower than that in stage VI.
Lower loss of SMA compared with MPR might be ascribed to the
preferable pH value and better mass transfer in batch experiment.
It should also be noticed from Fig. 4 that ten hours were required
for almost complete uptake of substrate in B1 while six hours
needed in B2 due to the higher content of biomass applied in B2.
The degradation of substrate was accompanied with production
of VFA as the key intermediates of anaerobic digestion. Linear
growth of TVFA up to 1743 and 2308 mg/L for B1 and B2 respec-
tively within the initial 12 h was obtained with the R2 nearly
0.97 (Fig. 4). The TVFA both maintained at 2400–2800 mg/L after
474 G. Luo et al. / Bioresource Technology 222 (2016) 470–477
Fig. 4. Methane production, substrate degradation and VFA content versus reaction time in batch experiments.
fermentation for 18 h. Accumulation of four types of short-chain
volatile fatty acids including acetate, propionate, butyrate and
pentanoate were detected with no ethanol measured. In B1, the
content of acetate, propionate, butyrate and pentanoate increased
to 843, 594, 398 and 611 mg/L respectively within 16 h and
remained at a close level afterwards (Fig. 4). Thus, an inflection
point of VFA accumulation could be identified to be around 16 h,
a value approaching to the HRT applied in stage VI (Table 1).
Besides, propionate and pentanoate occupied about 45–50% in
total of TVFA with a percentage of butyrate below 18% (Fig. S4).
Pentanoate could be converted into propionate and acetate
through anaerobic degradation. Thus, a fermentation process based
mainly on propionate with a butyrate-type supplement could be
confirmed in B1 or stage VI. However, in B2, the ratio of butyrate
to TVFA was maintained at 23–30% with a close proportion of pro-
pionate and pentanoate at 25–35% altogether after fermentation
for 6 h (Fig. S4). Thus, hybrid fermentation on the basis of butyrate
and propionate equally was demonstrated in B2 or stage VII. Fur-
thermore, the inflection point of VFA variation appeared in advance
at the 12th hour at which point the methane production almost
halted (Fig. 4). Butyrate and acetate were dominant products of
glucose fermentation at acidic condition (Tamis et al., 2015;
Zoetemeyer et al., 1982). A higher propionate yield of glucose fer-
mentation was obtained at pH greater than or equal to 7.0 (Fang
and Liu, 2002; Horiuchi et al., 2002). The anaerobic sludge applied
in B2 was collected at stage VII in which the effluent pH was below
6.0. Thus, decrease of pH resulted from accumulation of VFA due to
the withdrawal of external circulation might account for the
shifted metabolic pathway of glucose.
3.3. Kinetics and modeling
Three mathematical models were employed to obtain the
kinetic parameters. Fig. 4 shows the models matching the biogas
production and substrate degradation in B1 and B2. The estimated
values of parameters obtained from the models were listed in
Table 2. All the models exhibited a good fitting with the R2 both
above 0.94 (Table 2).
For batch test B1, TM could best describe the biogas production
and substrate degradation due to R2 both higher than 0.99. The Pm
and Rm gained with the LM was similar to the values with GM, both
lower than the values with TM. Interestingly, the maximum value
of k for biogas production and the minimum value of k for sub-
strate degradation both were obtained with the TM fitting. The
estimated parameters of Pm, Rm and k with TM were 182.5 mL/
gVSS, 16.8 mL/gVSS/h and 0.231 h respectively for biogas produc-
tion, and 3981.6 mg/L/gVSS, 1266.8 mg/L/gVSS/h and 0.027 h
respectively for substrate degradation. Similar results were
reported previously that the best agreement and highest value of
the three parameters were both achieved with the TM fitting the
biogas production by anaerobic digestion of grass (Li et al., 2012).
Besides, the TM also fit the biogas production well from anaerobic
digestion of sewage sludge (Donoso-Bravo et al., 2010). In this
study, the TM not only agreed with the biogas production but also
well described the substrate degradation of anaerobic sludge under
high OLR.
For batch test B2, however, the best fit with GM for biogas pro-
duction and substrate degradation was observed with the value of
R2 both above 0.99. In addition, the TM had the worst accordance
for both biogas production and substrate degradation. Likewise,
the values of the two parameters Pm and Rm with the TM fitting
were higher than the close values gained with the other two mod-
els. The predicated values of Pm and Rm acquired by the GM mod-
eling were 46.71 mL/gVSS, 7.29 mL/h/gVSS respectively for biogas
production, lower than those obtained with B1 by 72.0% and
34.9% respectively. The loss of Rm was close to the decrease of
SMA (37.0%). For substrate degradation, Pm and Rm of the GM
fitting were 1462.0 mg/L/gVSS and 448.9 mg/L/gVSS/h respec-
tively, less than those of B1 by 62.7% and 43.0% respectively. On
the contrary, the lag time k for biogas production and substrate
degradation in B2 was far longer than that in B1. Thus, lower value
of Pm and Rm, as well as the longer lag time of anaerobic sludge at
 G. Luo et al. / Bioresource Technology 222 (2016) 470–477
Table 2
Parameters obtained with the three models for methane production and substrate degradation.
Model Methane Production
2
Pm (mL/gVSS) Rm (mL/h/gVSS) k (h) R Pm (mg/L/gVSS)
B1 LM 162.7 10.8 0.008 0.977
GM 167.0 11.2 -0.126 0.991
TM 182.5 16.8 0.231 0.998
B2 LM 46.0 7.3 2.144 0.978
GM 46.7 7.3 1.970 0.990
TM 51.3 7.6 0.898 0.948
stage VII indicated the decreased activity of sludge after deteriora-
tion of the reactor. In previous studies, the modified Gompertz
model was usually used for simulation of hydrogen production
(Pan et al., 2008). Successful application of the GM for evaluation
of inactivation on anaerobic methane production by heavy metals
has also been reported by Altas (2009). According to the equation
of the GM, when the k is ignorable and t = 0, P converge to
Pm/exp(e), which is a positive value. While the k > 0 and t = 0, P
converge to 0 (Donoso-Bravo et al., 2010). Consequently, this equa-
tion exhibited better fitting for experiments data in B2 due to the
longer lag phase of sludge at stage VII (Table 2). Thus, this study
revealed that the modified Gompertz model also could be applied
to estimate methane production and substrate degradation by
anaerobic sludge at acidifying state.
3.4. Microbial community structure
In this study, high throughput sequencing was conducted to
elucidate the effect of external hydraulic circulation on the micro-
bial community including bacteria and archaea in IC reactor at high
OLR.
For bacteria, the value of OTU, Chao and Shannon at 3% cutoff
level was 791, 2964 and 2.89 respectively for S1, and increased
to 899, 3558 and 3.29 respectively for S2, indicating that with-
drawal of external hydraulic circulation led to an increase in rich-
ness and diversity. At phylum level, Firmicutes, Thermotogae,
Bacteroidetes and Proteobacteria were detected with abundance
above 1% in all the two samples (Fig. S5). In S1, the phylum
Firmicutes predominated absolutely since its abundance reached
up to 82.5% with proportion of other phyla below 6%. However,
the phylum Proteobacteria, Firmicutes and Bacteroidetes were dom-
inant in S2 with abundance of 51.7%, 32.8% and 10.5% respectively.
Besides, proportion of Thermotogae decreased from 5.2% in S1 to
1.5% in S2. Dominance of Proteobacteria, Firmicutes and Bacteroide-
tes in simultaneous denitrification and methanogenesis granular
was found in previous report (Yi et al., 2016). Proteobacteria was
common in wastewater treatment plant (Zhang et al., 2012) and
also be reported to be dominated in EGSB reactor treating high-
nitrate wastewater (Liao et al., 2013). Some species belonging to
the Firmicutes and Thermotogae are known as syntrophic acetate-
oxidizing bacteria (Hao et al., 2011). In addition, the order Clostridi-
ales with abundance of 1.7% in S1 and 1.1% in S2 was detected, of
which some bacteria were reported to be butyrate and propionate
oxidizer (Muller et al., 2010). Decreasing abundance of Clostridiales
in S2 may be responsible for the accumulation of propionate and
butyrate at stage VII. At genus level, the Streptococcus had highest
abundance of 76.4% in S1 (Fig. 5), but decreased to 19.1% in S2
remarkably. Streptococcus plays an important role in an anaerobic
system under short HRT or high upflow velocity (Hung et al.,
2007). Badiei et al. (2012) reported that nearly 50% of the isolates
present were identified as Streptococcus in an anaerobic hydrogen
producing reactor. In comparison, the abundance of Streptococcus
(76.4%) in stage VI was relatively high, which might be related with
the short HRT and high upflow velocity applied in stage VI in this
475
Substrate Degradation
Rm (mg/L/h/gVSS) k (h) R2
3892.5 770.8 0.130 0.969
3918.3 787.2 0.033 0.969
3981.6 1226.8 0.027 0.995
1458.3 418.0 0.950 0.997
1462.0 448.7 0.940 0.9996
1489.4 506.8 0.133 0.948
study. Streptococcus, belonging to the order Lactobacillales, could
produce lactic acid as the major metabolic end product of carbohy-
drate fermentation (Pfeiler and Klaenhammer, 2007). Streptococcus
was reported to be able to ferment glucose for acid formation and
its acid production activity decreased at lower pH (de Soet et al.,
2000). In this study, decreased abundance of Streptococcus in stage
VII (19.1%) might be mainly due to the decline of pH resulting from
withdrawal of the external circulation which inhibited the activity
of Streptococcus. Further from Fig. 5, the Raoultella with rising
abundance from 1.4% in S1 to 35.8% in S2 had replaced the Strepto-
coccus as the most dominant genus in S2. Besides, abundance of
Pilibacter and Aeromonas also increased to 8.7% and 5.8% in S2
respectively. Raoultella could serve as potential H2 producer from
anaerobic glucose fermentation with acetate as main liquid end
product (Marone et al., 2014) and was detected in the glucose-
fed sludge (Sun et al., 2015).
For archaea community, the number of OTU, Chao and Shannon
for S1 was 1209, 3479 and 4.7 respectively, while 1202, 4751 and
4.6 respectively for S2. The phylum Euryarchaeota occupied over
99% of total archaea in S1 and S2. For methanogenic archaea, 12
genera of methanogens was detected in S1 while 8 genera in S2.
Among these genera, Methanobacterium predominated in S1
(56.8%) and S2 (76.3%), followed by Methanocorpusculum with
abundance of 36.7% in S1 and 20.7% in S2 (Fig. 5). These two genera
were both reported to be hydrogenotrophic methanogens (Yi et al.,
2016). Enrichment of Methanobacterium in S2 might be due to its
stronger tolerance to wide range of pH values (Jablonski et al.,
2015). Hydrogenotrophic methanogens belonging to the order of
Methanobacteriales could still exist when the anaerobic reactor
running unstably at high OLR with the pH value below 5 (Kundu
et al., 2013). Two genera of acetoclastic methanogen detected in
this study were Methanothrix and Methanosarcina with total
abundance of 1.1% in S1 and 0.6% in S2. Declining proportion of
acetoclastic methanogen might be attributed to the unsuitable
pH and inhibition of high amount of acetate (Kundu et al., 2013).
Fig. 5. Relative abundance of archaea (A) and bacteria (B) genera.
476 G. Luo et al. / Bioresource Technology 222 (2016) 470–477
Thus, absolute predominance of hydrogen-utilizing methane pro-
ducer over acetoclastic methanogen indicated that the methane
produced from H2 consumption played an important role in the
formation of methane, consistent with the presence of potential
syntrophic acetate-oxidizing bacteria in bacteria phylum Firmi-
cutes and Thermotogae. Hydrogenotrophic Methanobacterium was
reported to dominate in an EGSB reactor treating pre-hydrolyzed
pig manure when OLR over 10 kgCOD/m3/d was applied
(Wang et al., 2015a). Based on the above results of bacteria and
archaea community, significant shift of microbial community was
observed as the deterioration of IC reactor by withdrawal of
external hydraulic circulation.
4. Conclusions
Performance, kinetics and microbial community in an IC reactor
with high OLR and the role of external hydraulic circulation were
investigated. At the OLR high to 18.94 kgCOD/m3/d, IC reactor
could remove 85% COD with 4.49 L/L/d methane produced. With-
drawal of external circulation led to decreasing and unstable
COD removal below 60%, indicating deterioration or acidification
of the reactor. Substrate metabolic pathway shifted to the hybrid
fermentation. Modified Gompertz instead of Transference model
could best describe the methane production and substrate degra-
dation in the acidification stage. Withdrawal of external circulation
caused the genera of Methanobacterium and Methanocorpusculum
dominated in archaea.
Acknowledgements
We gratefully acknowledge generous support provided by
National Natural Science Foundation of China (51378251 and
51408298), the Joint Innovation Project for Production-Study-
Research in Jiangsu Province (BY2015060-01), and Nanjing
University & Yancheng Academy of Environmental Protection
Technology and Engineering (NYHBKFKT-2014-09), China.
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.2016.10.
023.
References
Ahring, B.K., Sandberg, M., Angelidaki, I., 1995. Volatile fatty-acids as indicators of
process imbalance is anaerobic digesters. Appl. Microbiol. Biotechnol. 43 (3),
559–565.
Altas, L., 2009. Inhibitory effect of heavy metals on methane-producing anaerobic
granular sludge. J. Hazard. Mater. 162 (2–3), 1551–1556.
Badiei, M., Jahim, J.M., Anuar, N., Abdullah, S.R.S., Su, L.S., Kamaruzzaman, M.A.,
2012. Microbial community analysis of mixed anaerobic microflora in
suspended sludge of ASBR producing hydrogen from palm oil mill effluent.
Int. J. Hydrogen Energy 37 (4), 3169–3176.
Chen, Y., Cheng, J.J., Creamer, K.S., 2008. Inhibition of anaerobic digestion process: a
review. Bioresour. Technol. 99 (10), 4044–4064.
Cui, P., Zhou, X., Zhang, Y., 2011. The feasibility study of cotton pulp wastewater
treatment with IC anaerobic reactor. Proc. Environ. Sci. 11 (1), 686–692.
De Soet, J.J., Nyvad, B., Kilian, M., 2000. Strain-related acid production by oral
Streptococci. Caries Res. 34 (6), 486–490.
Ding, J.N., Wang, D.Z., 2005. Influence of external circulation on sludge
characteristics during start- up of internal circulation reactor. J. Cent. South
Univ. Technol. 12 (4), 425–429.
Donoso-Bravo, A., Pérez-Elvira, S.I., Fdz-Polanco, F., 2010. Application of simplified
models for anaerobic biodegradability tests. Evaluation of pre-treatment
processes. Chem. Eng. J. 160 (2), 607–614.
Fang, H.H.P., Liu, H., 2002. Effect of pH on hydrogen production from glucose by a
mixed culture. Bioresour. Technol. 82 (1), 87–93.
Gray, N.D., Miskin, I.P., Kornilova, O., Curtis, T.P., Head, I.M., 2002. Occurrence and
activity of Archaea in aerated activated sludge wastewater treatment plants.
Environ. Microbiol. 4 (3), 158–168.
Guo, X., Miao, Y., Wu, B., Ye, L., Yu, H., Liu, S., Zhang, X.X., 2015. Correlation between
microbial community structure and biofouling as determined by analysis of
microbial community dynamics. Bioresour. Technol. 197, 99–105.
Habets, L.H.A., de Boerstraat, T., 1999. Introduction of the IC Reactor in the Paper
Industry. The Netherlands, Technical report, Pqques BV.
Han, S.K., Kim, S.H., Shin, H.S., 2005. UASB treatment of wastewater with VFA and
alcohol generated during hydrogen fermentation of food waste. Process
Biochem. 40 (8), 2897–2905.
Hao, L.P., Lu, F., He, P.J., Li, L., Shao, L.M., 2011. Predominant contribution of
syntrophic acetate oxidation to thermophilic methane formation at high acetate
concentrations. Environ. Sci. Technol. 45 (2), 508–513.
Hao, L.P., Lu, F., Li, L., Shao, L.M., He, P.J., 2012. Shift of pathways during initiation of
thermophilic methanogenesis at different initial pH. Bioresour. Technol. 126
(12), 418–424.
Horiuchi, J.I., Shimizu, T., Tada, K., Kanno, T., Kobayashi, M., 2002. Selective
production of organic acids in anaerobic acid reactor by pH control. Bioresour.
Technol. 82 (3), 209–213.
Hung, C.H., Lee, K.S., Cheng, L.H., Huang, Y.H., Lin, P.J., Chang, J.S., 2007. Quantitative
analysis of a high-rate hydrogen-producing microbial community in anaerobic
agitated granular sludge bed bioreactors using glucose as substrate. Appl.
Microbiol. Biotechnol. 75 (3), 693–701.
Jablonski, S., Rodowicz, P., Lukaszewicz, M., 2015. Methanogenic archaea database
containing physiological and biochemical characteristics. Int. J. Syst. Evol.
Microbiol. 65, 1360–1368.
Jiang, J., Wu, J., Zhang, J., Poncin, S., Li, H.Z., 2014. Multiscale hydrodynamic
investigation to intensify the biogas production in upflow anaerobic reactors.
Bioresour. Technol. 155 (4), 1–7.
Kobayashi, T., Yasuda, D., Li, Y.Y., Kubota, K., Harada, H., Yu, H.Q., 2009.
Characterization of start-up performance and archaeal community shifts
during anaerobic self-degradation of waste-activated sludge. Bioresour.
Technol. 100 (21), 4981–4988.
Kundu, K., Sharma, S., Sreekrishnan, T.R., 2013. Changes in microbial communities
in a hybrid anaerobic reactor with organic loading rate and temperature.
Bioresour. Technol. 129 (2), 538–547.
Li, L.H., Kong, X.Y., Yang, F.Y., Li, D., Yuan, Z.H., Sun, Y.M., 2012. Biogas production
potential and kinetics of microwave and conventional thermal pretreatment of
grass. Appl. Biochem. Biotechnol. 166 (5), 1183–1191.
Liao, R.H., Shen, K., Li, A.M., Shi, P., Li, Y., Shin, Q.Q., Wang, Z., 2013. High-nitrate
wastewater treatment in an expanded granular sludge bed reactor and
microbial diversity using 454 pyrosequencing analysis. Bioresour. Technol.
134 (4), 190–197.
Lu, X., Zhen, G., Estrada, A.L., Chen, M., Ni, J., Hojo, T., Kubota, K., Li, Y.Y., 2015.
Operation performance and granule characterization of upflow anaerobic
sludge blanket (UASB) reactor treating wastewater with starch as the sole
carbon source. Bioresour. Technol. 180, 264–273.
Marone, A., Izzo, G., Mentuccia, L., Massini, G., Paganin, P., Rosa, S., Varrone, C.,
Signorini, A., 2014. Vegetable waste as substrate and source of suitable
microflora for bio-hydrogen production. Renew. Energy 68 (68), 6–13.
Morris, B.E.L., Henneberger, R., Huber, H., Moissl-Eichinger, C., 2013. Microbial
syntrophy: interaction for the common good. FEMS Microbiol. Rev. 37 (3), 384–
406.
Muller, N., Worm, P., Schink, B., Stams, A.J.M., Plugge, C.M., 2010. Syntrophic
butyrate and propionate oxidation processes: from genomes to reaction
mechanisms. Environ. Microbiol. Rep. 2 (4), 489–499.
Pan, C.M., Fan, Y.T., Xing, Y., Hou, H.W., Zhang, M.L., 2008. Statistical optimization of
process parameters on biohydrogen production from glucose by Clostridium sp.
Fanp2. Bioresour. Technol. 99 (8), 3146–3154.
Pfeiler, E.A., Klaenhammer, T.R., 2007. The genomics of lactic acid bacteria. Trends
Microbiol. 15 (12), 546–553.
Pires, A.C.C., Cleary, D.F.R., Almeida, A., Cunha, A., Dealtry, S., Mendonca-Hagler, L.C.S.,
Smalla, K., Gomes, N.C.M., 2012. Denaturing gradient gel electrophoresis and
barcoded pyrosequencing reveal unprecedented Archaeal diversity in mangrove
sediment and rhizosphere samples. Appl. Environ. Microbiol. 78 (16), 5520–5528.
Ravel, J., Gajer, P., Abdo, Z., Schneider, G.M., Koenig, S.S.K., McCulle, S.L., Karlebach,
S., Gorle, R., Russell, J., Tacket, C.O., Brotman, R.M., Davis, C.C., Ault, K., Peralta, L.,
Forney, L.J., 2011. Vaginal microbiome of reproductive-age women. Proc. Natl.
Acad. Sci. U.S.A. 108, 4680–4687.
Sun, L., Wan, S.G., Yu, Z.B., Wang, Y.H., Wang, S.F., 2012. Anaerobic biological
treatment of high strength cassava starch wastewater in a new type up-flow
multistage anaerobic reactor. Bioresour. Technol. 104, 280–288.
Sun, Q., Li, A.J., Li, M.X., Hou, B.L., 2015. Effect of pH on biodiesel production and the
microbial structure of glucose-fed activated sludge. Int. Biodeter. Biodegr. 104,
224–230.
Tamis, J., Joosse, B.M., van Loosdrecht, M.C.M., Kleerebezem, R., 2015. High-rate
volatile fatty acid (VFA) production by a granular sludge process at low pH.
Biotechnol. Bioeng. 112 (11), 2248–2255.
Wang, H.Y., Tao, Y., Gao, D.W., Liu, G., Chen, C.H., Ren, N.Q., van Lier, J.B., de Kreuk,
M., 2015a. Microbial population dynamics in response to increasing loadings of
pre-hydrolyzed pig manure in an expanded granular sludge bed. Water Res. 87,
29–37.
Wang, J., Xu, W., Yan, J., Yu, J., 2014. Study on the flow characteristics and the
wastewater treatment performance in modified internal circulation reactor.
Chemosphere 117, 631–637.
Wang, J., Yan, J., Xu, W., 2015b. Treatment of dyeing wastewater by MIC anaerobic
reactor. Biochem. Eng. J. 101, 179–184.
 G. Luo et al. / Bioresource Technology 222 (2016) 470–477 477

Xu, F., Miao, H.F., Huang, Z.X., Ren, H.Y., Zhao, M.X., Ruan, W.Q., 2013. Performance
and dynamic characteristics of microbial communities in an internal circulation
reactor for treating brewery wastewater. Environ. Technol. 34 (20), 2881–2888.
Yi, X.H., Wan, J.Q., Ma, Y.W., Wang, Y., 2016. Characteristics and dominant microbial
community structure of granular sludge under the simultaneous denitrification
and methanogenesis process. Biochem. Eng. J. 107, 66–74.
Zhang, T., Shao, M.F., Ye, L., 2012. 454 Pyrosequencing reveals bacterial diversity of
activated sludge from 14 sewage treatment plants. ISME J. 6 (6), 1137–1147.
Zhang, Y., Ma, Y., Quan, X., Jing, Y., Dai, S., 2009. Rapid startup of a hybrid UASB-AFF
reactor using bi-circulation. Chem. Eng. J. 155 (1–2), 266–271.
Zhao, Z., Zhang, Y., Chen, S., Quan, X., Yu, Q., 2014. Bioelectrochemical enhancement
of anaerobic methanogenesis for high organic load rate wastewater treatment
in a up-flow anaerobic sludge blanket (UASB) reactor. Sci. Rep. 4, 6658.
Zoetemeyer, R.J., Matthijsen, A.J.C.M., Vandenheuvel, J.C., Cohen, A., Boelhouwer, C.,
1982. Anaerobic acidification of glucose in an upflow reactor. Biomass 2 (3),
187–199.