Chemical Engineering Journal 244 (2014) 587596

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Chemical Engineering Journal

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Characterization of methanogenesis, acidogenesis and hydrolysis

in thermophilic methane fermentation of chicken manure
Qigui Niu a, Toshimasa Hojo a, Wei Qiao a,b, Hong Qiang c, Yu-You Li a,d,
a

Graduate School of Environmental Studies, Tohoku University, 6-6-06 Aza-Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan
State Key Laboratory of Heavy Oil Processing, China University of Petroleum, PR China
c
College of Resources and Environment, Northwest A&F University, Yangling 712100, China
d
Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Japan
b

h i g h l i g h t s

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

 Thermophilic methane fermentation

of chicken manure was studied at 10%

TS.
 Ammonia inhibition was evaluated by
long time operation of continuous
experiment.
 Methanogenesis was more sensitive
to ammonia than acidogenesis and
hydrolysis.
 Microbial community shifts were
characterized by 16S rDNA cloning
library.

a r t i c l e

i n f o

Article history:
Received 9 April 2013
Received in revised form 14 November 2013
Accepted 26 November 2013
Available online 4 December 2013
Keywords:
Thermophilic methane fermentation
Methanogenesis
Wide range of ammonia
Microbial community
Chicken manure

a b s t r a c t
The thermophilic methane fermentation of chicken manure (CM) with a total solids (TS) of approximately
10% was investigated with regard to ammonia inhibition. A gradual increase in total ammonia nitrogen to
6000 mg/L was observed in the case of raw CM fermentation. A distinct rise in VFA accumulation combined with a low methane production of 0.29 L/gVS occurred at a TAN of 4000  5000 mg/L. Biogas production completely ceased when TAN reached 8000 mg/L after the addition of NH4HCO3. The high
sensitivity of methanogenesis to TAN and free ammonia (FA) was quantitatively conrmed. The IC50 of
TAN for methanogenesis, acidogenesis and hydrolysis was 5058, 5305 and 5707 mg/L at a pH value of
8.1 0.2. Similar results but with a lower IC50 were obtained for FA inhibition during fermentation.
The microbial community analysis revealed signicant differences in hydrogenotrophic methanogens
and aceticlastic methanogens before and after inhibition.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Chicken manure (CM) is a typical agriculture waste with a high
fraction of biodegradable organic matter. In 2008, about 13 million
tons of CM was produced in Japan [1]. This huge amount of CM is
mainly treated by incineration and composting. However, due to

secondary pollutant emissions, both of these methods are controversial [2].

Methane fermentation, a widely used technology to stabilize organic waste and simultaneously generate energy in the form of
biogas [3], has been proposed as a method for treating CM. Thermophilic fermentation in particular has been attracting more and

Corresponding author. Address: Graduate School of Engineering, Tohoku University, 6-6-06 Aza-Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan. Tel.: +81
227957464; fax: +81 227957465.
E-mail address: yyli@epl1.civil.tohoku.ac.jp (Yu-You Li).
http://dx.doi.org/10.1016/j.cej.2013.11.074
1385-8947/ 2014 Elsevier B.V. All rights reserved.

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Q. Niu et al. / Chemical Engineering Journal 244 (2014) 587596

more attention due to the high rate of biogas production, the short
hydraulic retention time (HRT) and improved pathogen destruction. Thermophilic fermentation has been applied to many cases,
such as sludge, municipal solid waste, cattle manure, poultry manure and corn grain ethanol industry waste treatment [46]. Compared with the mesophilic process, however, the thermophilic
process is more sensitive to changes in pH, VFA, ammonia and
toxic substrates. Nitrogen overloading has been shown to have a
signicant negative inuence on methanogenic activity, and can
render a reactor unstable. The TAN concentration of 3000
4000 mg/L is regarded to be at the edge of inhibition. Even at a
low TAN of 1700 mg/L, inhibition occurrence has been reported
[7]. Hashimoto reported that both thermophilic and mesophilic
processes are inhibited at a TAN of 2500 mg/L [8]. Thermophilic
fermentation has been reported to be unsuccessful for the relatively low nitrogen content in pig waste [9]. The fermentation of
swine manure as the sole substrate has been shown in earlier reports to be unsuccessful, mainly due to the high content of ammonia in the waste [8]. It has been shown that FA is a crucial fraction
of TAN for inhibition [10].
Individual volatile fatty acids (VFAs) can be used as process stability indicators in methane fermentation. Among them, butyrate
and isobutyrate acid have been shown to provide especially sensitive results [11]. Acetic and propionic acid were the most abundant
VFAs in manure digestion. VFA accumulated as Ammonia inhibition occurred, in effect indicating the extent of the inhibition.
It should be noted that CM is a high nitrogen content organic
waste with nitrogen levels much higher than those of cow manure,
food waste, pig manure and waste active sludge [12]. Hydrolysis in
thermophilic is faster than mesophilic which in itself makes CM
thermophilic fermentation a higher risk process [13]. More importantly, nitrogen inhibition is the result of chronic accumulation
during the operation of the reactor. The high concentration of
ammonium and volatile fatty acids (VFAs) tend to occur simultaneously, resulting in a stable pH. It was decided that the only
way to get convincing results on the chronic inhibition effect is
to operate the reactor in a long term.
Moreover, neither the whole performance nor the mechanism
of ammonium inhibition of thermophilic CM fermentation has
yet to be revealed in detail. Alternative methods of reducing the
nitrogen loading are by diluting CM to a low TS of 0.53% [14] or
mixing the CM with other livestock manure or sludge [15]. These
methods tend to increase the amount of nal discharged waste
water and result in a more complex process system. The effectiveness of high solid and sole CM fermentation needs further
investigation.
In the present research, a 240 days thermophilic fermentation
process using a continuous stirred tank reactor (CSTR) fed with
high solid CM was performed to investigate the followings: (1)
the methane yields from raw CM and ammonia stripping CM, (2)
the inhibition predisposing factor of high solid and high nitrogen
CM fermentation, (3) the effect of nitrogen inhibition on methanogenesis, acidogenesis and hydrolysis, (4) the microorganism community evolution before and after inhibition.

2. Material and methods

2.1. CM properties
Original CM with TS of 44.3% was kept in the refrigerator at 4 C.
Raw CM was diluted to 10 2% TS content with tap water. The diluted CM was shredded into slurry and was provided for the CSTR
reactor. The shredded CM was stored in a substrate tank with 4 C
cooling water circulation to avoid microbial activity. The raw CM
was pretreated to reduce nitrogen through ammonia fermentation

Table 1
Characteristics of raw CM and ammonia stripping CM.
Constituent

Unit

Average (n = 6)

SD ()

Raw CM element analysis

TS
(%)
VS
(%)
SS
(%)
VSS
(%)
T-COD
(mg/L)
TN
(mg/L)
TAN
(mg/L)
C
%
H
%
N
%
O
%
S
%

11.2
8.27
10.1
7.55
102,600
6450
3850
35.2
4.83
5.44
30.12
0.84

0.53
0.83
0.11
0.67
3200
810
200
0.45
0.05
0.24
0.18
0.10

Ammonia stripping CM
TS
(%)
VS
(%)
SS
(%)
VSS
(%)
T-COD
(mg/L)
TN
(mg/L)
TAN
(mg/L)

8.93
6.13
0.79
5.59
94400
3590
2500

0.144
0.197
0.049
0.046
8230
570
100

T-COD: total COD, TN: total nitrogen, TAN: total ammonia nitrogen, C:C element in
dry CM.

and ammonia stripping. The ammonia stripped CM, hereafter referred to as pretreated CM, and had a lower nitrogen substrate than
the Raw CM. Both the pretreated CM and raw CM were used in the
experiments respectively. The Total Solid (TS), Total Volatile Solid
(TVS), Total COD (TCOD), NH
4 N and Total Nitrogen (TN) were
analyzed to ascertain the stability of the substrate stability. The
characteristics of CM are given in Table 1.
2.2. CSTR operation procedure
A schematic diagram of the experimental device is provided in
Fig. 1. A lab-scale continuous stirred tank reactor (CSTR) with a
working volume of 12 L (total 15 L) was operated under thermophilic (55 1 C) conditions. The reactor was warmed by water circulation and agitated with a motor. A wet gas meter was used to
measure the daily biogas amount. The substrate tank was stirred
with 200300 RPM to keep the CM at a uniform semisolid state.
The TS content of the feedstock was adjusted to about 10% by adding tap water. The HRT was set at 30 days. The daily feedstock
amount was 0.4 L with OLR 0.21 kg/gd. The peristaltic inuent
pump with a timer was used to control the feeding at 12 times
per day to reduce feeding shock. Each feed was lower than 1% of
the reactor working volume. Seed sludge with TS of 3.1% was sampled from thermophilic anaerobic digestion of municipal sewage
treatment plant.
2.3. Analytical methods
The analyses of pH, alkalinity, COD, total ammonia nitrogen
(TAN), TS and TVS were performed according to Japan standard
methods [16]. The VFA concentration was determined by gas chromatography (Agilent-6890) equipped with a DB-WAXetr capillary
column (30 m 0.53 mm 1 lm) and an FID detector. The carrier gas
was Helium, with a pressure of 30.4 kPa, injected at a rate of
399 mL min1. The temperature of the injection port and detector
were 250 C and 250 C, respectively. The oven temperature was
set at 125180Cat a speed of 15 C min1.
Biogas was calibrated to that under standard conditions (0 C;
1.013 bar). The biogas composition was measured by a gas chromatograph (SHIMADZU GC-8A) equipped with a thermal

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Q. Niu et al. / Chemical Engineering Journal 244 (2014) 587596

Fig. 1. Schematic diagram of continuous experiment.

conductivity detector (TCD) and a 2 m stainless steel column

packed with Porapak Q. Helium was the carrier gas at 30 mL min1.
The column temperature was 70 C and the injector and detector
temperatures were 100 C and 70 C, respectively. The elemental
composition of C, H, O, N, and S were analyzed by an elemental
analyzer (Nario EL III CHNS).
The microbial community was analyzed by 16SrDNA gene cloning and sequencing. Genomic DNA was extracted from samples
with an Ultra Clean Soil DNA Isolation Kit (MO-BIO). The amplication of 16SrDNA was performed with the primers EUB 8F [17] and
Univ-1500R [18]for bacteria and A109F [19]and 1059R [20] for archaea. Thermal cycling of PCR consisted of 30 s denaturing at 94 C,
40 s of annealing at 50 C, and extracting at 72 C for 1 min with 30
cycles for archaea and 23 cycles for bacteria. The PCR products
were rstly puried by Micro Spin S-400 HR (Amersham Pharmacia GE, USA). The puried DNA was cloned with the TOPO TA
Cloning Kit (Invitrogen, USA) and transformed into Escherichia
coli DH5a competent cells. Cloned DNA fragments were obtained
and spread on plates. After an incubation period of 24 h at 37 C,
the white ones were randomly picked out and transferred to LB
with another 6 h of continuous incubation. Insert check was performed using vector of M13 primer. The successful ones were used
for sequencing. Similarity searches for the assembled sequences
were performed using the NCBI Blast search program within the
GenBank database (http://www.ncbi.nlm.nih.gov/blast/).
2.4. Denition of conversion ratio
The conversion ratio of hydrolysis, acidogenesis and methanogenesis were calculated based on a COD balance in the whole
anaerobic process as shown in Eqs. (1)(3). TCODin was the total
inuent COD. SCOD was the centrifuged efuent supernatant
COD and SCODin was the inuent SCOD. CODCH4 was calculated
based on the principle of 0.35 m3-CH4/kg-COD under standard conditions. CODVFA was the total VFA concentration of C2C5 and expressed as HAC. CODVFAin was the inuent CODVFA.

Hydrolysis %

SCOD  SCODin CODCH4

TCODin  SCODin

Acidogensis %

CODVFA  CODVFAin CODCH4

TCODin  CODVFAin

Methanogenesis %

CODCH4
TCODin

Free ammonia (FA) is pH and TAN concentration dependent and

can be calculated according to the following equilibrium equation
[21]:

NH3 H2 O $ NH4 OH

NH3

TAN

10pH

4
!1
5

0:090182729:92

Tk

10

The effects of TAN and FA on hydrolysis, acidogenesis and methanogenesis were described and calculated using the extended
Boltzmann equation:

Y A2

A1  A2
1 expxx0

dx

ICi X0 dx  LN

A1  A2
100  i  A2  1

6

7

A1 initial value (left horizontal asymptote), A2 nal value (right

horizontal asymptote), x0 center (point of inection), dx width (the
change in X corresponding to the most signicant change in Y values), ICi the concentration of i% inhibition thermophilic reactor
performance
3. Results
3.1. Performance of methane fermentation during a long term
operation
The whole experiment was divided into 3 phases. In phase 1,
pretreated CM was used to investigate low nitrogen CM fermentation performance. In phase 2, raw CM was used and long term
tested to analyze nitrogen accumulation and its inhibition effects.
Phase 2 was subdivided into three stages phase 2a, phase 2b and
phase 2c based on the average performance. In phase 3, NH4HCO3
was added together to the raw CM to increase the TAN to an extreme high concentration. All of the experimental data were used
to establish an inhibition model, calculate the inhibition threshold
and describe the inhibition mechanism. The pH, alkalinity, biogas
production rate, VFA and ammonia concentration are shown in
Fig. 2.
In the phase of startup, the biogas production rate, the methane
content, pH and total VFA concentration were stable after 32 days,
indicating the digester had successfully started up. During phase 1,
the TAN concentration varied from 2000 mg/L to 3500 mg/L and
the VFA kept below 3000 mg/L. TAN concentrations above
3000 mg/L were considered a risk to reactor stability [22]. After a
run of 32 days, the reactor achieved a VS conversion of 61.1% and
a biogas conversion ratio of 0.35 L/g VS with a methane content
over 60%. The pH value was maintained at 8.18.3. The alkalinity
was stable at 10,000 mg/L.

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Q. Niu et al. / Chemical Engineering Journal 244 (2014) 587596

Fig. 2. The time course of biogas production, pH, VFA, and TAN content.

In phase 2a, from day 75 to day 120, high nitrogen content CM

was fed into the reactor. In this stage, while the TAN gradually
climbed to approximately 4000 mg/L, the VFA maintained low concentrations between 4000 mg/L and 6000 mg/L. The alkalinity and
pH always remained stable. Biogas production dropped to 0.29 L/gVS, which marked a reduction of approximately 17% compared to
phase 1. At this point, the methane content was reduced to 50%,
suggested the reactor had begun to be subjected to inhibition.
In phase 2b from day 120 to day 160, the TAN gradually increased to 6000 mg/L. A sharp VFA accumulation from 4000 mg/L
to 20000 mg/L occurred at a TAN concentration of 5000 mg/L after
running for 200 days. Meanwhile, biogas production reduced
0.18 L/g VS, equivalent to 51.4% of that in phase 1. The methane
content varied in a low percentage band between 40% and 50%.
The pH kept between 8.1 and 8.3. The sharp increase in VFA concentration is attributable to the occurrence of inhibition. The suppression still continued in the next phase. A lower methane production
0.13 L/gVS with methane content varying between 33% and 50%
was observed through phase 2c. VFA accumulation was found to
be steady over 20,000 mg/L and even reached to 25,000 mg/L. The
high VFA residue in the reactor also indicated insufcient methanogenesis activity for the consumption of even readily consumable
substrates. Throughout all of phase 2, with a running time of over
125 days, the TAN increased from 3689 mg/L to 6000 mg/L. Correspondingly, TCOD removal efciency decreased from 55% to 26%.
In phase 3, NH4HCO3 was added from day 200 in order to increase the TAN concentration. The pH increased from 7.5 to 8.32
and then decreased to 7.4 within 20 days. The biogas production
tended to cease when the methane content dropped below 30%.
The VFA was kept at a high concentration above 25000 mg/L.
Through phase 2a and phase 3, the addition of raw CM and NH4HCO3 into the reactor gradually increased the TAN concentration

from 3689 mg/L to 70009000 mg/L. Consequently, readily consumable carbohydrate removal ratio dropped from 77% to 59%,
and the protein content dropped from 21% to 310%.
Throughout these three phases for 240 days, TAN continued to
accumulate, except during the rst stage when low nitrogen pretreated CM was used. After prolonged exposure to ammonia, the
TAN concentration reached 8000 mg/L resulting in a serious inhibition of the methane yield with a high VFA accumulation of
25000 mg/L. The total performance of CSTR is summarized in
Table 2.
Our results suggest that it is not possible to avoid inhibition
from ammonia in the thermophilic fermentation of raw CM with
high solid (about 10%) inuent. Long term operation was shown
to result in a high nal TAN concentration well in excess of the stable digestion limitation. Ammonium is a byproduct of the digestion
of organics and is produced together with biogas production. The
high biogas conversion and low TAN concentrations were in conict, and this was especially true in the case of high organic content
waste digestion. The reactor operation strategy was adjusted to accept low solid inuent rather than high solid.
3.2. The stoichiometry for methane fermentation of CM
The elemental composition of the raw CM was analyzed by dry
matter (see Table 1). The stoichiometric biochemical reaction
formula,
CnHaObNc + (n  0.25a  0.5b + 1.75c)
H2O ? (0.5n +
0.125a  0.25b  0.375c) CH4 + (0.5n  0.125a + 0.25b  0.625c)
CO2 + cNH4 + cHCO3 [23] was used to describe the element balance
and biogas conversion efciency. The stoichiometric formula was
identied as:

C7:5 H12:4 O4:8 N 3:89H2 O ! 3:7CH4 2:8CO2 NH4 HCO3

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Q. Niu et al. / Chemical Engineering Journal 244 (2014) 587596

Table 2
Average performance of thermophilic reactor during the steady and inhibition stages.
Parameters

Unit

Phase 1

Phase 2a

Phase 2b

Phase 2c

Phase 3

T-COD removal efciency

Protein removal efciency
Carbohydrate removal efciency
TAN
Gas production
CH4
CO2

(%)
(%)
(%)
(mg/L)
(L/g-VS)
(%)
(%)

3075 d
66.6
36.0
82.1
2830
0.32
61.75
37.6

75120 d
55.0
21.0
77.0
3700
0.28
51.01
47.1

120160 d
39.0
12.0
71.0
5170
0.18
39.27
56.8

160200 d
26.0
14.0
67.0
6000
0.13
40.59
57.9

200240 d
<19.0
3.010.0
59.0
70009000
0.05
34.17
63.8

CH4-COD/T-COD
VFA-COD/T-COD
S-COD/T-COD
P-COD/T-COD

(%)
(%)
(%)
(%)

66.5
4.5
6.1
23.0

55.1
4.7
6.2
33.8

38.6
10.3
15.1
36.1

26.2
30.3
5.5
37.8

13.5
29.6
3.2
53.7

CH4-COD/T-COD: COD as CH4 per total COD.

VFA-COD/T-COD: COD as VFA per total COD.
S-COD/T-COD: (SCOD-COD as VFA) per total COD.
P-COD/T-COD: particle COD per total COD.
Gas production (L/g-VS): L/g-VSinuent.

Considering the biochemical reaction formula, degrading 1 kg

VS of CM can produce 0.74 m3 biogas, 0.42 m3 methane (CH4,
56%) and 70.93 g of ammonia nitrogen. These theoretically calculated results are consistent with the experimental results of the
steady stage phase 1 and the slightly inhibited phase 2a.
3.3. COD mass balance at different TAN levels
Table 2 summarized the average mass balance in different
phases based on the COD at different TAN levels. In phase 1, feeding with pretreated CM, with TAN varying from 2000 to 3500 mg/L,
about 66.5% of the COD was converted into biogas, 4.5% into VFA,
6.1% into S-COD and 23% remained in SS. In phase 2a, TAN was
within 35005000 mg/L, 55.1% of COD converted into biogas,
4.7% to VFA, 6.2% to S-COD and 33.8% remained in SS. In phase
2b, TAN climbed to 7000 mg/L, about 38.6% of COD transferred to
biogas, and the COD in the VFA increased by 10.3%. In phase 2c,
the VFA concentration was found to be notably high. At this point,
however, about 30.3% COD was in the form of VFA. At this stage,
the methanogenesis activity was seriously affected. Only 26.2% of
COD transferred to CH4, whereas it was about 40% of the stable
phase 1. In the following operation, the addition of NH4HCO3 resulted in an increased in the TAN concentration from 7000 to
9000 mg/L. A signicant increase of particulate COD approximately
from 23% (in phase 1) to 53.7% (in phase 3) was also noted, indicating that not only methanogenesis but also the hydrolysis reaction
had been suppressed.
In phase 1 and phase 2a, when ammonia stripping CM was used,
18.6 L/d of biogas was recovered from one kg of substrate with TS
of 8.97%, whereas 23.3 L/d of biogas was produced feeding with
one kg of raw CM with TS of 12.7% at a TAN concentration below
4000 mg/L. Our study illustrated that successful operation was
possible with the pretreated CM, yielding an OLR of
2.0 0.1 kg VS/m3 and 0.32 0.01 m3/kg VS of methane.

efciency were evaluated under different TAN concentrations to

assess the performance with and without inhibition.
Fig. 3 illustrates the effect of TAN on TCOD, protein and carbohydrate removal efciency. The results clearly demonstrated that it
was possible to obtain a stable TCOD removal efciency of over
60% and a carbohydrate removal efciency of 80% with a TAN concentration of less than 4000 mg/L. At a TAN concentration of
6000 mg/L, the carbohydrate removal efciency was 60%. However, stable protein conversion was achieved only when the TAN
was lower than 3000 mg/L. At TAN concentrations exceeding
3000 mg/L, a reduction in rapid protein removal was observed.
The protein removal efciency decreased from 40% at a TAN of
1500 mg/L to just 10% at a TAN of 6000 mg/L. The threshold toxicity levels for TCOD, carbohydrate and protein degradation are
approximately at 4000 mg/L, 4000 mg/L and 3000 mg/L, respectively. Protein conversion was 71.4% lower under inhibition conditions than stable conditions, only a 23.5% reduction in

4. Discussion
4.1. Effect of TAN on TCOD, protein and carbohydrate conversion
Since the carbohydrates, protein and lipids in organic waste are
known to have the largest contribution for methane fermentation,
it is important to reveal the extent to which they were degraded in
the fermentation process. Our focus was especially on protein and
carbohydrate degradation. The CM had a high protein content of
25% TS. In this study, protein, carbohydrate and TCOD removal

Fig. 3. Effect of TAN on the T-COD, protein and carbohydrate removal efciency.

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Q. Niu et al. / Chemical Engineering Journal 244 (2014) 587596

carbohydrate conversion was observed. These results indicate that

protein degradation is more sensitive to ammonia loading than
carbohydrate and TCOD degradation.

to thousands depending on the substrate and the operation method. In this study, the signicantly inhibition concentration of FA
was about 1800 mg/L from the steady stage to the inhibited stage.

4.2. Effects of TAN and FA on biogas production and VFA accumulation

4.3. Effect of TAN on methanogenesis, acidogenesis and hydrolysis

Fig. 4a illustrates the effect of the TAN concentration on the gas

production/composition and on the VFA accumulation concentration. Inhibition was observed when the ammonia concentration increased to levels above 4000 mg/L. At low TAN concentrations
(<4000 mg/L), the VFA concentration was lower than 6000 mg/L
(with acetic acid 94%) and no signicant uctuations in the amount
of biogas were observed. However, the biogas composition was
quite sensitive to TAN and slight decreases in the amount of biogas
production when the TAN concentration was higher than approximately 4000 mg/L were observed. Signicant decreases in methanogenesis were observed at the TAN concentrations up to
4500 mg/L, and a remarkable amount of VFA accumulation was observed (higher than 25,000 mg/L with acetic acid 82%) at TAN concentrations exceeding 5000 mg/L. The reactor tended to be
unstable when the TAN concentration was higher than 6000 mg/
L. Gas production almost stopped when TAN was about 9000 mg/L.
Fig. 4b illustrates the VFA accumulation and gas production under different FA levels. As reported, FA is more toxic than ionized
ammonia because of its capability to penetrate through the cell
membrane, and the subsequent intra-cellular accumulation leads
to the production of a toxic cytosolic enzyme and/or a proton
imbalance [24]. In our investigation, FA primarily caused a reduction in gas production, and a high FA concentration of 1800 mg/L
began to inhibit methanogenesis. A sharp VFA accumulation occurred at an FA concentration of 2000 mg/L, indicating a strong
inhibition on CM fermentation. Simultaneously, a sharp biogas
drop was observed when FA reached 2000 mg/L.
Even though the mechanism of ammonia toxicity in anaerobic
microorganisms is still largely understood, previous studies indicated that the FA inhibition concentration varied from hundreds

The methane fermentation of organic waste is a process involving three distinct stages; hydrolysis, acidogenesis, and methanogenesis. The methane fermentation process proceeds efciently
only when the degradation rates of all three stages are matched.
Different types of microorganisms characterize the three stages
and each presents a different sensitivity to ammonia inhibition.
The three stages of the biochemical reaction ratio are presented
in Fig. 5. A stable COD conversion during thermophilic digestion
was able to be maintained when TAN was below 4000 mg/L. The
COD, hydrolysis, acidogenesis and methanogenesis conversions
were 82%, 76%, 73% and 71%, respectively under stable conditions.
When the TAN concentration exceeded 4000 mg/L, methanogenesis conversion was the rst to drop. The half conversion ratio of
methanogenesis appeared at a TAN of 5058 mg/L. The acidogenesis
conversion began to decrease when the TAN concentration exceeded 5000 mg/L. The half conversion ratio of acidogenesis was
around 6000 mg/L. The hydrolysis ratio decreased from 76% at a
TAN of 5076 mg/L to 30% at a TAN of 6240 mg/L. When TAN
reached 6240 mg/L, the methanogenesis ratio decreased to 20%
while acidogenesis and hydrolysis ratio decreased to 25% and
32%. It could be concluded that methanogenesis was rstly and
readily inhibited, and that acidogenesis and hydrolysis conversion
declined in turn.
The three stage sequence of occurrences involved in inhibition
supported the principle of imbalance between the acetogenic and
methanogenic microorganisms, which differ signicantly in terms
of their physiology, nutritional needs, growth kinetics and sensitivity to environmental conditions. Hydrolysis and acidication could
run under a higher OLR and a low pH with a high biochemical reaction rate.

Fig. 4. Effects of TAN (a) and FA (b) on VFA and gas production.

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Q. Niu et al. / Chemical Engineering Journal 244 (2014) 587596

Fig. 5. Simulation curve of TAN (a) and FA (b) effected on hydrolysis, acidogenesis and methanogenesis conversion ratio.

Table 3
Results of model fetting and calculation of IC10, IC50 and IC75 for Eq. (7).
Parameter

A1

TAN
Hydrolysis
Acidogenesis
Methanogenesis

A2

89.5 4.4
76.7 3.5
70.2 3.2

FA
Hydrolysis
Acidogenesis
Methanogenesis

89.5 7.5
80.7 3.7
70.2 6.1

x0

dX

IC10 (mg/L)

IC50 (mg/L)

IC75 (mg/L)

30 7.6
29 3.9
9.1 4.9

5494 202.6
5233 96.1
5277 112.2

311.7 1169.4
298.1 120.1
311.7 138.9

4817
4778
4244

5707
5305
5058

6292
5996
5603

20.2 17.9
16.0 6.7
4 9.6

2073 60.5
2101 28.4
2022 49.8

80.3 55.8
80.3 30.8
80.3 43.3

1944
1961
1839

2120
2115
2012

2291
2249
2083

4.4. Inhibition modeling

The parameters and the calculated IC10, IC50 and IC75 are summarized in Table 3. The tting curves of hydrolysis, acidogenesis,
and methanogenesis are shown in Fig. 5a. Based on the simulation
results, the threshold of TAN on hydrolysis, acidogenesis, and
methanogenesis was over 8300 mg/L. With TAN inhibited methane
fermentation, hydrolysis was the most tolerant step while methanogenesis was the most sensitive and also the rst inhibited when
exposed to high nitrogen levels. The results also illustrate that following the increase of TAN, acidogenesis and hydrolysis were
inhibited in sequence and resulted in the failure of digestion. Those
results are consistent with the termination of gas production indicated in Fig. 4.
Fig. 5b illustrates the effect of FA inhibition on hydrolysis, acidogenesis, and methanogenesis. Obvious FA inhibition was noted
at levels of around 2000 mg/L among all three steps. The threshold
of FA on hydrolysis, acidogenesis, and methanogenesis were over
2500 mg/L. The IC10, IC50 and IC75 of FA for methanogenesis were
lower than those for both acidogensis and hydrolysis. In our study,

A quantitative resolution of ammonia inhibition is usually

based on a batch fermentation experiment and tends to be mainly
focused on the methanogenesis stage. Both hydrolysis and acidogenesis usually attract less attention. In this study, the long term
experimental results were calculated to establish a chronic inhibition model rather than one based on a batch test. Furthermore, the
hydrolysis, acidogenesis, and methanogenesis inhibition were considered to illustrate total methane fermentation.
Parameters dx determined the change in X, which corresponds
to the most signicant change in Y values and describes the variation inhibited extent. In this study, extended value of ICi was
calculated by Eq. (7). In this model, the inhibition effects of TAN
and FA are described by IC50 [25]. Both IC10 and IC75 were calculated to assess the initial inhibition and serious inhibition. A tting
curve was plotted to simulate long term experiments data using
sigmoidal program in Origin software, version 8.1.

Table 4
Archaeal community in the thermophilic reactor of different stages.
Sampling stage (day)

Phylogenetic group description

No. of clones

No. of OTUs

Percentage (%)

Uitilization information

Before inhibition (day 35)

Methanoculleus marisnigri JR1

Methanothermobacter thermautotrophicus str.
Methanosarcina mazei Go1
Methanobacterium sp.

42
5
6
1

1
1
1
1

77.8
9.3
11.1
1.9

H2 + CO2, formate utilizing

H2 + CO2, formate utilizing
Acetate, H2 + CO2 utilizing
H2 + CO2, formate utilizing

Total

54

100.0

Methanothermobacter thermautotrophicus str.

Methanobacterium sp.

19
1

1
1

95
5

Total

20

100.0

After Inhibition (day 120)

H2 + CO2, formate utilizing

H2 + CO2, formate utilizing

594

Q. Niu et al. / Chemical Engineering Journal 244 (2014) 587596

Table 5
Changes of bacterial community before and after inhibition.
Sampling stage (day)

Phylogenetic group

Before inhibition (day 35)

Firmicutes

Description

No. of OTUs

No. of clones

Percentage (%)

Natranaerobius thermophilus
Mahella australiensis
Peptoniphilus sp.
Alkaliphilus
Halothermothrix orenii
Ammonifex degensii
Moorella thermoacetica
Lactobacillus
Kurthia sp.
Tepidanaerobacter sp.
Thermoanaerobacter ethanolicus
Clostridium thermocellum
Dolosigranulum pigrum
Enterococcus

5
3
3
2
2
2
2
1
1
1
1
1
1
1

53
16
16
7
7
6
4
3
2
1
1
1
1
1

37.6
11.3
11.3
5.0
5.0
4.3
2.8
2.1
1.4
0.7
0.7
0.7
0.7
0.7

Citricoccus sp.
Corynebacterium
Propionibacterium
Streptomyces

2
2
2
1

3
3
3
1

2.1
2.1
2.1
0.7

Riemerella anatipestifer
Leadbetterella byssophila
Cytophaga hutchinsonii

1
1
1

3
2
1

2.1
1.4
0.7

Hylemonella gracilis

2.8

Actinobacteria

Bacteroidetes

Proteobacteria
Synergistetes
Anaerobaculum hydrogeniformans

1.4

39

141

100.0

Natranaerobius thermophilus
Clostridium thermocellum
Desulfotomaculum
Clostridium sporogenes
Halothermothrix orenii
Pelotomaculum thermopropionicum
Dethiobacter alkaliphilus
Turicibacter sanguinis
Ruminococcaceae bacterium
Syntrophothermus lipocalidus
Thermoanaerobacter
Enterococcus casseliavus
Eremococcus coleocola
Erysipelothrix rhusiopathiae
Moorella thermoacetica
Clostridium clariavum
Alkaliphilus metalliredigens
Pelotomaculum thermopropionicum

3
7
3
9
2
3
3
2
1
1
2
1
1
1
1
1
1
1

32
31
18
12
8
4
4
3
2
2
2
2
1
1
1
1
1
1

24.1
23.3
13.5
9.1
6.0
3.0
3.0
2.3
1.5
1.5
1.5
1.5
0.8
0.8
0.8
0.8
0.8
0.8

Pseudomonas stutzeri
Methylophaga sp.

1
1

1
2

0.8
1.5

Corynebacterium casei

2.3

Total
Firmicutes
After Inhibition (day 120)

Proteobacteria

Actinobacteria
Synergistetes
Anaerobaculum hydrogeniformans
Total

a high IC50 (5058 mg/L) was obtained, and a high threshold

(excessing 8300 mg/L) was noted for the thermophilic methane
fermentation for methanogenesis.
4.5. Microbial community analysis with and without inhibition
To reveal the evolution of the microbial community before and
after ammonia inhibition, with the reactor sampled at days 35 and
120, the cloning analysis of bacteria and archaea were performed,
respectively. The cloning analysis results are summarized in Table 4
(archaea) and Table 5 (bacteria).

0.8

48

133

100.0

Under the stable fermentation condition, Methanoculleus marisnigri JR1 utilizing H2 + CO2, formate and dihydric alcohol was the
dominated methanogen. In the inhibited stage, Methanothermobacter thermautotrophicus str. increased to dominate methanogen
with 95% of archaea. Methanosarcina mazei Go1 was acetate acid
utilizing which occupied 11.1% of archaea in the steady stage but
it was not detected in the inhibited stage, proving that the VFA
accumulation involved is largely acetic acid accumulation as
showed in Fig. 4. The assumption that acetate utilizing
methanogens is the rate limiting step was consistent with the results in this study. The cloning sequencing results illustrated that

Q. Niu et al. / Chemical Engineering Journal 244 (2014) 587596

M. thermautotrophicus str. was the most tolerant archaea in the

reactor. Similar results were also reported that acetate consuming
methanogens were much more sensitive than hydrogen utilizing
methanogens [26].
A summary of the bacterial community is provided in Table 5,
indicating the dominant phylogenetic group of Firmicutes, which
occupied 84% and Actinobacteria (7%), Bacteroidetes (4.2%), Proteobacteria (2.8%), Synergistetes (1.4%) without inhibition, respectively.
While on day 120 with the inhibition of TAN concentration over
IC50, the phylogenetic group Firmicute, represented 84% and
Actinobacteria (2.3%), Proteobacteria (2.3%), Synergistetes (0.8%)
were observed with fewer phylogenetic groups and main
compositions which differed from the stable stage. As previously
reported, Mahella australiensis [27], Clostridium thermocellum
[28], Clostridium sporogenes [29], Clostridium clariavum [30],
Leadbetterella byssophila [31]and Cytophaga hutchisonii [32] are
able to cause hydrolysis the particulate COD of cellulose,
starch, lipid and protein. M. australiensis [27], C. thermocellum
[28], C. sporogenes [29], C. clariavum [30], Alkaliphilus [33],
Halothermothrix orenii [34], Moorella thermoacetica [35] and
Lactobacillus are able to cause acidogenesis. Ammonia production
and ammonia inhibition of CM fermentation in different stages is
showed in Fig. 6. Among the occupied phylogenetic group
Firmicutes, Natranaerobius it was the utilization of fructose,
cellobiose, ribose, trehalose which contributed to the hydrolysis
with a signicant before and after inhibition changing from
37.6% to 24.1%. Peptoniphilus sp. was the protein utilized,
representing 11.3% at the steady stage but not detected during
inhibition at day 120. At this stage, a sharp decrease in protein
removal efciency was also noted (see Fig. 3). A comparison of
the two different samples of the microbial community indicates
that TAN inhibition caused the construction of the community
evaluated in both archaeal community and bacterial community.

Fig. 6. Ammonia production and inhibition schematic diagram of methane

fermentation on CM.

595

5. Conclusions
(1) A feasibility of CM fermentation was obtained feeding with
ammonia stripping CM and feeding with raw CM with TAN
below 4000 mg/L producing 0.74 m3/kg VSdegraded biogas
and 70.93 g/kg VSdegraded of ammonia nitrogen.
(2) The VFA accumulation was obviously at a TAN of 4000
5000 mg/L, and biogas was ceased at a TAN over 8000 mg/L.
(3) The IC10, IC50 and IC75 of TAN and FA on methanogenesis
were much lower than those for both acidogenesis and
hydrolysis.
(4) 16S r DNA cloning library resulted that aceticlastic M. mazei
Go1 occupied 11.1% of the methanogens in the steady stage
but not detected after inhibition, thus revealing serious VFA
accumulation.

Acknowledgements
The authors would like to acknowledge the technical cooperation in this investigation from Hitachi Engineering and Serves Co.
Ltd. The rst author was supported by China Scholarship Council
(CSC) for a scholarship.
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