Process Biochemistry 48 (2013) 901–911

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Review

Ammonia inhibition in anaerobic digestion: A review

Orhan Yenigün, Burak Demirel ∗
Institute of Environmental Sciences, Boğaziçi University, Bebek, Istanbul 34342, Turkey

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

Article history: Even though ammonia is an essential nutrient for bacterial growth, it may inhibit methanogenesis during
Received 27 February 2013 anaerobic digestion process if it is available at high concentrations. Therefore, ammonia is regarded as a
Received in revised form 11 April 2013 potential inhibitor during anaerobic digestion, particularly when dealing with complex type of substrates
Accepted 17 April 2013
such as manure or the organic fraction of municipal solid waste (OFMSW). Ammonia is produced through
Available online 22 April 2013
biological degradation of nitrogenous matter. Ammonium ion (NH4 + ) and free ammonia (NH3 ) are the two
principal forms of inorganic ammonia nitrogen. Both forms can directly and indirectly cause inhibition in
Keywords:
an anaerobic digestion system. Particularly, free ammonia (FAN) is a powerful inhibitor in an anaerobic
Ammonia
Anaerobic digestion
digester above threshold concentrations. Process inhibition is related to the particular characteristics of
Inhibition the substrate to be anaerobically digested, pH, process temperature (mesophilic or thermophilic), type
Recovery of the seed sludge (inoculum), the reactor configuration and to the concentrations of ammonium and
ammonia. In this paper, ammonia inhibition in anaerobic digestion systems and the recovery efforts after
inhibition are discussed. Furthermore, the impacts of ammonia inhibition on the microbial population
available in anaerobic digesters, namely bacteria and Archaea, are also evaluated in detail.
© 2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 901
2. Early studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 901
3. Mesophilic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 902
4. Thermophilic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 905
5. Mesophilic vs thermophilic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 905
6. Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 906
7. Recovery after inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 908
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 909
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 909
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 909

1. Introduction modeling, and process microbiology. Furthermore, the activity and

fate of two distinct groups of microorganisms, namely acidogens
Anaerobic digestion processes have gained extreme importance and methanogens, also make the study of anaerobic digestion more
within the last three decades, since biogas, a form of renewable challenging [1]. However, anaerobic digestion processes are vul-
energy, can be produced through biological treatment of wastes and nerable to inhibition by certain accumulating chemicals, among
wastewaters with different characteristics. Research activities on which ammonia (NH3 ) and ammonium ion (NH4 + ) are the most
various aspects of anaerobic digestion have recently taken a more significant inhibitors. Although some ammonium is beneficial for
accelerated pace, particularly focusing on the effects of changes bacterial growth, undesirably high concentrations may be reached
in the operational and environmental parameters on the process during the breakdown of proteins available in the substrate.
performance and stability, inhibition and toxicity, optimization,
2. Early studies

∗ Corresponding author. Tel.: +90 212 359 46 00; fax: +90 212 257 50 33. Anaerobic digestion has been used to treat municipal sludge,
E-mail addresses: yeniguno@boun.edu.tr (O. Yenigün), animal wastes and industrial wastes of high organic content
burak.demirel@boun.edu.tr, demirelb@gmail.com (B. Demirel). since the beginning of the twentieth century. The conventional

1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2013.04.012
902 O. Yenigün, B. Demirel / Process Biochemistry 48 (2013) 901–911
substrates were primary sludge or slurried organic wastes. After
mid 50s, thickened sludges were introduced to, so called, high-
rate digesters, however frequent reactor failures were observed.
High alkalinity concentrations of up to 6500 mg/l and pH values
as high as 7.4, first led to the suggestion that the bacterial activ-
ity was hindered due to some toxic effect of the alkalinity [2].
Another common observation was the rapid increase of volatile
fatty acid (VFA) concentrations observed simultaneously during
reactor failures. McCarty and McKinney showed that the decrease
in bacterial activity was actually due to salt toxicity [3]. Use of
various salts in the digesters for pH control was a common exer-
cise. Among those used, sodium, potassium and ammonium salts
were found to cause toxic effects [2–5]. It was generally agreed
that for high rate digesters, total ammonia nitrogen (TAN), i.e. free
ammonia nitrogen + ammonium nitrogen concentrations of around
1700–1800 mg/l caused reactor failure [2,6]. McCarty and McKin-
ney further proposed that the inhibition/toxicity was due to free
ammonia (FA) in solution rather than the ammonium ions, equilib-
rium concentrations being dependent on pH and temperature [3].
They also found out that 150 mg/l free ammonia nitrogen (FAN)
concentration was completely inhibitory to anaerobic digestion.
Melbinger and Donnellon studied the relationship between organic
loading rate (OLR) and ammonia inhibition [6]. The authors found
out that the bacteria could be acclimated to ammonium, if it was fed
at slowly increasing concentrations, thus in such high rate digesters
TAN concentrations of 2700 mg/l could exist without signs of slow
down or failure. In another study with piggery wastes as the sub-
strate, partial inhibition was observed at 3000 mg/l total ammonia
(TA), i.e. free ammonia + ammonium ion concentration [7]. Further
research on the acclimation of Archaea to higher TAN concentra-
tions revealed that levels up to 5000 mg/l could be tolerated in
digested sewage sludge and 3075 mg/l in piggery manure [8]. These
results were confirmed by Braun et al. [9]. All early studies men-
tioned above were concentrated on the causes of inhibition and
relationships with pH, loading rates, VFA and salt concentrations,
synergistic and antagonistic effects and what could be done to
increase bacterial toleration.
During the 70s, the importance of energy recovery and renew-
able energy became increasingly apparent. In time, as some
advantages of anaerobic treatment over aerobic treatment were
recognized, the necessity of more research focusing on other
aspects of the anaerobic digestion process became evident. Among
these were studies related to process stability with respect to
process parameters, biogas recovery, microbiological aspects and
process modeling. Many different types of wastes and co-digestion
practices with various mixtures were considered. Digestion under
mesophilic and thermophilic conditions was also compared. Nearly
in all these studies, stretching the limits of certain experimental
conditions led to process slow down or failure, mainly due to inhibi-
tion/toxicity caused by the accumulation of various inhibitor/toxic
chemicals [10]. In time, a great amount of work has been particu-
larly carried out about inhibition and toxicity.
Ammonia (NH3 ) and ammonium (NH4 + ), apart from being
present in the sludge/slurry to be digested, also accumulate dur-
ing breakdown of proteins and are the foremost inhibitors to the
anaerobic digestion process. The important factors here are initial
concentrations, process temperature, pH, organic loading rate and
acclimation of inoculum, all of which have direct or indirect effect
on inhibitory concentrations. A summary of selected works about
ammonia inhibition in anaerobic digestion from literature covering
both mesophilic and thermophilic processes is given in Table 1.
3. Mesophilic digestion
Mesophilic anaerobic digestion generally refers to the pro-
cess temperatures between 30 and 40 ◦ C. Most of the full scale
anaerobic digesters are operated under mid-mesophilic condi-
tions, around 35 ◦ C. As mentioned above, TAN concentrations of
around 1700–1800 mg/l were completely inhibitory with unaccli-
mated inoculum, although with acclimation, inhibitory TAN levels
could increase up to 5000 mg/l. Another concern was the process
stability [11]. It was possible to maintain a stable digestion of a
synthetic acetic acid substrate at inhibitory TAN levels in excess
of 5000 mg/l, adjusting pH to 8.0, with corresponding 256 mg
FAN/l. Stable digester operation was therefore possible with lower
methane yield. Slight increases of TAN caused progressively lower
methane production and at a TAN level of 6700 mg/l, complete
inhibition was observed. In another study, a different synthetic
substrate with a chemical oxygen demand (COD) concentration of
10,000 mg/l was digested with slow additions of NH4 Cl [12]. The
digesters were flow-through and the pH was kept around 7.0–7.2.
When TAN concentration reached 7850 mg/l, slight inhibition was
observed (corresponding FAN concentration was 80–100 mg/l). A
50% inhibition at 11,780 mg TAN/l and pH 6.8 was the outcome.
Near complete inhibition was encountered at 18,300 mg/l TAN and
a pH of 6.6. In a similar study, during continuous NH4 Cl addition
to slurried poultry manure in a 5-liter laboratory digester, a TAN
concentration of 5980 mg/l depressed biogas yields by 27% at a
pH of 7.43 and 145 mg FAN/l [13]. Using potato juice as substrate
in batch reactors, ammonia inhibition of acclimated (to 2315 mg
TAN/l) granular sludge [14] was investigated to higher ammo-
nium concentrations by ammonium ion addition [15]. It was found
that at 11,831 mg TAN/l, with pH of 7.57, methanogenic activity of
0.04 g COD/g VS/day was maintained. Another noteworthy conclu-
sion here was the operation of a full scale upflow anaerobic sludge
blanket (UASB) reactor with TAN concentration between 5000 and
7500 mg/l, which had a maximum specific methanogenic activ-
ity of 0.1–0.15 kg COD/kg VS/day and an average sludge content of
40 kg VS/m3 .
Ammonia inhibition versus sludge retention time (SRT) was
studied with slug and continuous additions of NH4 Cl to anaerobic
acetate and propionate enrichment cultures [16]. High SRT systems
(40 days) were more tolerant to TAN concentration of 5000 mg/l in
comparison with low SRT systems (25 days). 55 mg FAN/l was the
maximum tolerable concentration in these experiments. Heinrichs
et al. employed acclimated sludge (to >1500 mg TAN/l) and a syn-
thetic substrate comprising acetic, propionic acids and mixtures as
well as lactic acid [17]. FAN levels causing 50% inhibition varied
between 106 and 183 mg/l. Digestion of diluted poultry manure
(10–14% total solids) in 5 l reactors gave satisfactory results in terms
of biogas yield at TAN concentrations of up to 7700 mg/l within a
pH range of 8.1–8.5 [18].
The substrates with high initial TAN and protein concentra-
tions such as sea-food industry wastes and wastewaters could
be treated anaerobically using acclimated sludges containing up
to 3000 mg/l TAN concentrations (corresponding to 150 mg FAN/l
at pH of 7.6) [19]. Working with macerated fish offal and cat-
tle slurry mixtures in continuous anaerobic digesters resulted in
reduced methane yields under 100 mg/l FAN concentration. How-
ever, the cause of inhibition was attributed to the presence of
long chain fatty acids (LCFAs) associated with the lipids in the
fish offal [20]. The same research group also studied batch anaero-
bic digestion with a variety of organic mixtures including slurries
of cattle manure, chicken manure, fish offal, fruit and vegetable
wastes and brewery sludge [21]. Successful reactor operation
and satisfactory methane yields were obtained in this study. The
digesters that contained chicken manure slurry exhibited depres-
sion in methane yields (up to 65%) at FAN concentrations of about
1000 mg/l, although a total reactor failure was not observed. In all
experiments, unacclimated bacterial cultures were used and the
digesters containing chicken manure had lag periods of up to 9
days.
Table 1

O. Yenigün, B. Demirel / Process Biochemistry 48 (2013) 901–911

A summary of selected works focusing on ammonia inhibition in anaerobic digestion.

Substrate Reactor type Loading rate Temperature pH TAN (critical conc. or as FAN (critical conc. or as Acclimation Reference
specified) specified)

Sludge Laboratory-scale – 30 ◦ C 7.2–7.4 >5000 mg/l – Yes [8]

batch reactors
Piggery manure Laboratory-scale – 30 ◦ C 7.2–7.4 >3075 mg/l Yes [8]
batch reactors
◦
MSW/Sludge Semi continuous – 39 C 8.0 2800 mg/l – No [24]
Synthetic wastewater UASB 1.2 kg COD/m3 /d 35 ◦ C 7.7–8.1 6000 mg/l 0.8 g/l Yes [27]
Slaughterhouse waste + CSTR 3.7 kg VS/m3 /d 34 ◦ C 7.5 4100 mg/l 337 mg/l Yes [31]
organic fraction of
MSW
Sewage sludge Semi continuous 2.0 kg VS/m3 /d 35 ◦ C 8.0 3000 mg/l 400 mg/l Yes [41]
Cattle manure CSTR – 45 ◦ C 7.4–7.9 6000 mg/l 0.7 g/l Yes [54]
Organic fraction of MSW High solids reactor 6.5 g VS/kg/d 55 ◦ C 7.0 2500 mg/l (100% inhibition) – Yes [56]
Non-fat dry milk CSTR 4 g COD/l/d 55 ◦ C 6.5–8.0 5.77 g/l (64% inhibition) – Yes [57]
Pig manure CSTR 9.4 g VS/l/d 51 ◦ C 8.0 11 g/l (50% inhibition) 1450 mg/l (50% inhibition) Yes [59]
Organic fraction of MSW Batch – Mesophilic– 215–468 mg/l (50% inhibition) – [67]
thermophilic

Critical concentration is defined as the concentration at which inhibition starts.

TAN, total ammonia nitrogen; FAN, free ammonia nitrogen.

903
904 O. Yenigün, B. Demirel / Process Biochemistry 48 (2013) 901–911
The effect of pH at various TAN levels was investigated dur-
ing high-solids sludge digestion [22]. The reactors were 120 ml
glass bottles. The pH range was chosen to vary between 6.5 and
9.0, while TAN concentration range varied from 0 to 6000 mg/l.
At all pH levels, 5000 mg TAN/l reduced the methanogenic activ-
ity by 50%. At pH of 9.0 and FAN concentration of 900 mg/l, the
digester could be operated satisfactorily and the authors con-
cluded that the TA, rather than FA, was a more significant factor
in inhibiting methanogenic activity in high-solids digestion. When
lag (acclimation) periods were investigated with respect to TAN
and FAN concentrations, it was found that within a pH range of
6.5–9.0, acclimation periods and ammonium ion concentrations
were not correlated, whereas acclimation periods increased with
increasing FAN levels. This finding suggested that the FA was a
more sensitive factor for the inhibition of an unacclimated bac-
terial system. Another high-solids digestion study was reported,
in which the substrate was chicken manure, with total solids
ranging from 5.0 to 21.7% [23]. In reactors with unacclimated
sewage sludge, the methanogenesis was severely inhibited after
250 mg/l FAN. Municipal solid waste and sludge were digested at
a slightly higher mesophilic temperature of 39 ◦ C with unadapted
seed sludge [24]. The TAN concentration causing 50% inhibition was
around 2400 mg/kg (141 mM), and the total failure took place at
2800 mg/kg (165 mM).
Anaerobic glucose degradation in batch reactors was inhibited
by about 70% at 3500 mg/l TAN concentration and at pH of 8.0 [25].
Although a seed sludge, which was acclimated to 3100 mg TAN/l
was used, glucose consuming acidogenic bacteria could not stand
these TAN concentrations. In another study with a pH value of 8.1,
stable reactor operation was possible with 587 mg FAN/l (4718 mg
TAN/l), using fishery effluents as substrate [26]. Here ammonia
adapted anaerobic filter reactors were used and NH4 Cl was added
to test the extent of inhibition. Using a synthetic substrate com-
posed of acetic, propionic, butyric acids and yeast extract, ammonia
inhibition was investigated in five laboratory digesters started-up
with different seed sludges [27]. Ammonium hydroxide was grad-
ually added to elevate TAN concentrations from 1000 mg/l up to
6000 mg/l, and similarly, the initial pH of 7.7 was gradually raised
to 8.1, with FAN concentration reaching to 800 mg/l. At the end of
450 days of experiment, all five reactors were stably in operation
although decreasing COD removal efficiencies were obtained.
Sludge from juvenile salmon hatcheries was treated in a lab-
oratory size anaerobic digester using inoculum from a digester
operated using saline fish farming sludge [28]. A severe inhibi-
tion was reported at TAN of 6390–7460 mg/l at pH being around
7.4–7.5, corresponding to 197–230 mg FAN/l at pH 7.5. How-
ever, the concentrations of LCFA and VFAs were also high and a
shared responsibility of inhibition was the conclusion. Alfalfa silage
(minced and mixed with water) was used as the substrate in a study,
where three 10 l anaerobic digesters were operated under different
loading conditions [29]. The pH varied between 7.2 and 7.8 and ini-
tial TAN concentration was 1600 mg/l, which went up to 5000 mg/l,
causing severe inhibition in one of the digesters in which effluent
recirculation was employed.
Anaerobic digestion of the solid fraction of pig slurry was inves-
tigated using the seed from a sewage sludge digester [30]. FAN
concentrations in the batch digesters reached as high as 388 mg/l.
A combination of diluted solid slaughterhouse wastes and organic
fraction of municipal solid waste was treated in laboratory digesters
with gradual increase of organic loading rates and with ammonium
acclimated seed sludge [31]. At pH values varying between 7.5 and
8.0, stable reactor operation could be achieved with TAN levels of
4100 mg/l.
Waste sludge from a high load activated sludge plant treating
slaughterhouse wastes was co-digested with various meat indus-
try wastes (cow manure, ruminal wastes and pig-cow waste slurry)
at different mixing ratios in 1 l batch reactors [32]. The seed was
a digested material from a conventional anaerobic digester, and
it was not particularly adapted to high ammonia concentrations.
It was found out by the authors that 50% inhibition of methane
production occurred at a co-digestion ratio of 90:10 waste sludge
to pig-cattle slurry with a TAN concentration of 1130 mg/l (corre-
sponding to 70 mg FAN/l) and 80% inhibition at 65:35 waste sludge
to pig-cattle slurry ratio with a TAN concentration of 2770 mg/l
(230 mg FAN/l). Swine waste was used as the substrate in another
study, in which anaerobic digestion was carried out in laboratory
scale anaerobic squenching batch reactors [33]. The seed was a
sludge from a digester treating pre-acidified brewery wastewa-
ter. At a sub-mesophilic temperature of 25 ◦ C, the reactors with
4000 mg/l TAN produced 45% lower methane yields compared with
the reactors containing 1600 mg TAN/l (pH varying between 7.5 and
7.6). Raising the operating temperature to 35 ◦ C, the methane yields
of reactors increased but with only 13% lower methane yields for
the reactors containing 4000 mg/l TAN concentrations. During this
stage of operation, the methane yields were reported to change
between 0.31 and 0.36 l CH4 /g VSfed . Efficient digester performance
with a TAN concentration of 5200 mg/l could be obtained near the
end of the operational period.
Anaerobic digestion of meat and bone meal (animal food) was
carried out at different solid contents of up to 10% in laboratory
scale batch reactors [34]. Methane production was inhibited in
the digesters with 5 and 10% solid substrate. FAN concentrations
reached up to 1000 mg/l, and the authors concluded that at such
high FAN concentrations, Archaea could still sustain activity and
inhibition was reversible. Leather fleshing wastes were co-digested
with organic fraction of municipal solid waste using various carbon
to nitrogen (C:N) ratios and controlled pH values ranging between
4.5 and 8.5 [35]. Biogas yields were satisfactory in general except
for the reactor with low carbon to nitrogen ratio of 5 (TAN concen-
tration here was 3600 mg/l) and the reactor with leather fleshing
waste only (TAN concentration here was 4289 mg/l).
Organic municipal wastes were digested in laboratory-scale
reactors with gradual increases of TAN concentrations from 800
to 6900 mg/l [36]. At pH of 7.9 and 3300 mg TAN/l concentration,
the methane yields were optimum. However, as TAN went up to
5500 mg/l, 50% reduction in methane production was observed.
Wastewaters obtained from settling and filtering of piggery slurry
were treated anaerobically in serum bottles with additions of VFA,
nutrient solutions and inoculum sludge obtained from full-scale
anaerobic digesters [37]. FAN concentrations of around 40 mg/l
caused 50% inhibition in methanogenic activity. In a parallel study,
the liquid fraction of settled pig slurry was fed to an upflow anaero-
bic sludge blanket (UASB) reactor containing ammonia unadapted
seed sludge [38]. The OLR was gradually increased to prevent
ammonia inhibition and thus, FAN levels up to 375 mg/l did not
affect the COD removal efficiency.
Ground chicken feathers (380–440 g) were fed to 42 l anaerobic
digesters and co-digested with ammonia adapted swine manure
and slaughterhouse sludge (35 l each) at a sub-mesophilic tem-
perature of 25 ◦ C and a pH between 7.6 and 7.9 [39]. The initial
TAN concentrations of swine manure and slaughterhouse sludge
were 4800 mg/l and 3200 mg/l, respectively. Methane production
in digesters with feathers was substantially higher in comparison
to those in the control digesters without feathers. Besides, TAN con-
centrations increased to 6900 mg/l and 3500 mg/l in feather fed
digesters with swine manure and slaughterhouse sludge, respec-
tively. No inhibition could be observed. Glucose was used as the
substrate in another batch study, where inocula from various full-
scale anaerobic reactors were operated [40]. At pH values ranging
between 7.6 and 8.4, 50% inhibition was observed at a TAN con-
centration of about 5000 mg/l. Dewatered sludge was digested in
laboratory size reactors using inoculum from an anaerobic digester
 O. Yenigün, B. Demirel / Process Biochemistry 48 (2013) 901–911
of a conventional wastewater treatment plant [41]. Biogas produc-
tion was moderately inhibited at TAN levels of 3000 mg/l (400 mg
FAN/l at pH 8.0) and severe inhibition was observed when TAN con-
centration was 4500 mg/l with corresponding FAN concentration of
800 mg/l.
The impacts of transient overloads on the performance of
a laboratory-scale mesophilic anaerobic ammonium oxidizing
anaerobic baffled reactor was investigated through increasing
substrate concentration or influent flow rate using synthetic
wastewater [42]. The authors reported that when the concentra-
tions of ammonium and nitrite exceeded threshold, they became
inhibitors. During shock loading, the concentration of free ammonia
ranged between 8.2 and 97.1 mg/l. The effect of ammonia inhibition
was studied during the enhanced anaerobic treatment of digested
effluent from a full-scale CSTR using a laboratory-scale mesophilic
anaerobic reactor [43]. The reactor was operated within an OLR
range of 1.5–3.5 kg COD/m3 /d at a HRT of 1.5 d. It was reported
by the authors that the SCOD/TAN (soluble COD/total ammonia
nitrogen) ratio was a key parameter and the threshold value of the
SCOD/TAN was determined to be 2.4 (TAN concentration of about
1250 mg/l) at an influent pH of 8.5–9.0.
The impact of ammonia removal from piggery wastewater by
ammonia stripping was investigated as a pre-treatment method in
order to increase methane yield from anaerobic digestion of piggery
wastewater [44]. When ammonia was air-stripped at pH values of
9.5 and 10, significant improvements in methane yields from anaer-
obic digestion were achieved, also preventing the process failure.
Trace element demand of anaerobic digesters at elevated ammonia
concentrations fed with food waste was studied [45]. It was con-
cluded that both selenium (Se) and cobalt (Co) were required for
interspecies electron transfer at high ammonia levels to prevent
accumulation of propionic acid.
The effects of Fe, HCl and trace element addition on process
stability of mesophilic anaerobic digestion of pig slaughterhouse
waste was investigated using laboratory-scale CSTRs in order to
achieve higher OLRs [46]. The concentrations of TAN in all reac-
tors generally ranged from 1.5 to 2.5 g/l, while it increased to 3.5 g/l
in one of the reactors. The concentrations of FAN were calculated
to be between 0.05 and 0.22 g/l in all reactors. These values did not
cause inhibition in any of the reactors in this work. The authors also
reported that addition of Fe, HCl and trace element allowed oper-
ation of mesophilic CSTRs at higher OLRs around 2.25 kg VS/m3 /d.
Mesophilic anaerobic batch co-digestion of dairy manure, chicken
manure and wheat straw were investigated in a laboratory-scale
study, particularly focusing on the C:N ratio [47]. The authors
reported that the C:N ratios of 25, 30, and 35 provided low and
stable TAN and FAN concentrations of 712, 604, and 444 mg/l, and
9.1, 7.5, and 2.2 mg/l, respectively. On the contrary, the C:N ratio
of 15 resulted in higher TAN and FAN concentrations of 2614 and
223 mg/respectively. The optimization of the C:N ratio resulted in
a stable co-digestion process.
4. Thermophilic digestion
Thermophilic anaerobic digestion processes are carried out
within a temperature range of 45–65 ◦ C. Thermophilic process has
advantages over mesophilic digestion for achieving higher rates of
digestion, greater conversion of waste organics to gas, faster solid-
liquid separation, and minimization of bacterial and viral pathogen
accumulation [48]. Due to the high energy density of the sub-
strates and high loading rates, self-heating effects have brought
about an increase in operating temperatures from mesophilic to
sub-thermophilic and thermophilic temperatures more recently
[49]. The disadvantages of thermophilic operation are poor super-
natant quality and poor process stability [50]. One of the earlier
905
studies investigating the effect of various operating parameters on
the thermophilic anaerobic digestion of cattle wastes concluded
that at high loading rates and pH levels above 7.8, the process could
be inhibited by TAN concentrations of above 1700 mg/l [51]. This
was in agreement with inhibitory ammonia concentrations found
in earlier mesophilic studies. Diluted cow manure was digested in
a semi-continuous 120 l reactor, where the inoculum was fresh
cow manure [52]. Inhibition started at 1700 mg TAN/l concen-
tration. Cattle manure was used as the substrate in continuously
fed laboratory scale reactors in another work [53]. NH4 Cl was
gradually administered for adaptation and the pH was kept con-
stant. The first signs of inhibition occurred at a TAN concentration
of 4000 mg/l, corresponding to 900 mg FAN/l. The process insta-
bility due to ammonia led to VFA accumulation, which lowered
the pH. Therefore, the decreased FAN concentration eventually
resulted in a stable, but lowered methane yield, which was called
by the authors as the “inhibited steady state”. A parallel study
by the same researchers investigating the effect of temperature
changes within the thermophilic range revealed that with adapted
inoculum, 6000 mg TAN/l could be tolerated at 45 ◦ C [54]. The key
conclusion here was that the decreasing the process temperature
overcame ammonia inhibition. In a follow-up research, digestion
of swine manure was investigated in laboratory scale batch and
continuously stirred tank reactors (CSTRs) [55]. A severe inhibition
and a very low methane yield were observed at a TAN concentra-
tion of 6000 mg/l and the methane yield decreased with increasing
temperature (within the thermophilic range). It was concluded
that a threshold of 1100 mg/l FAN concentration was required for
introducing inhibition.
Biodegradable organic fraction of municipal solid waste was
digested in a pilot scale high-rate anaerobic digester at different
mass retention times and gradually increasing ammonia concen-
trations [56]. The start of ammonia inhibition, 50% inhibition and
total reactor failure occurred at 1000, 1500 and 2500 mg/l TAN
concentrations, respectively. Impacts of stepwise increases in TAN
concentrations were studied on anaerobic digestion of soluble non-
fat dry milk [57]. A TAN concentration of 4920 mg/l caused 39%
inhibition in specific methanogenic activity.
Diluted cattle manure containing 1300–1400 mg TAN/l was
digested in laboratory-scale CSTRs [58]. TAN concentrations were
gradually elevated with pulse additions of NH4 Cl and tryptone. A
38% reduction in methane yield was associated with 6200 mg/l
TAN concentration. Pig manure was digested in laboratory scale
CSTRs with inoculum adapted to high concentrations of ammonia
[59]. With periodic pulse additions of NH4 Cl, ammonia inhibition
levels were investigated at pH ranges between 7.91 and 8.03. A
50% decrease in methane yield was observed at 11,000 mg TAN/l,
corresponding to 1450 mg FAN/l. At these inhibitory levels, VFA
concentrations did not vary in comparison to those in the control
reactor (without the pulse additions), a result in contradiction to
those in previous studies [53,60].
5. Mesophilic vs thermophilic
Some investigators have exclusively studied the effect of tem-
perature on anaerobic digestion in relation to ammonia inhibition.
Lower biogas production at thermophilic temperatures in compar-
ison to mesophilic temperatures was attributed to FA inhibition
[61]. Hashimoto studied the anaerobic digestion of beef cattle
manure by gradual additions of NH4 Cl [60]. In unadapted digestion,
ammonia inhibition started at 2500 mg TAN/l both for mesophilic
and thermophilic operations. However, as FA increased with tem-
perature, the calculated FAN concentrations were 30 mg/l for the
mesophilic and 200 mg/l for the thermophilic digesters. In adapted
operations, 4000 mg TAN/l could be tolerated.
906 O. Yenigün, B. Demirel / Process Biochemistry 48 (2013) 901–911
The organic fraction of household waste was digested in con-
tinuous CSTRs at mesophilic and thermophilic temperatures, with
ammonia unadapted inoculum [62,63]. Ammonium ion was added
to the assays at various concentrations. At pH of 7.5, 220 mg
FAN/l caused a 50% inhibition of mesophilic methane production,
whereas 690 mg FAN/l caused a 50% inhibition at thermophilic con-
ditions. As for peptone deamination and methane generation, 50%
inhibition was observed with 88–92 mg FAN/l in the mesophilic
and 251–297 mg FAN/l in the thermophilic assays. Poultry manure
was digested in serum vials at gradually increasing ammonium con-
centrations [64]. At about 4000 mg TAN/l, independent of operating
temperatures, 50% inhibition took place.
Effect of stepwise and pulse temperature increase from 35 to
55 ◦ C was investigated in pilot-scale and laboratory-scale reactors
[65]. The substrates were maize silage and pig manure, and ammo-
nia adapted bioslurries were used as seed. At 45 ◦ C, the critical TAN
concentration was found to be 5500 mg/l. Temperature increases
caused process disturbances such as drops in methane yields and
propionic acid accumulation. Two laboratory scale CSTRs operat-
ing at 35 and 55 ◦ C digested secondary residuals obtained from
high rate anaerobic digestion of brewery wastewaters [66]. FAN
concentrations climbed up to 640 mg/l in the thermophilic reac-
tor, but could be lowered to 358 mg/l by decreasing pH from 7.6 to
7.3, before any sign of inhibition. Both reactors exhibited similar
methane yields rendering the mesophilic reactor superior in terms
of energy consumption.
The organic fraction of municipal solid waste was digested in
reactors with supernatant recycling [67]. The methane yield was
inhibited by 50% at FAN concentrations of 215 and 468 mg/l under
mesophilic and thermophilic conditions, corresponding to 3860
and 5600 mg TAN/l, respectively.
6. Microbiology
Anaerobic microbial communities can be classified into
two domains, namely bacteria and Archaea [68]. Four major
metabolic groups are involved in a stable ongoing anaerobic
conversion process. These groups are hydrolytic-fermentative
bacteria, proton-reducing acetogenic bacteria, hydrogenotrophic
methanogens and acetoclastic methanogens [69]. During anaerobic
degradation of a particulate substrate, carbohydrates, proteins and
lipids are firstly hydrolyzed to organic monomers by hydrolytic-
fermentative bacteria. The carbonic products of these reactions
are either acetate or propionate and butyrate, which are subse-
quently converted to acetate and hydrogen by acetogenic bacteria.
During methanogenesis, methane is produced from acetate by ace-
toclastic methanogens or from hydrogen and carbon dioxide by
hydrogenotrophic methanogens. Methyl groups (methylamines,
methanol) are also converted into methane in this stage. A classifi-
cation of the methanogens reported in this paper is given in Table 2
[68]. The performance of an anaerobic reactor is directly associ-
ated with the structure of the microbial community present within
the reactor. The operational and environmental parameters of the
process eventually influence the fate of the microbial community.
Therefore, the concentrations of ammonia, coming directly with the
substrate or produced during anaerobic degradation, is a parame-
ter that has to be monitored carefully in order to achieve a stable
and efficient process, since excess concentrations of ammonia have
been often reported to inhibit the activity of the anaerobic microbial
communities in anaerobic reactors treating a variety of substrates.
A summary of selected works from literature is also given in Table 3.
Methanobacterium formicicum was reported to be partly
inhibited at a TAN concentration of 3000 mg/l at pH 7.1, while com-
plete inhibition took place at 4000 mg TAN/l [7]. Methanospirillum
hungatei exposed to ammonia lost up to 98% of the cytoplasmic K+
Table 2
The classification of methanogens.
Class I. Methanobacteria (known to grow on H2 /CO2 and formate as C source)
Order I. Methanobacteriales
Family I. Methanobacteriaceae
Genus I. Methanobacterium
Genus II. Methanobrevibacter
Family II. Methanothermaceae
Class II. Methanococci (known to grow on H2 /CO2 and formate as C source)
Order I. Methanococcales
Family I. Methanococcaceae
Family II. Methanocaldococcaceae
Class III. Methanomicrobia (known to grow on H2 /CO2 and formate as C source)
Order I. Methanomicrobiales
Family I. Methanomicrobiaceae
Genus IV. Methanogenium
Family II. Methanocorpusculaceae
Family III. Methanospirillaceae
Genus I. Methanospirillum
Order II. Methanosarcinales (known to be acetato- and methylotrophic)
Family I. Methansarcinaceae
Genus I. Methanosarcina
Family II. Methanosaetaceae
Genus I. Methanosaeta
via an ammonia/K+ exchange reaction [70]. NH4 OH or methylamine
additions were most effective resulting in depletion of K+ at alka-
line pH, indicating that ammonia was the chemical species crossing
the membrane. The authors concluded that methanogenesis was
sensitive to both pH of the cytoplasm and the medium.
The impact of TAN concentration between 680 and 2601 mg/l
on methanogenic sludge was investigated in batch experiments
[71]. The accumulation of acetate at a TAN concentration at
2601 mg/l showed that, above the threshold concentration of
1700 mg/l, ammonia-N had relatively more negative effect on
acetate-consuming methanogens than on hydrogen consuming
methanogens; although the opposite was true for TAN levels below
this threshold.
Transport of K+ in M. hungatei pretreated with ammonia was
studied and it was observed that the methanogenic activity was
lost, but it could be recovered through addition of Ca+2 or Mg+2
[72]. Ca+2 or Mg+2 addition seemed to activate cells so that they
could make ATP and transport K+ . The toxicity of ammonia to the
growth of several methanogenic bacteria was evaluated in terms of
an ammonia/potassium exchange reaction and in terms of inhibi-
tion of methanogenesis [73]. Growth of Methanobrevibacter smithii,
Methanobrevibacter arboriphilus and Methanobacterium strain G2R
was normal in media containing up to 400 mM NH4 Cl (5.55 g/l TAN),
with no observation of inhibition of methane production. How-
ever methane synthesis from Methanothrix concilii was completely
inhibited at TAN levels of 560 mg/l, while methane formation
from Methanosarcina barkeri was not inhibited at 2800 mg TAN/l.
Another observation was that certain cations countered the toxic
effects which ammonia had on methane synthesis, notably Ca2+ in
M. concilii and Na+ in M. barkeri.
During thermophilic anaerobic digestion of livestock wastes,
inhibition at high ammonia concentrations was reported to influ-
ence the formation of methane from H2 and CO2 [74]. Ammonia
had only a minor effect on methane formation from acetate, which
could be shown by the independence of the specific growth rate of
acetate-consuming methanogens from a TAN concentration up to
4500 mg/l. The effect of NH4 Cl on methanogenesis by pure cultures
of M. hungatei, M. barkeri, Methanobacterium thermoautotrophicum,
and M. formicicum at a pH of 6.5 was investigated [75]. M. barkeri, M.
thermoautotrophicum, and M. formicicum were found to be resistant
to ammonia concentrations over 10,000 mg TAN/l, while M. hun-
gatei was more sensitive with 50% of inhibition of methanogenesis
at 4200 mg TAN/l.
 O. Yenigün, B. Demirel / Process Biochemistry 48 (2013) 901–911
Table 3
A summary of research findings for impact of ammonia on Archaea in anaerobic digestion.
Substrate Temperature pH Critical TAN conc.
(◦ C) or as specified
(mg/l)
– 38 ◦ C – 4000 (100% inhibition)
– – 6.5 4200 (50% inhibition)
– 60 ◦ C 6.9 4000
7.0 6000 (50% inhibition)
Swine waste 25 ◦ C – ≥3500
Synthetic wastewater 35 ◦ C 8.0 6000 (100% inhibition)
Synthetic wastewater 35 ◦ C 7.7 – >100 (100% inhibition)
Sodium acetate – – 7000 (acclimated)
5000 (non-acclimated)
Cattle excreta + olive 37/55 ◦ C – 1300
mill waste
The effect of ammonia on the maximum growth rate (m ) of
hydrogen-consuming methanogens was studied at different pH
and temperature values [76]. The maximum inhibited growth rate
of hydrogenotrophic methanogens present in sludge appeared to be
0.126 h−1 at pH and temperature of 7.0 and 37 ◦ C, respectively. The
increase in pH from 7.0 to 7.8 at 37 ◦ C seemed to enhance ammonia
inhibition. During anaerobic digestion of dairy cattle manure, the
methanogenic community was inhibited at high TAN concentra-
tions as indicated by changes in relative rates of acetate utilization
[77]. The maximum growth rate (m ) of acetoclastic methanogens
was investigated during anaerobic digestion of poultry manure at
TAN concentrations from 7700 to 10,400 mg/l and a pH of between
7.80 and 7.93 [78]. It was reported that pH and the TAN concentra-
tion were the predominant inhibition factors for the acetoclastic
methanogens.
The effects of ammonia on pure cultures of thermophilic
hydrogen-utilizing methanogens including M. thermoautotroph-
icum, Methanobacterium thermoformicicum, Methanogenium sp. and
a putative M. thermoautotrophicum were investigated in batch tests
[79]. For all strains, initial inhibition started at 3000–4000 mg TAN/l
and 50% decrease in growth rates were observed at around 6000 mg
TAN/l. In addition, slow growth and aggregate formation of M.
thermoformicicum could be observed at 9000 TAN/l. A further con-
clusion of this study was that the thermophilic hydrogen utilizers
are less sensitive towards ammonia then their mesophilic counter-
parts. During thermophilic anaerobic digestion of livestock waste,
ammonia concentrations of 4000 mg N/l or more was found to
inhibit digestion of cattle manure. In another previous study, M.
barkeri was found to be more sensitive to excess ammonia lev-
els than Methanobacterium bryantii [80]. TAN concentration higher
than 5000 mg/l was reported to inhibit thermophilic anaerobic
digestion of cattle manure in UASB reactors [81]. The authors also
stated that the acetoclastic methanogens showed a higher sensitiv-
ity to the presence of ammonia in comparison to hydrogenotrophic
methanogens.
During operation of a full-scale anaerobic sequencing batch
reactor (ASBR) of 600 m3 volume for treating swine waste,
methanogenic population changes were monitored at a HRT of
15 days and at a TAN concentration of about 3600 mg/l [82].
The 16S ribosomal RNA (rRNA) levels of the acetate-utilizing
methanogens of the genus Methanosarcina was observed to
decrease from 3.8 to 1.2% (expressed as a percentage of the
total 16S rRNA levels) during this period, while the 16S rRNA
levels of Methanosaeta concilii remained below 2.2%. Methane
production and the reactor performance were not affected as
the 16S rRNA levels of the hydrogen-utilizing methanogens of
the order Methanomicrobiales increased from 2.3 to 7.0%. The
authors concluded that the anaerobic digestion of swine waste
907
Critical FAN conc. Organisms Molecular Reference
or as specified affected/present technique
(mg/l) applied
– Methanobacterium formicicum – [7]
– Methanospirillum hungatei – [75]
– Methanobacterium [79]
Thermoformicucum (present)
– Methanomicrobiales 16S rRNA gene [82]
analysis
Methanosarcina
>700 (100% inhibition) Methanosarcina FISH/DGGE [85]
Methanosaeta-related species FISH/DGGE [86]
– Methanosarcinaceae spp. FISH [93]
Methanococcales spp.
– Methanosarcina PCR/16S rRNA [94]
gene analysis
could be sustainable at TAN levels exceeding 3500 mg/l, due
to the ability of the hydrogen-utilizing methanogens of the
order Methanomicrobiales to be active at these high TAN lev-
els.
Microbial diversity of two different laboratory-scale anaero-
bic reactors (namely an UASB and a hybrid bed) treating a young
landfill leachate with TAN concentrations up to 2700 mg/l, was
investigated using FISH (fluorescent in situ hybridization) and
DGGE (denaturing gel gradient electrophoresis) techniques [83].
During low acetate levels in the reactors, the stability of operation
was supported by the abundance of Methanosaeta, and in both reac-
tors the presence of Methanobacteriaceae was also detected, while
other methanogenic species were not encountered. The effect of
ammonia on methanogenic activity of anaerobic films enriched by
methylaminotrophic methane producing Archaea was studied in
batch tests operated at a pH of 7.5 and a temperature of 37 ◦ C [84].
The concentrations of ammonia used in the experiments were 48.8,
73.8, 98.8, 148.8, 248.8, 448.8, and 848.8 mg FAN/l, respectively.
The findings indicated that then highest methanogenic activity
took place at 48.8 mg FAN/l, and above 148.8 mg FAN/l inhibition
could easily be observed. The lowest methanogenic activity was at
848.8 mg FAN/l.
During investigation of high free ammonia (FAN) concentra-
tions on the performance of UASB reactors operated with synthetic
wastewater and within a TAN concentration ranging from 1000
to 6000 mg/l, the results of FISH analysis showed that propionate
degrading acetogenic bacteria was more sensitive to free ammonia
than Archaea and they were significantly inhibited at a FAN of above
200 mg/l [27]. The impact of FAN concentrations up to 750 mg/l
was investigated in laboratory-scale UASB reactors and the
methanogenic population was monitored by using FISH and DGGE
[85]. The FISH results indicated the abundance of Methanosarcina-
like acetoclastic methanogens in reactors. Increase in FAN level
above 700 mg/l resulted in disintegration of large Methanosarcina
clusters. It was also reported by the same authors that, after a
start-up period of 140 days in mesophilic laboratory-scale UASB
reactors, when FAN concentration was gradually increased from
50 to 130 mg/l, Methanosaeta-related species coming from the
seed sludge have noticeably lost their activities and their filamen-
tous forms deteriorated when FAN level exceeded 100 mg/l [86].
Although some Methanobacterium and Methanospirillium-related
hydrogenotrophic methanogens could then be detected, at the
end of start-up period mainly Methanosarcina-like acetoclastic
methanogens were abundant in reactors. The authors concluded
that the coccus shaped Methanosarcina species were more resis-
tant to elevated FAN levels than rod shaped Methanosaeta cells as
an advantage of their high volume to surface ratio and formation
of big clusters.
908 O. Yenigün, B. Demirel / Process Biochemistry 48 (2013) 901–911
The effects of ammonia concentration and zeolite addition on
the specific methanogenic activity (SMA) of anaerobic sludges were
investigated in batch tests conducted at a pH of 6.8–7.2 and at
35 ◦ C [87]. Piggery, malting production and urban sludges obtained
from full-scale anaerobic reactors were employed in this work. It
was reported that piggery sludge was mostly affected by ammo-
nia concentrations and addition of zeolite at doses from 0.01 to
0.1 g/g VSS reduced the inhibitory effect of ammonium. The micro-
bial population detected by 16S rRNA technique was composed of
Methanococcaceae, Methanosarcina, and Methanosaeta.
Mesophilic anaerobic digesters of 3.6 l were continuously run
at 35 ◦ C to treat poultry waste [88]. Increasing ammonia levels
and organic loading rate did not result in a microbial community
shift, however, Crenarchaeota archaeal populations were detected
in reactors by PCR instead of Euryarchaeota methanogens. The
impact of carbon textile fibers (CTF) addition to thermophilic
methanogenic bioreactors was investigated under increasing
ammonia concentrations [89]. The reactors were operated at a pH
of 7.8 and synthetic garbage slurry was used as substrate. Under
3000 mg TAN/l, the methanogenic archaea was dominated by
Methanobacterium sp. and Methanosarcina sp. as detected by 16S
rRNA gene analysis. In the control reactor without CFT addition,
Methanosarcina sp. was inhibited and washed out at a TAN con-
centration of 1500 mg/l, while Methanobacterium sp. continued
to proliferate. According to the results obtained, the authors
concluded that CFT enables proliferation of the methanogens by
preventing ammonia inhibition. In a recent work, Methanosarcina
sp. was reported to be quite robust to ammonia toxicity when
compared with other methanogens [90]. Methanosarcina sp. could
tolerate a TAN of up to 7000 mg/l. Bioaugmentation of syntrophic
acetate-oxidizing culture in biogas reactors fed with whole stillage
and cattle manure [91]. Mesophilic laboratory-scale anaerobic
reactors were operated under gradually increasing ammonia
concentrations from 1.5 to 11 g TAN/l, and a fixed volume of syn-
trophic acetate-oxidizing culture was daily added to the reactors,
however, addition of the culture did not seem to affect the stability
and performance of the biogas reactors, and the reactors eventu-
ally deteriorated at high ammonia concentrations. In a previous
study, it was reported that during a gradual increase in ammonia
concentration up to 7 g TAN/l in a mesophilic semi-continuous
laboratory-scale anaerobic digester fed with source-separated
organic fraction of municipal solid waste at a HRT of 30 days,
a shift from the acetoclastic methanogenesis to syntrophic
acetate oxidation took place when the TAN concentration rose
above 3 g/l [92].
In a recent study, the effect of ammonium and acetate on
methanogenic pathway and methanogenic community was inves-
tigated using batch tests and FISH technique [93]. When the
culture wax acclimatized to acetate and ammonia, thermophilic
cultures were reported to change their acetate bioconversion
pathway from syntrophic acetate oxidation with subsequent
hydrogenotrophic methanogenesis to acetoclastic methanogene-
sis mediated by Methanosarcinaceae spp. The adapted mesophilic
culture showed no pathway shift. At high ammonia levels of 7 g
TAN/l, the acetoclastic Methanosarcinaceae spp. was determined
to be the dominant methanogen in nonacclimatized thermophilic
culture. Mesophilic and thermophilic Methanococcales spp. from
nonacclimatized cultures were found to be tolerant to ammonia
concentrations up to 5 g TAN/l.
7. Recovery after inhibition
Previous experimental studies indicated that the ammonia inhi-
bition could be removed through lowering of pH and subsequent
decrease of free ammonia concentration, suggesting that, although
digester toxicity might indirectly be related to free ammonia
concentration, it was directly related to unionized volatile acid
concentration [11].
Serum bottle experiments were conducted at 35 ◦ C to evalu-
ate the reversibility characteristics of methane formation systems
exposed to ammonium concentrations of 4000, 8000, and
24,000 mg/l TAN [95]. The authors observed that the ammo-
nium toxicity was very reversible and if the ammonium in the
supernatant were removed, the system could rapidly recover to
full biogas production. The threshold dose for ammonium was
reported to be less than 2500 mg/l. In another early study, it
was reported that the TAN concentrations at 1900–2000 mg/l
caused failure of methanogenesis. However, after an adaptation
period, methanogenesis seemed to be possible even at higher TAN
concentrations [14]. After the adaptation period, the maximum
specific methanogenic activity at a TAN concentration of 2315 mg/l
was higher than the maximum specific methanogenic activity at
1900 mg TAN/l.
Anaerobic digestion of potato juice at extreme concentrations
of ammonia was investigated in a laboratory-scale study using
reactors with working volumes of 4.5 l operated at 30 ◦ C [15].
The highest TAN concentration, at which methanogenesis was
found to be possible, was 11.8 g/l. The authors stated that after
being adapted, which meant having gained the ability to gener-
ate methane at ammonia concentrations exceeding the threshold
level, the microbial community could produce methane again at a
TAN concentration of 11.8 g/l, showing that toxicity was reversible
even at extremely high ammonia concentrations. Furthermore, the
acidogenic population seemed to be hardly affected within a TAN
concentration range of 4051–5734 mg/l, while the methanogenic
population lost 56.5% of its activity under these conditions.
Anaerobic treatment of sea food processing wastewater was
investigated using an industrial pilot plant operated at 37 ◦ C
[19]. During the operation, TAN and FAN concentrations varied
between 1 and 3 g/l, and 25 and 150 mg/l, respectively. Higher
protein content in influent wastewater increased the forma-
tion of FA, and when the free ammonia concentrations reached
300 mg/l, inhibition took place. When the protein content in
the influent wastewater was decreased, inhibition could then be
reversed and normal treatment efficiencies could be achieved after
10 days.
The influence of ammonium on anaerobic treatment of poultry
manure was investigated in a laboratory-scale work [96]. It was
observed that within a NH4 Cl concentration range of 2 and 10 g/l,
production of methane was not affected, but at higher concentra-
tions from 10 to 30 g/l, methane production significantly decreased.
The authors tried to recover the system through addition of 10%
(w/v) powdered phosphorite ore and this external supplemen-
tation increased production of biogas and methane up to NH4 Cl
concentration of 30 g/l. However, at concentrations above 50 g/l,
addition of phosphorite even did not help to recover inhibition of
methanogenesis.
During thermophilic anaerobic digestion of swine manure, a low
methane yield of 67 ml CH4 /g VS was achieved at a TAN level of 6 g/l
and several methods were investigated to increase the methane
yield [97]. Through addition of 1.5% (w/w) activated carbon, 10%
(w/w) glauconite or 1.5% (w/w) activated carbon and 10% (w/w)
glauconite, the methane yield increased to 126, 90 and 195 ml
CH4 /g VS, respectively.
Thermophilic pilot-scale biogas reactors were operated using
the organic fraction of MSW as substrate in order to investigate
ammonia inhibition [98]. The authors reported that the inhibition
took place at a TAN concentration of 1200 mg/l. Dilution of digester
content with water or adjustment of substrate C:N ratio of 30–40
were suggested to mitigate ammonia inhibition. In another work
[35], adjustment of C:N ratio and pH to maximize biogas production
 O. Yenigün, B. Demirel / Process Biochemistry 48 (2013) 901–911
and keep TAN and FAN concentrations under control gave best
results when C:N ratio was 15 at a pH of 6.5.
During thermophilic batch and CSTR anaerobic treatment of cat-
tle manure, dilution of the biomass with water, reactor effluent
and manure seemed to improve the recovery speed and stability of
an ammonia-inhibited biogas digester fed with cattle manure [99].
Among these recovery options, dilution with manure provided a
high production of methane, while dilution with reactor effluent
seemed to provide the most stable recovery process.
In a more recent work, 500 mg/l FA was reported to result in
inhibition during mesophilic batch anaerobic digestion of meat
and bone meal at TS concentrations of 10% [34]. According to the
authors, with a decrease of pH from 8.0 to 7.5, inhibition could be
reversed and the methanogens could adapt themselves to higher
FA concentrations up to 998 mg/l.
Thermophilic anaerobic batch experiments were conducted
using digested piggery effluent in order to mitigate FA inhibition
[100]. Based on the experimental findings, the authors reported
that the reduction of pH from initial value of 8.3–6.5, and zeo-
lite treatment ranging from 10 to 20 g/l seemed to be effective
options to mitigate ammonia inhibition during thermophilic anaer-
obic treatment of piggery wastewater.
During thermophilic pilot-scale anaerobic digestion of vari-
ous substrates such as food waste, fruit and vegetable waste,
paper waste, and green waste up to OLRs of 7–10 kg VS/m3 /d and
retention time up to 19 days, FA accumulation/inhibition was
encountered in the system [101]. A carbon to nitrogen (C:N) ratio
of 32 in feedstock seemed to result in 30% less ammonia formation
in the digester than a C:N ratio of 27. The authors recommended
changing the C:N ratio, and a higher OLR in order to reduce and
overcome ammonia inhibition.
The feasibility of continuous ammonia removal in an anaerobic
digestion process was evaluated using a hollow fiber membrane
contactor module [102]. Mesophilic laboratory-scale anaerobic
reactors were run using slaughterhouse waste and within TAN
concentrations from 6 to 7.4 g/l. Use of hollow fiber membrane
contactor helped in reduction of FAN concentration by about 70%,
providing a more stable operation.
8. Conclusions
Anaerobic digestion process is not only an efficient tool for waste
management, but also an important technology for recovery of bio-
gas from organic substrates as a source of renewable energy as long
as the process is carefully controlled and monitored. On the other
hand, inhibitory compounds such as FAN can be formed during
biological degradation of various substrates. Above threshold con-
centrations, FAN is a powerful inhibitor in an anaerobic digester,
and can easily cause process instability indicated by a decrease in
both biogas and methane yields, which can eventually lead to fail-
ure of the reactor. Therefore, control and monitoring of ammonia
concentrations in substrate and digester, pH and process tempera-
ture enable a safe and stable process, particularly during anaerobic
digestion of substrates such as manure (especially poultry manure,
cow manure and piggery waste). As long as the microbial commu-
nity in the anaerobic digester is gradually acclimated to increasing
levels of ammonia, it has been shown that the digester can operate
even at very high concentrations of ammonia without jeopardiz-
ing its safety. Pre-treatment of substrate before anaerobic digestion
process will also decrease the possible adverse impacts of ammo-
nia beforehand. If the process is inhibited by high concentrations of
FAN, recovery options such as dilution of the substrate, dilution of
the reactor contents, adjustment of process pH, adjustment of C:N
ratio of the substrate, external addition of compounds like zeolite,
glauconite, and activated carbon can be employed to increase
909
biogas and methane yields and to achieve a stable and efficient
process.
Acknowledgment
Orhan Yenigün acknowledges the support provided by Dr. Pra-
tim Biswas, Head of the Department of Energy, Environmental and
Chemical Engineering, at the Washington University in St. Louis,
USA, in the preparation of this manuscript during his sabbatical
leave from Boğaziçi University.
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