1925 © IWA Publishing 2017 Water Science & Technology | 76.

8 | 2017

Methods of ammonia removal in anaerobic digestion:

a review
Niclas Krakat, Burak Demirel, Reshma Anjum and Donna Dietz

ABSTRACT
Niclas Krakat (corresponding author)
The anaerobic digestion of substrates with high ammonia content has always been a bottleneck in
Reshma Anjum
the methanisation process of biomasses. Since microbial communities in anaerobic digesters are Donna Dietz
Department of Bioprocess-Engineering,
sensitive to free ammonia at certain conditions, the digestion of nitrogen-rich substrates such as Leibniz Institute for Agricultural Engineering and
Bio-Economy Potsdam,
livestock wastes may result in inhibition/toxicity eventually leading to process failures, unless Max-Eyth-Allee 100, D-14469 Potsdam,
Germany
appropriate engineering precautions are taken. There are many different options reported in
E-mail: nkrakat@atb-potsdam.de
literature to remove ammonia from anaerobic digesters to achieve a safe and stable process so that
Niclas Krakat
along with high methane yields, a good quality of effluents can also be obtained. Conventional Department of Biotechnology,
University of Applied Sciences,
techniques to remove ammonia include physical/chemical methods, immobilization and adaptation Lohbrügger Kirchstraße 65,
21033 Hamburg,
of microorganisms, while novel methods include ultrasonication, microwave, hollow fiber Germany
membranes and microbial fuel cell applications. This paper discusses conventional and novel Burak Demirel
Institute of Environmental Science,
methods of ammonia removal from anaerobic digesters using nitrogen-rich substrates, with
Boğ aziçi University,
particular focus on recent literature available about this topic. Bebek,
Istanbul 34342,
Key words | ammonia removal, anaerobic digestion, biogas, nitrogen elimination, nitrogen-rich Turkey
substrates

INTRODUCTION

Anaerobic digestion (AD) is an effective biotechnology in
waste and wastewater treatment processes to reduce waste
volume and to produce agricultural fertilizers as well as bio-
methane, a source of renewable energy. AD processes
require a low energy input and have a relatively lower
amount of sludge production in comparison to aerobic
waste treatment systems. The biogas process is divided
into four phases: hydrolysis, acidogenesis, acetogenesis
and methane formation, which are carried out by different
groups of microorganisms. However, the conflicting needs
between microorganisms regarding nutrients, environ-
mental conditions and growth kinetics, especially between
the fast growing acid formers (Bacteria) and the slow grow-
ing methane formers (Archaea), often cause process
instability. Correspondingly, the microorganisms react in a
varying degree to the presence of inhibitory substances in
wastes and wastewater. The dysfunction of one single
microbial group participating in the anaerobic food chain
affect the whole digestion process.
Inhibitors in AD processes include organics or inorganics in
nature or a combination thereof. Among these compounds,
chlorophenols, halogenated aliphatics, N-substituted aromatics,
doi: 10.2166/wst.2017.406
long chain fatty acids, lignins and lignin related compounds
are potential organic inhibitors. Inorganic inhibitors are
ammonia, sulfide, light metal ions (Na, K, Mg, Ca, Al) and
heavy metals (Anjum et al. ). However, the limit of tox-
icity concentrations vary considerably, since AD processes
are very complex. Particularly, the interactions of microbial
communities and their ability to adapt to substrate and pro-
cess conditions are often unpredictable. Therefore, the
inhibition of AD systems due to excess ammonia levels
have often been reported and studied in literature. This
paper provides an overview of research activities in AD con-
ducted on ammonia mitigation from animal manure, with
particular attention to recent methodologies.
NITROGEN-RICH SUBSTRATES
Nitrogen, at an optimal level, is a nutrient source for micro-
organisms and ensures the buffering capacity in AD
processes. However, substrates with high nitrogen fractions
inhibit the degradation, which may lead to reduced biogas
quality and quantity. Hereof, a wide range of inhibiting

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 1926 N. Krakat et al. | Ammonia removal in anaerobic digestion
ammonia concentrations were reported in the literature,
ranging from 1,700 mg/L to 14,000 mg/L (Anjum &
Krakat ). But also, ammonium has been known for
having a toxic impact on the microbial biocoenosis. Concen-
trations greater than 3,000 mg/L NHþ 4 showed an inhibitory
effect by 40% on the biogas process (Braun ).
During the AD process, the organic nitrogen in the form
of proteins, amino acids and uric acids is hydrolyzed to inor-
ganic ammonia. The protein degradation process is very
slow and the released ammonia tends to accumulate
(Zeshan et al. ). Substrates known to have high nitrogen
contents are animal wastes, municipal wastes (bio-wastes),
meat processing wastes and dairy wastes. Besides being an
inhibitor in the AD process, free ammonia is also an
environmental pollutant. Free ammonia and the increased
eutrophication, respectively, is toxic for most fish species,
it decreases dissolved oxygen, causes corrosion and reduces
disinfection efficiencies (Lauterböck et al. ).
As the human population grows, the demand of meat
and dairy products also increases. Animal by-products,
including chicken poultry, swine and cattle manure grows
as well. Particularly, the global population of poultry indus-
tries has been growing rapidly. The average stock of poultry
is almost 23 billion worldwide, producing about 587 billion
tons of ammonium-rich excreta per year (Anjum et al. ).
A part of this manure is used as fertilizer, but the major por-
tion must be disposed of properly. Untreated, the animal
wastes cause environmental problems, such as malodor,
ammonia volatilization and groundwater contamination.
Slaughterhouse wastes are by-products of the meat pro-
cessing industries. They contain the inedible parts of
animals, such as bones, blood, viscera and feathers. Due to
their high lipid and protein content, slaughterhouse wastes
offer a sustainable treatment method to generate energy in
combination with the use of residues as fertilizers (Edström
et al. ). However, to meet the current European regu-
lations for the disposal or use of animal by-products,
slaughterhouse wastes must be pretreated. Other com-
pounds with high nitrogenous content are dairy effluents,
wastewater from food industry processing whey, cheese
and casein (Kovács et al. ). The composition and proper-
ties of feedstock is consequential to evaluate further
treatment. Although AD offers a solution to the waste man-
agement with many advantages, the nitrogen problem still
persists.
Many possibilities to control ammonia inhibition have
been studied and reported in literature. Some methods
were practical and applicable in large scales, while some
are still in research at laboratory scale. Nevertheless, each
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Water Science & Technology | 76.8 | 2017
method has its pros and cons, depending on the inoculum,
type and characteristics of the substrate, reactor configur-
ation, and environmental and operational conditions.
AMMONIA INHIBITION MECHANISM
Only a proportional fraction of the organic nitrogen is bio-
logically degraded to inorganic ammonia (NHþ 4 -N/NH3-N).
Gallert & Winter () reported that only about 1/3 and
1/2 of the total Kjeldahl nitrogen was converted to ammonia
during mesophilic and thermophilic degradation. Bujoczek
et al. () referred to an organic nitrogen conversion
rate ranging between 62.6% and 80.3%. Yabu et al. ()
reported that only 34–38% of the total nitrogen of the gar-
bage was converted to ammonia.
In aqueous solution, the total ammonia exists in two
principal forms: the ionized form of the ammonia ion
(NHþ 4 ) and the free, unionized gaseous form of ammonia
(NH3). The dissociation equilibrium of ammonia in aqueous
solutions (Equation (1)) depends on pH and temperature.
With a rise in pH and temperature, the equilibrium shifts
to NH3.
NH4þ ↔ NH3 þ Hþ (1)
The uncharged free ammonia molecule (NH3) is
reported to be the major component causing inhibition
during AD processes (Angelidaki & Ahring ; Kayhanian
; Salminen & Rintala ; Siles et al. ). Assuming
that the total ammonia in aqueous solution is present only
as NH3 (aq) and NHþ 4 , the free ammonia concentration
(Equation (2)) is estimated as follows (Hansen et al. ):
!1
NH3 10pH
¼ 1þ (2)
NH4þ 10(0:09018þ (2729:92=T(K)))
where NH3 is the concentration of free ammonia, NHþ 4 is
the total ammonia concentration and T is the temperature
in Kelvin (K).
Among the anaerobic degrading microorganisms,
methanogens (Euryarchaeota) are reported to be the most
affected groups by elevated ammonia levels (>1,800 mg/L)
and the first to be inhibited (Kayhanian ; Chen et al.
; Niu et al. ). However, hydrogenotrophic methano-
gens were reported to be dominant in engineered habitats
with low ammonium concentrations (Krakat et al. a,
b). As for the bacterial consortium, mostly the
 1927 N. Krakat et al. | Ammonia removal in anaerobic digestion
dominance of the Firmicutes phylum over the Proteobac-
teria phylum was reported in stable AD processes,
although the dominance of Firmicutes, Bacteroidetes, and
Proteobacteria was also reported (De Vrieze et al. ).
Furthermore, the hydrolysis and acidogenesis conversion
ratio declined when the total nitrogen concentration was
higher 5,000 mg/L (Niu et al. ).
Several effect mechanisms of ammonia inhibition,
mainly studied with pure cultures of methanogens, were pro-
posed: (1) free ammonia inhibits the methane synthesizing
system directly, (2) free ammonia causes proton imbalance,
because ammonia diffuse passively into the cells of metha-
nogens, (3) free ammonia changes the intracellular pH,
(4) free ammonia increases the maintenance energy require-
ment, and (5) free ammonia inhibits specific enzymatic
reactions (Wittmann et al. ; Kayhanian ; De
Vrieze et al. ).
When the methanization is interrupted, the concen-
tration of volatile fatty acids (VFA) accumulates. In turn,
the pH remains stable unless there is enough buffering
capacity (alkalinity) available in the system and, thus, the
concentration of free ammonia decreases. That results in
an ‘inhibited steady-state’, a condition with significant
losses of the biogas and methane production yields (Fotidis
et al. ). It was reported by Yenigün & Demirel (),
that the ammonia inhibition was reversible for the methano-
genesis phase. The microbial community even increases its
tolerance towards ammonium after recovery. However, in
contrary, Niu et al. () reported that the former steady-
state could not be recovered after a thermophilic reactor
was loaded with a very high amount of nitrogen.
Methanogens are primarily classified as acetoclastic,
hydrogenotropic and methylotrophic organisms (Liu &
Whitman ). There is contradictory information reported
in literature regarding the sensitivity of the methanogens to
environmental conditions. Acetoclastic methanogens are
acetate consumers, while hydrogenotropic methanogens
are capable of utilizing H2, CO2 and formate to produce
methane. Some studies in literature observed that acetoclas-
tic methanogens were more sensitive to ammonia inhibition
(Borja et al. ; Calli et al. ; Niu et al. ), while
others observed a lower tolerance by hydrogenotropic
methanogens (Karakashev et al. ; Chen et al. ;
Song et al. ). At elevated ammonia levels, the microbial
community shifts from the acetoclastic methanogenesis to
syntrophic acetate oxidation (Schnürer & Nordberg ;
Westerholm et al. a). The syntrophic acetate oxidation
involves a two-step reaction consisting of acetate oxidation
to hydrogen and carbon dioxide by syntrophic acetate-
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Water Science & Technology | 76.8 | 2017
oxidizing bacteria, followed by conversion of these products
to methane by hydrogenotropic methanogens (Westerholm
et al. a).
Some studies reported that thermophilic microbial con-
sortia could tolerate double the amount of free ammonia
compared to mesophilic ones (Gallert & Winter ;
Gallert et al. ), while others studies revealed that
thermophilic conditions with high free ammonia concen-
trations were unstable and could be more easily inhibited
(Braun et al. ; Hashimoto ; Angelidaki & Ahring
).
The toxicity limits of free ammonia described in litera-
ture differed significantly, with concentration ranging
between 50 to 1,500 mg NH3-N/L (Braun et al. ;
Koster & Lettinga ; Angelidaki & Ahring , ;
Gallert & Winter ; Hansen et al. ; Kayhanian
; Bujoczek et al. ; Siles et al. ). Nevertheless,
ammonium thresholds greater than 12 g/L were also
reported as tolerable concentrations for microorganisms
detected in thermophilic fermented chicken dung (Anjum
& Krakat ). The degradation process was either partly
or totally inhibited at given concentrations and conditions.
Many research groups have published results of ammonia
thresholds. A summary of these results was provided in
different reviews (Rajagopal et al. ; Yenigün & Demirel
). The varying results about methanogens sensitivity and
ammonia toxicity limits may be attributed to (1) differences
in the types of the substrates used, (2) varied inoculum
sources, (3) environmental conditions such as pH, tempera-
ture, and (4) operational conditions such as substrate
loading rate, retention time and acclimation periods micro-
organisms (De Baere et al. ; Angelidaki & Ahring
; Liu & Sung ).
STRATEGIES TO OVERCOME AMMONIA INHIBITION
The conventional biological method of ammonia elimin-
ation is nitrification and denitrification. These processes
require a significant energy input and carbon sources.
Therefore, they are not suitable in combination with AD
processes. Moreover, the process is generally performed
on wastewater containing low nitrogen concentration
(Ahn ; Lin et al. b). But also less common
approaches such as break point chlorination and membrane
based technologies are used.
Some studies focused on the adjustment of feedstock
C/N ratios to guarantee an optimal microbial growth
(Kayhanian ; Siles et al. ; Zeshan et al. ).
 1928 N. Krakat et al. | Ammonia removal in anaerobic digestion
Kayhanian () reported a C/N ratio of 27 to 32 as suffi-
cient for the degradation of biodegradable organic fraction
of municipal solid waste. Zeshan et al. (), however, pre-
pared simulations using bio-degradable feedstocks and
identified a C/N ratio 32 as appropriate to minimize or to
avoid ammonia inhibition at dry thermophilic ADs, while
a C/N ratio 27 was less adequate. Siles et al. () men-
tioned, that the biogas production from the degradation of
glucose-containing synthetic solutions decreased signifi-
cantly when the C/N ratio was below 4.42.
A common approach is to dilute the substrate to a total
solids (TS) level of 0.5%–3% (Chen et al. ). Even though
no additional equipment was required, this approach proved
to be economically unattractive, since dilution seriously
decreased the digester’s gas production level and also
resulted in an increased waste volume and increased dewa-
tering costs, respectively (Kayhanian ).
The anaerobic population of wastewater reactors and
biogas plants may overcome the inhibitory effects of ammo-
nia by acclimation or long-term adaptation (Angelidaki &
Ahring ; Beinersdorf et al. ). Yet adaptation pro-
cesses may take a long time and, thus, they are not
suitable for a fermentations in which ammonia concen-
trations vary strongly and shock loadings may occur.
Other processes based on a physical-chemical reaction
are namely stripping and chemical precipitation. The ammo-
nia can either be reduced in a pretreatment step, during AD
or as a post-treatment of the AD effluent (Serna-Maza et al.
). For instance, stripping is a conventional method,
which is applicable as a pre-treatment step (Zhang et al.
; Markou ), during the digestion (Yuan et al. )
and as post-treatment (Lei et al. ). A successful air strip-
ping as pretreatment step was performed by Zhang et al.
() to increase the methane yield from AD of piggery
wastewater. The ammonia stripping pre-treatment showed
that the removal of ammonia seemed to be dependent on
pH and aeration rate, and particularly, it could be an
option at alkaline pH levels to prevent digester failure
during anaerobic treatment of raw piggery wastewater.
Bousek et al. () recently investigated effects of ammonia
stripping in anaerobic digesters with high ammonia levels.
The authors concluded, that the side stream air stripping
could be a promising tool to decrease high ammonia levels
in anaerobic digesters. However, this approach might pose
adverse impact on the microbial community due to inhi-
bition through oxygen exposure. Abouelenien et al. ()
reported that nitrogen can successfully be eliminated by
air stripping combined with gas washing in sulfuric acid to
capture ammonia when chicken dung manure was digested.
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Thereby, a recirculation of the stripped liquid fraction to the
fermenter could be a solution in large-scale applications to
control the ammonia concentration (Nie et al. ). How-
ever, other carrier gases such as biogas and hot steam can
also be used for elimination processes.
Acclimation
Numerous studies have been conducted to find practical sol-
utions to overcome ammonia inhibition. These strategies are
discussed in the following parts.
Bioaugmentation
The addition of ‘stress-resistant’ – or ‘efficient biogas produ-
cing’ microorganisms in bioreactors to improve the AD
process, known as bioaugmentation, has been used for the
last 15 years (Nzila ). One of the advantages of bioaug-
mentation is the decreasing of the adaptation period of
bioreactors. The challenge for the bioaugmented culture is,
however, to adapt and to survive under reactor conditions,
which may be not similar to their optimal growth con-
ditions. Moreover, added cultures must be able to assert
themselves to compete with the established microbial
populations.
Lü et al. () studied the combined effect of acids and
ammonia on acetoclastic methanogens, hydrogenotrophic
methanogenes and syntrophic acetate oxidizers (SAO)
during a thermophilic AD process. It was reported that, fore-
most, the stress of acetate and ammonia was synergetic.
Methanogenic pathways were regulated by acetate and
ammonia, jointly. Therefore, the microbial population struc-
ture in a thermophilic anaerobic culture shifted, depending
on the combined effect of acetate and ammonia. At lower
acetate concentrations (50 mmol/L), acetoclastic methano-
gens were dominant, regardless of the ammonia
concentration. When the acetate concentration was high
(150 mmol/L and 250 mmol/L) and ammonia levels were
moderate (1–4 g/L), acetate was mainly degraded by the
acetoclastic methanogens.
Generally, the efficiency of acetate degradation by both
SAO and hydrogenotrophic methanogens were lower than
that of the acetoclastic methanogens. However, at elevated
ammonia concentrations, SAO and hydrogenotropic
methanogens were more robust and could continue to
function.
Nielsen et al. () studied the effect of bioaugmenta-
tion during a two-stage thermophilic AD of cattle manure
containing large fraction of biofibers with two different
 1929 N. Krakat et al. | Ammonia removal in anaerobic digestion
strains of hydrolytic hyperthermophilic bacteria, Caldicellu-
losiruptor lactoaceticus and Dictyoglomus. Even though the
free ammonia concentration was lower than the reported
inhibition concentration, the improvement of the methane
yield was modest, only about 9–10%, compared to that in
the control reactors without bioaugmentation.
Westerholm et al. (b) explored the enhancement of
the biogas yield by bioaugmentation with SAO cultures
containing Clostridium ultunense sp., Syntrophaceticus
schinkii, Tepidanaerobacter acetatoxydans and Methanocul-
leus sp. The cultures were added daily to stirred tank
reactors fed with a mixture of cattle manure and whole stil-
lage. To achieve elevated ammonia concentrations, the
whole stillage was substituted by egg albumin powder. Sur-
prisingly, SAO activities were also detected at low acetate
and ammonia concentrations. This was attributed to the
long hydraulic retention time adjusted. At higher dilution
rates, the acetoclastic methanogens dominated. Overall,
the results of reference and bioaugmented reactors were
similar, which indicated that the bioaugmentation with
SAO and hydrogenotropic cultures did not have any domi-
nant influence on the acetate degradation pathway.
Moreover, the bioaugmentation did not improve the reac-
tors’ operation during periods of increasing ammonia levels.
A similar study was conducted by Fotidis et al. () in
mesophilic up-flow anaerobic sludge blanket reactors fed
with synthetic medium containing glucose and NH4Cl as
carbon and ammonium nitrogen source, respectively. A
SAO co-culture (Clostridium ultunense spp. in living
association with Methanoculleus spp.) was used for bioaug-
mentation. Again, an identical performance of reference and
bioaugmented reactors was observed indicating that the
bioaugmentation process did not affect the methane pro-
duction at high ammonia levels. This could be attributed
to very slow growth rates of SAO co-cultures and to the chal-
lenge to immobilize used co-cultures. To elucidate the
reason for the unsuccessful bioaugmentation, the SAO
co-culture was co-cultivated with hydrogenotrophic
Methanoculleus bourgensis in fed-batch reactors. The total
incubation period of SAO co-culture was lowered by 33%
with M. bourgensis and increased the growth rate by 42%.
This indicated that Methanoculleus spp. as partner in the
SAO co-culture was the rate limiting factor in the consor-
tium. Presumably, Methanoculleus spp. reduced the
hydrogen partial pressure less effectively as Methanoculleus
bourgensis and, therefore, did not bring any advantage for
the biomethanization process.
An improved bioaugmentation was conducted with
Methanoculleus bourgensis by Fotidis et al. () in
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continuous stirred tank reactors (CSTR) fed with pig and
cattle manure (70–90%) and organic waste (10–30%).
Methanoculleus bourgensis was a fast growing hydrogeno-
trophic methanogen that could produce methane at
elevated ammonia concentration (4 g NHþ 4 N=L). In com-
parison to the reference reactors, the methane yield of the
bioaugmented reactors showed an immediate improvement
by an increase of 31.8%. The bioaugmentation was success-
fully performed without interrupting the continuous
operation of the reactor and without changing the ammo-
nia-rich feedstock. It was assumed that a ‘critical biomass’
of ammonia tolerant methanogens was necessary, which
was a minimum amount of microorganisms needed to pro-
mote the desired microbial activity in the reactor. The
advantages of bioaugmentation include no changes in reac-
tor operational parameters such as pH and temperature, and
a low cost. However, choosing the suitable microorganisms
and wash-out occurrences are potential disadvantages of
bioaugmentation.
Anaerobic ammonia oxidation
A further strategy to remove nitrogen form process liquids is
the anaerobic ammonium oxidation (Anammox) process
catalyzed by chemolitoautotrophic Planctomycetes. The
process deals in the oxidation of ammonia to di-nitrogen
gas with nitrite as an electron acceptor (Kimura et al.
). Commonly, NHþ 4 N is removed by nitrification to
nitrate, followed by denitrification. In Anammox-based pro-
cesses, only about 50% of ammonium is oxidized to nitrite,
when oxygen is present as terminal election acceptor, after
which, in oxygen free environments, the produced nitrite
reacts with the remaining ammonium to form nitrogen gas
(N2). This approach is preferably used for the elimination
of nitrogen from wastewaters with low C/N ratios and
high ammonium levels with low amounts of organics,
respectively (Zhang et al. a). Therefore, aerobic single
reactor systems for high ammonia removal over nitrite treat-
ment processes (SHARON) are frequently used which
involve part conversion of ammonium to nitrite often
coupled with a second reactor in which Anammox occurs.
Alternatively, the completely autographic nitrogen removal
over nitrite (CANON) process may be used, which involves
nitrogen removal within one single reactor under oxygen
limited conditions (Khin & Annachhatre ).
The SHARON-Anammox process does not require the
addition of chemical oxygen demand (COD) and, thus, the
combined system saves 50% of oxygen and 100% on the
external carbon source, reducing CO2 emissions by about
 1930 N. Krakat et al. | Ammonia removal in anaerobic digestion
100%. Overall, this approach is 90% less expensive than
conventional processes (Dijkman & Strous ).
The CANON process is autotrophic and, therefore,
requires no added COD. The entire nitrogen removal can
be achieved in a single reactor with little aeration reducing
space and energy requirements. The CANON process con-
sumes 65% less oxygen and 100% less reducing agents
compared to conventional removal process (Khin &
Annachhatre ). However, the Anammox process offers
many advantages and disadvantages for the ammonium
removal (van der Star ).
Anammox – advantages:
• No requirement for external oxidants
• No CO2 emission (CO2 is consumed by autotrophic
Anammox-bacteria instead of produced)
• Lower energy costs due to minimized aeration costs.
Anammox – disadvantages:
• Anammox organisms grow notoriously slowly (doubling
time is approximately 10–15 days under known optimal
conditions).
• Anammox bacteria react very sensitively to oxygen, even
at low concentrations, with growth inhibition and cell
death as a consequence.
• High sensitivity to changing environmental conditions.
Zeolites/clay minerals
Natural zeolites crystalline, hydrated alumina silicates of
alkali and alkaline earth cations, are large mineable deposits
in many parts of the world. Especially in AD processes, the
porous materials of natural zeolites provided a support and
immobilization material enabling the anaerobic reactor to
retain high biomass concentrations and significantly
reduced retention times. Moreover, the properties of zeolite
also showed a great capacity for metal adsorption, which
was useful for removing toxic materials causing inhibition
in AD processes (Zhang et al. b). So, the high ion
exchange capacity of zeolites, their large reserves, the short-
age of competing minerals and the relatively low market
price make zeolite an attractive mineral to be applied in
large scales (Lin et al. ).
Two mechanisms of zeolites were advantageous for the
AD process: as an ion exchange element for NH4-N and the
adsorption of NH3. Due to the presence of Naþ, Kþ, Ca2þ
and Mg2þ cations, zeolite has been used as an ion exchange
element for NH4-N- removal in AD (Montalvo et al. ).
Since the cations are loosely bound, they can be easily
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exchanged by other surrounding cations, for example by
ammonium ions. These cations stimulated the microbial
growth and they were antagonistic to ammonia inhibition.
However, when high levels of these cations were exchanged
and released at the adsorption process, they were toxic for
the microorganisms (Lin et al. ). Another mechanism
proposed that the zeolite adsorbed NH3 on active areas of
the material (Milán et al. ).
Zeolites can either be used in their natural form or
modified. Since the natural zeolites have a lower adsorption
capacity, they usually need to be modified to improve their
adsorption capacity and purity (Huang et al. ). One of
the possible modification methods is a sodium chloride
(NaCl) solution treatment. The surface of modified zeolite
became rougher and more irregular, compared with the
natural zeolite that, in turn, increased the surface area,
total pore volume and average pore diameter significantly
(Lin et al. ). After that modification, more Naþ ions
were available and they were readily exchanged by
ammonium ions on zeolite surfaces. NaCl modification
also replaced Ca2þ and Mg2þ ions on zeolite, resulting in
the production of large pores and cavities in the zeolite. In
this study, the modified zeolite exhibited 58% higher
ammonium adsorption capacity and faster adsorption rates.
The doses and types of zeolites to be employed is an
important parameter. When the amount of zeolite is too
high, the apparent viscosity will increase and thereby will
hinder the mass transfer between the substrate and microor-
ganisms (Milán et al. ).
Milan et al. () compared the effect of different
homoionic zeolites on piggery wastewater by anaerobic
fixed bed reactors (Na, Ca, K and Mg). From all these
ions, homoionic sodic zeolite showed the best performance,
with 91% and 58% ammonia removal capacity after 10 h
and 30 h of operation, respectively. According to the exper-
imental results obtained, the exchange capacity was in the
order Na-Zeo > Ca-Zeo > K-Zeo > Mg-Zeo. This order of
exchange capacity was also confirmed by Lin et al. ().
Especially Ca-Zeo and Mg-Zeo were affected by the high
concentration of suspended solids and the viscosity of the
influent, which led to a low mobility of these cations in
the liquid phase. Moreover, Lin et al. () stated that
Naþ was only dominant when the ammonium concen-
tration was low (under 500 mg N/L). At high ammonium
ion concentration (>1,000 mg N/L), Ca2þ replaced Naþ as
dominant ions for ammonium adsorption. Depending on
the pH, the adsorption mechanism shifted from ion
exchange to molecular adsorption. The significance of mol-
ecular adsorption was negligible at pH < 8. At a higher pH,
 1931 N. Krakat et al. | Ammonia removal in anaerobic digestion
the molecular adsorption contributed more to the ammonia
removal, and became significant at pH 11, owing to the
absence of ionized ammonium in the solution.
A series of experiments treating swine manure with zeo-
lite doses between 0.2 and 10 g/L showed that the AD
process was favored by the addition of natural zeolite at
doses between 2 and 4 g/L and increasingly inhibited at
doses beyond 6 g/L (Milán et al. ). A digestion failure
was observed at zeolite doses of 10 g/L. This was explained
by the decrease of total volatile fatty acid/alkalinity ratio
and the simultaneously increasing pH value.
Since the thermophilic AD of animal wastes or manure
was very challenging, mainly due to the ammonia inhibition,
relatively fewer studies were conducted at this temperature.
By increasing the ammonia concentration gradually up to
5 g/L, Angelidaki & Ahring () studied the effect of
addition of bentonite and the waste product bentonite-
bound oil (BBO), each with 1.1% bentonite, on the AD of
cattle manure under thermophilic conditions. Bentonite is
a clay mineral with a characteristically layered structure,
allowing water, numerous elements and organic matter to
enter the layer spaces. In a continuous process, as the ammo-
nia concentration was gradually increased, the methane
production decreased in all reactors. However, bentonite
and BBO added reactors recovered and reached the same
level as before the inhibition. In batch culture experiments,
the addition of bentonite shortened the lag-phase.
Kotsopoulos et al. () studied the effect of zeolite
addition on the thermophilic AD of pig wastes. The prelimi-
nary experiments showed that the obvious differences in
biogas production were observed at zeolite doses of 0, 4,
8, and 12 g/L, where the degradation process also ceased
after 15 days. Especially, the addition of 8 g/L and 12 g/L
of zeolites increased the methane production significantly.
The total ammonia concentrations only slightly decreased
when increased zeolite doses were added. However, the
free ammonia concentration increased in the treatments
with zeolite. The pH value remained, in any case, within
the optimal range for the AD (6.8–7.5). Thus, it was also con-
cluded in this study that the positive effect of zeolite
addition was not only the result of the ion exchange capacity
of zeolite, but also due to the immobilization surface for the
microorganisms.
The growth rate of microorganisms immobilized on zeo-
lites was higher compared to those in suspension, as
calculated by Borja et al. (). The kinetic constant and
maximum growth rate in the zeolite loaded reactor were
35% and 59% higher compared to the control reactor with-
out zeolite. The maximum volumetric methane production
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rate was also increased by approximately 62% when bio-
mass was added. This result was confirmed by Nikolaeva
et al. () using up-flow anaerobic fixed bed digesters
packed with waste tire rubber. Particularly the combination
of waste tire rubber and zeolite improved the values of the
maximum methane yield and kinetic constants by 11.1%
and 29.4%, respectively.
Different removal strategies also were investigated by
Ho & Ho () to mitigate ammonia inhibition on thermo-
philic AD of piggery wastewater. The pH was adjusted and
the effects of different additives (biomass, natural zeolite
and humic acid) were investigated, individually or in combi-
nation. The enhancement with biomass and humic acid did
not show any desirable result, with or without pH adjust-
ment. On the other hand, the addition of 10–20 g/L of
zeolite showed an increase in methane production. The
enhancement effect of zeolite was greater on the pH-
unadjusted piggery wastewater (pH 8.1) than on the
pH-reduced wastewater (pH 6.5). The high ammonia con-
centration of piggery wastewater used in this study, about
1,740 mg NH4-N/L, presumably also required a high
amount of zeolite addition. However, the ammonium-nitro-
gen concentration remained unchanged after 10 days of
batch digestion, which suggested that the applied zeolite
concentrations were inadequate in reducing the high ammo-
nia-nitrogen concentrations. Since the addition of zeolite
was nonetheless effective in increasing methane production,
it was hypothesized that a combination of microbial immo-
bilization and stimulation by unknown exchanged cations
released from the zeolite were likely factors that contributed
to the beneficial effects.
The effect of different amounts of zeolites coupled with
acclimatized inoculum was studied by Kougias et al. ()
in mesophilic batch reactors. Using well digested swine
manure as inoculum, poultry manure was anaerobically
digested with 5 and 10 g/L of natural zeolites. The results
showed that a significant increase of methane production
was observed when zeolite was added to the system. The
best results were obtained by the addition of 10 g/L zeolite,
with a 109.75% increase of methane production compared
to the reference reactor without zeolite. The VFA concen-
trations were also noticeably lower in reactors loaded with
zeolite, indicating a stable digestion process.
The effect of zeolite addition on the mesophilic AD of
swine manure with 10% TS was investigated (Lin et al.
). Based on the assumption that the ammonium adsorp-
tion capacity of zeolite was 13.3 mg N/g zeolite, 60 g/L of
zeolite was added to the reactor. The results showed that
the zeolite addition led to a faster start-up and a better
 1932 N. Krakat et al. | Ammonia removal in anaerobic digestion
performance in term of biogas production and methane yield,
especially at high ammonium concentrations. The total
ammonia nitrogen (TAN) was rapidly absorbed by zeolite in
the first 5 days. However, the calculated zeolite adsorption
capacity was lower than the maximal capacity. This might
be due to two reasons: the competition with Naþ and Kþ
ions co-existing in the digester and the ammonium mass trans-
fer might be hindered and retarded by the high TS
concentration and the sedimentation of zeolite particles cov-
ered by solid substrates. The zeolite addition did not have
any significant influence on the microbial biodiversity.
In an another study, Huang et al. () proposed a pro-
cess to simultaneously remove ammonium nitrogen and
phosphate from simulated swine wastewater by modified
zeolite and struvite crystallization. Recently, Wang et al.
() used struvite precipitation (SP) to combat ammonia
toxicity in a two-stage anaerobic digester fed with protein-
rich feedstocks. The authors observed that the SP treatment
was successful in removal of TAN along with better
methane yields (Wang et al. ).
To minimize the operational costs, an effective way to
regenerate exhausted zeolite, especially one that can adapt
to condition of higher ammonia loading from manure waste-
water, is of great significance. Zeolite regeneration is
generally realized using brine solutions with different brine
compositions or by a biological process, where ammonium
adsorbed on the zeolite is transferred to gaseous nitrogen
species by nitrification-denitrification (Guo et al. ). How-
ever, if organic matter adsorbs proportionately, the effluent
may contain high concentrations of salt and COD.
A study was conducted by Guo et al. () using differ-
ent combinations of alkaline (NaOH) and salt (NaCl)
concentrations from batch desorption tests to expanded
fixed-bed column. The regeneration efficiency was depen-
dent on alkaline concentrations (pH), salt strength and
flow rate. The desorption rate increased significantly from
70% to 90–95% with increasing alkaline concentration
between 0.032 and 0.1 mol/L. At this lower alkaline concen-
tration, the desorption rate also increased with increasing
salt concentration (from 10 to 58.5 g/L). No influence on
ammonium desorption was observed, when the salt concen-
tration was further increased for more than 20 g/L at
alkaline concentration greater than 0.1 mol/L. The elution
curves showed best results at flow rates in the range of
2.5–3.0 bed volumes per hour. For most cases in this
study, complete ammonia elution was achieved in 10 bed
volumes. Later, the ammonia retained in the regenerant
can be recovered by air or steam stripping and, conse-
quently, the regenerant can be recycled. This work also
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provides a comprehensive table presenting a comparison
of regeneration parameters and corresponding performance
between different literatures.
Deng et al. () described an effective and economic
method to regenerate exhausted zeolite. The study included
the assessment of ammonia exchange capacity, zeolite regen-
eration efficiency and economic analysis. The ammonia
exchange capacity of natural zeolite increased initially with
increasing ammonium ion and then reached a plateau.
This exchange, however, was also influenced by pH. At
high pH-values (9.5 and 10.5), a significant decrease in
ammonia exchange capacity was observed, probably due to
the transformation of ammonium ion to unionized ammonia
that was then inaccessible for the ion exchange. The regener-
ation efficiency was strongly affected by the mass ratio of
Naþ ions and zeolite-NH4þ-N. The alkaline regeneration at
pH 12 decreased the mass ratio to 4.2, and only 10 g/L
NaCl was needed for the recovery (85% in 2 h in continuous
columns tests). The economic analysis showed that this alka-
line regeneration saved chemical costs over 10 times as
compared with a conventional regeneration method at pH 9.
A combination of ammonia and phosphate removal was
investigated by Huang et al. (). Using modified zeolite,
which was natural zeolite modified with magnesium salts,
as adsorbent for ammonium ions, the magnesium ions
released from zeolite served as the source of SP for the
removal of phosphate. Both TAN and phosphate were effec-
tively removed. The removal efficiencies were maximum at
pH-values 8.5–9, where 82% total ammonium and 98%
phosphate were removed with 110 g/L of modified zeolite
and 40 minutes reaction time from simulated swine waste-
water. However, the presence of various cations (K2þ,
Ca2þ, Na2þ and Mg2þ) had a significantly negative effect
on the removal of ammonia-nitrogen. This method has yet
to be tested using real animal wastes.
In a relatively recent work, Zheng et al. () developed
a bio-zeolite fixed-bed reactor using different materials to
mitigate ammonia inhibition during AD of livestock wastes
with high ammonium contents. The novel fixed-bed reactor
designed using chlorinated polyethylene (CPE) material was
eventually recommended by the authors to improve AD of
livestock wastes of high ammonium content, since CPE
fixed zeolite favored the adsorption of ammonia and immo-
bilization of microorganisms.
Zeolite – advantages:
• Shortened lag-phase
• Low cost
• Relative easy to operate
 1933 N. Krakat et al. | Ammonia removal in anaerobic digestion
• Immobilization surface, enhance growth
• Adsorption of ammonia (not in every study)
• Somehow counteract ammonia inhibition, without redu-
cing ammonia concentration
• Ability to recover inhibited process
• Enhance methane production (not in every study).
Zeolite – disadvantages:
• Adsorption capacity in some studies was quite low, i.e.
application for high strength NH4-N possible?
• In some publications, no reduction of ammonia was
observed
• Regeneration (high dose chemicals), i.e. high costs
• Presence of other cations can decrease ammonium
exchange capacity.
OTHER METHODS
Ultrasonication
Ultrasonication has been widely used to degrade organic
compounds from wastewater (Wang et al. ). It evokes
cavitation in the organic material by rapid bubble formation
in the liquid phase. When the bubbles collapsed, instant
high temperature and pressure were produced, leading to
the formation of radicals and degradation of the particulate
matters (Luste & Luostarinen ). In the removal of
ammonia nitrogen, most of the free ammonia molecules
enter into cavitation bubbles in which they are transformed
into nitrogen and hydrogen molecules via pyrolysis under
instant high temperature and high pressure in the cavitation
bubbles (Wang et al. ). Luste & Luostarinen ()
studied the effect of ultrasound and thermal pretreatments
on dairy cattle slurry. Both the CH4-yield and NHþ 4 N con-
centration of ultrasound tested (6,000 kJ/kg TS) cattle slurry
was about 20% higher compared to the untreated slurry. The
ammonium content increased afterwards due to the
increased amount of soluble organic materials. A decrease
in ammonia concentration was reported by Cho et al.
(). Applying ultrasonication technique at two different
frequencies (28 and 40 kHz) to simulated and real livestock
wastes, the ammonia removal efficiency was tested at
diverse pH-values (10–12), duration (5–60 min) and temp-
W
erature (30–72 C) ranges. The elevated pH was necessary
to shift the equilibrium to NH3, since only NH3 could be
stripped. Interestingly, the temperature increase of the
samples treated in the same duration of time was similar
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at both ultrasonication intensities. A high removal rate was
obtained at lower frequency of 28 kHz and pH 11 for
15 min. The higher ammonia removal efficiency at lower fre-
quency was assumed due to the non-thermal effect, which
contributed to different convective phenomena (micro-
streaming, micro-convection, shock waves, micro-jets). The
non-thermal effects were the dominant mechanism in the
ultrasound assisted ammonia stripping, accounting for
64% of the total ammonia removal rate. The experiments
with the livestock waste showed an ammonia removal rate
of up to 55% and an increase in the methane yield by
58%. An additional aeration did not bring any positive
increase of results.
Ultrasonication – advantages:
• Enhance solubilization, thereby increasing hydrolysis
rate and simultaneously reducing ammonia.
Ultrasonication – disadvantages:
• No/low reduction of ammonia concentration
• Rarely applied in actual plant
• Released other toxic compounds when too intense
• Destruction of microorganisms.
Microwave
Similar to the ultrasonication technique, the ammonia
removal by microwave radiation is also based on the shift
of the ammonia-nitrogen equilibrium to molecular ammonia
(NH3) and its subsequent evaporation by microwave radi-
ation. In order to treat coke-plant wastewater containing
high level of ammonia (5,000 mg/L at pH 11), Lin et al.
(a) developed an effective method to remove the ammo-
nia by microwave radiation. To find an optimal operating
condition, the experiments were conducted under different
pH levels, radiation time, with and without aeration, and
initial ammonia concentration. The aeration did not show
any significant influence on the ammonia removal. How-
ever, a fundamental difference between conventional
heating and microwave radiation could be observed accord-
ing to the NH3 transfer into the gas phase. Throughout, the
elimination efficiency was increased when microwave radi-
ation was applied.
MW radiation seems to be an effective strategy for the
removal of high ammonia concentrations from wastewater
in a short time. 10 min MW radiation (750 W) led to a
decreasing ammonia concentration from 5,000 mg/L to
350 mg/L at a pH-value of 11. However, both biological
and economic disadvantages may be considered, as
 1934 N. Krakat et al. | Ammonia removal in anaerobic digestion
microorganisms can be destroyed and costs for high power
consumption can rise.
Hollow fiber membrane
Using hollow membrane contactor, Lauterböck et al. ()
tried to counteract ammonia inhibition in slaughterhouse
wastes with ammonium concentration ranging from 6 to
7.4 g/L. A module consisted of seven hollow fibers, with
the microporous hydrophobic membrane material made
from polypropylene without outer casing, were directly sub-
merged into the fermentation broth. Simultaneously,
sulfuric acids were circulated through the lumen of the
fibers and the nitrogen was recovered in the form of an
ammonium sulfate solution. This technology allows a gas-
eous transfer between two liquid phases (NH3 rich feed
and the acidic absorption solution), with the gas filled
pores of the membrane as the actual transfer area. The driv-
ing force for the mass transfer is the difference in NH3
partial pressure between the two liquid phases (Lauterböck
et al. ). The ammonia was continuously extracted by the
membrane contractor. The results showed that NHþ 4 -N was
significantly reduced by the hollow fibers contactor. More-
over, the pH was decreased as NHþ 4 -N was removed and
thus lowering the free ammonia concentration by 70%. It
was also observed that the VFA concentrations were lower
in the membrane reactor and the biogas yield was higher.
In addition to these methods, Kim et al. () investi-
gated microbial fuel cells (MFCs) in order to remove TAN
and residual COD from the effluent of an anaerobic reactor
fed with swine wastewater as substrate in batch mode. The
authors observed that MFCs could be useful to remove
TAN. Cerrillo et al. () employed a combination of an
AD system with a microbial electrolysis cell (MEC) coupled
with an ammonia stripping unit to improve the quality of the
effluent. It was concluded by the authors that this combined
AD-MEC system could be used to prevent nitrogen over-
loads and to achieve a high quality of effluents along with
the possibility of nutrient recovery. Zhang & Angelidaki
() employed a submersible microbial desalination cell
in a CSTR to reduce ammonia levels. As a result of batch
experiments for 30 days, the authors found out that the con-
centration of ammonia in the CSTR decreased from 6 to
0.7 g N/L.
MAP/struvite precipitation
SP has been widely applied to remove ammonium-nitrogen
from wastewaters (Darwish et al. ). The ammonium
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removal by adding magnesium salt and phosphate to
form equal molar concentrations of MgNH4PO4•6H2O
(MAP) under alkaline conditions is a useful method to pro-
duce struvite. Magnesium, ammonium and phosphate are
required in equimolar quantities (1:1:1) to form MAP. For
optimum recovery of ammonia; however, slight excess of
M and P are required (Çelen & Türker ). MAP has a
comparable composition of Mg, P and N as commercial
fertilizers in soil. Those MAP fertilizers are most com-
monly applied in granular form in the field (Degryse et al.
). However, wastewaters also contain large amounts
of phosphorus, and there are two reasons to remove P
from wastewater. Firstly, the removal of P is necessary to
avoid and prevent eutrophication in the environment.
Secondly, phosphate is a limited source, and thus recycling
P is vital for the sustainable production of food in future
(Cordell et al. ).
Different processes are used for the precipitation of
MAP. According to reagents used, most commonly
MgCl2, is applied while the pH is adjusted with NaOH.
These Mg salts react quickly with phosphorus during crys-
tallization process so that high quality struvite is formed
(Britton et al. ). Alternatively, MgO or Mg(OH)2 can
be used, which is cheaper than Mg salts and, furthermore,
the need for NaOH is less during the process. However,
the reaction time is slower than those of Mg salts resulting
in a product containing excess MgO of Mg(OH)2 (Capde-
vielle et al. ). The solubility of MAP crystals in water
decreases with increasing pH below 9 (Bhuiyan et al.
).
CONCLUSIONS
To prevent process failures due to ammonia toxicity, differ-
ent conventional strategies such as stripping, chemical
precipitation, adjustment of C:N-ratios, immobilization and
adaption of microorganisms, bioaugmentation, dilution of
substrates and/or co-digestion of nitrogen rich wastes have
been suggested.
Many studies also reported about using zeolite. Benefits
such as the ability to recover process failure and easy oper-
ation were discussed, but the possibility of low adsorption
in some cases and high costs for regeneration were the
pronounced drawbacks. Among the novel methods, ultra-
sonication and microwave techniques were reported to
provide ammonia reduction. Also MFC/microbial desalina-
tion cell seemed to provide high TAN-removals from AD
systems along with good effluent qualities.
 1935 N. Krakat et al. | Ammonia removal in anaerobic digestion
ACKNOWLEDGEMENTS
N. K., D. D. and R. A. are thankful for grants provided by the
German Federal Ministry of Food and Agriculture (BMEL,
grant no. 22026411) with Fachagentur Nachwachsende
Rohstoffe e. V. (FNR) as promotor.
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