Process Biochemistry 40 (2005) 1285–1292

www.elsevier.com/locate/procbio

Effects of high free ammonia concentrations on the

performances of anaerobic bioreactors
Baris Callia,*, Bulent Mertoglua, Bulent Inancb, Orhan Yenigunc
a
Department of Environmental Engineering, Marmara University, 34722 Goztepe, Istanbul, Turkey
b
National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
c
Institute of Environmental Sciences, Bogazici University, 80815 Bebek, Istanbul, Turkey
Received 15 September 2003; accepted 16 May 2004

Abstract

UASB reactors inoculated with different seed sludges were operated for 450 days under high ammonia concentrations to investigate
inhibition effects. Reactors were fed with synthetic wastewater providing an organic loading of about 1.2 kg COD m3/day and having total
ammonia nitrogen (TAN) concentrations gradually increasing from 1000 to 6000 mg/l. As, corresponding free ammonia nitrogen (FAN)
concentrations elevated up to 800 mg/l, COD removal efficiencies in the range of 78–96% were determined. High removal efficiencies
revealed that seed sludges taken from anaerobic reactors treating considerably low ammonia containing wastewaters have adapted to elevated
free ammonia concentrations successfully. However, propionate accumulation in some reactors and decrease in eubacterial signals detected
by fluorescent in situ hybridization (FISH) indicated that propionate degrading acetogenic bacteria are more sensitive than methanogenic
archaea to free ammonia. Differences in the efficiencies of reactors were correlated with the quantity of resistant and sensitive microorganisms
dominated in the reactors.
# 2004 Elsevier Ltd. All rights reserved.

Keywords: Acetogenic bacteria; Ammonia; Butyrate; Inhibition; Propionate; UASB reactor

1. Introduction In literature, ammonia inhibition was reported to occur

There are various wastes containing high ammonia con-
centrations near or above inhibition levels for anaerobic
treatment. These are the organic fraction of municipal solid
waste [1–3], waste activated sludge [4], wastewaters origi-
nating from potato starch [5], dairy [6] and seafood proces-
sing [7] industries, young landfill leachate [8,9] and animal
manures [10–12]. However, noticeably different inhibition
threshold concentrations were reported in these studies in
consequence to differences in parameters which directly
affect the form of ammonia such as pH and temperature
and the adaptation level of anaerobic sludge used.
* Corresponding author. Environmental Engineering Department,
Faculty of Engineering, Marmara University, 34722 Goztepe, Istanbul,
Turkey. Tel.: +90-216-347-40-90; fax: +90-216-348-02-93.
E-mail address: bcalli@eng.marmara.edu.tr (B. Calli).
above pH 7.4 in the range of 1500–3000 mg/l total ammonia
nitrogen (TAN), whereas at concentrations in excess of
3000 mg/l, ammonia was claimed to be toxic irrespective
of pH [5,13]. These studies have been carried out using
anaerobic digester sludge, in which TAN concentrations
were typically below 1000 mg/l, as a seed material. By
using the seed sludge taken from swine manure digester
acclimated to 2400 mg/l TAN, methane production occurred
even at 5000 mg/l without any sign of inhibition. This was
also verified when the methanogenic activity, even with
10%, was maintained at 11,800 mg/l TAN after acclimation
of the granular sludge which was initially completely inhib-
ited at 1900 mg/l [14].
The inhibitory effects of ammonia, as far as is
known, influence mainly the phase of methanogenesis in
anaerobic reactors [5,10,15–17]. Within two distinct
methanogenic groups, acetate consuming methanogens
were in most cases found to be more sensitive than

0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2004.05.008
1286 B. Calli et al. / Process Biochemistry 40 (2005) 1285–1292
hydrogen utilizing ones [2,4,10]. Although some pure
culture experiments supported this result [18], in one of
the studies under thermophilic conditions, hydrogen uti-
lizing methanogens in contrast were defined as the sensi-
tive group [19].
Two different mechanisms were attributed to ammonia
inhibition of methanogens. According to the first mechan-
ism, activities of methane synthesizing enzymes are
directly inhibited by free ammonia. In the second one,
hydrophobic free ammonia molecules diffuse passively
into the cell and are rapidly converted to ammonium owing
to the intracellular pH conditions. Ammonium accumu-
lated inside the cell may be toxic by altering the intracel-
lular pH [20,21].
To investigate the inhibitory effects of ammonia nitrogen
on anaerobic treatment, long-term adaptation studies should
be carried out by controlling critical parameters such as pH,
temperature, retention time and organic loading rate. In this
study, to investigate the ammonia inhibition in details, which
was observed several times throughout the anaerobic treat-
ability of a young landfill leachate in our previous study [9],
laboratory scale UASB reactors were operated under high
ammonia levels for a long-term period. Evaluations of their
influences on the performances of anaerobic reactors are
presented.
2. Materials and methods
Five laboratory scale identical UASB reactors inoculated
with different seed sludges were operated for 450 days under
constant organic loading rate of about 1.2 kg COD m3/day
and gradually increasing influent ammonia concentrations
from 1000 to 6000 mg/l TAN.
Fig. 1. Experimental set-up consisting of five UASB reactors.
The concentration of free ammonia nitrogen (FAN) was
calculated by using the following formulae [17,22,23]
TAN
FAN ¼ (1)
1 þ 10ðpKa pHÞ
2729:92
pKa ¼ 0:09018 (2)
T þ 273:15
where, pKa is the dissociation constant for ammonium ion,
8.95 at 35 8C; T the temperature, 8C
Each reactor had an effective volume of 4.3 l. Their
diameters and effective heights were 10 and 55 cm, respec-
tively. There were three sampling ports located along with
the height of the reactors. Reactors were fed from the
influent funnels connected to the bottom and located above
the effluent port level. Synthetic wastewater in the feed
tank was pumped to these funnels with peristaltic pumps
providing 4 days hydraulic retention time in each reactor.
To homogenize the solution and prevent precipitation, the
feed tank was intermittently stirred with a mechanical
mixer. Meanwhile, a constant temperature room was used
to maintain the temperature at 35  2 8C. Configurations
and dimensions of the reactors and the details of the
experimental set-up are shown in Fig. 1.
Three of the seed sludges inoculated to the reactors
were taken from full scale anaerobic reactors treating corn
processing, alcohol distillery and snack food processing
wastewaters. The fourth one was taken from a laboratory
scale reactor treating young municipal solid waste landfill
leachate [9]. The fifth seed sludge was obtained by con-
centrating the biomass in the aforementioned leachate.
Sources, physical forms and volatile to total suspended
solids (VSS/TSS) ratios of seed sludges are given in
Table 1.
A simple synthetic wastewater was fed to the reactors
which was adapted from a dilution solution used in metha-
 B. Calli et al. / Process Biochemistry 40 (2005) 1285–1292 1287

Table 1
Seed sludges and their characteristics
Reactor Source Form VSS/TSS
R1 Full scale EGSB reactor treating corn processing wastewater Granular (broken) 0.83
R2 Lab scale UASB reactors treating Kömürcüoda landfill leachate Flocculent 0.39
R3 Full scale UASB reactor treating alcohol distillation wastewater Granular (broken) 0.94
R4 Concentrated biomass from Kömürcüoda landfill leachate Flocculent 0.17
R5 Full scale UASB reactor treating snack food processing wastewater Granular (tiny) 0.54

nogenic activity assays [24]. It was composed of 3230 mg/l
acetic acid, 570 mg/l propionic acid, 550 mg/l butyric acid,
150 mg/l CaCl22H2O, 120 mg/l Na2S9H2O and 200 mg/l
yeast extract and prepared with dechlorinated tap water to
supply some of the trace elements that anaerobic micro-
organisms require. As ammonium source, 25% NH4OH
solution was used and added in proper amounts parallel
to gradually increasing concentrations. The pH of the feed
was adjusted to 7.7 with a HCl (4.5%) and H3PO4 (12.5%)
mixture instead of HCl alone to prevent chloride inhibition
in the reactors. Furthermore, the pH of the feed was elevated
to 8.1 to increase the FAN concentration in addition to
increasing TAN loads.
In the influent and effluents of the reactors pH, total
alkalinity, COD and TAN were monitored three times in a
week, while volatile fatty acids (VFAs), TSS and VSS were
measured twice and soluble COD once a week. Meanwhile,
sludge concentrations in the reactors were monitored
monthly by taking samples from bottom sampling ports.
All analyses were carried out in accordance with Standard
Methods [25].
To determine the quantitative variations in eubacterial
and archaeal communities using a fluorescent in situ hybri-
dization (FISH) technique, monthly sludge samples were
taken from the bottom sampling ports of UASB reactors.
Sludge samples were immediately fixed and hybridized with
FLUOS labeled Eub338 and Cy3 labeled Arch915 oligonu-
cleotide probes according to procedure outlined in our
previous papers [26,27]. Visualization was carried out with
an Olympus BX50 microscope. Images of the slides were
taken with a digital camera.
3. Results and discussion
All five UASB reactors were fed with the synthetic
wastewater having a COD concentration of about
5000 mg/l and operated for 450 days under almost constant
loading rates in the range of 0.9–1.4 kg COD m3/day. In the
meantime, influent TAN concentrations were increased step
by step from 1000 to 6000 mg/l with 500 mg/l increments
(Fig. 2a). Influent pH was adjusted to 7.7 until day 302 and
then increased gradually up to 8.1 to elevate the FAN
concentration in the reactors (Fig. 2b).
In the start-up period until day 75, performances of the
reactors were unstable. Therefore, influent TAN concentra-
tion was kept around 1000 mg/l so as not to elevate the FAN
concentration above 50 mg/l (Fig. 2a). In this period, after

Fig. 2. Free ammonia concentrations increasing parallel to influent pH and total ammonia.
1288 B. Calli et al. / Process Biochemistry 40 (2005) 1285–1292

Fig. 3. COD removal efficiencies and effluent VFA concentrations of the reactors.

day 50, COD removal efficiencies of about 95% were
observed in R2 and R4. 80% efficiency in R3 and 70%
efficiencies in R1 and R5 at day 50 reached to 85% at day 75
and 90% at day 98, respectively (Fig. 3). Meanwhile,
reductions in acetic and propionic acid concentrations in
the effluents corresponding to improvement in reactor per-
formances indicated the termination of the start-up period
(Fig. 3).
Influent TAN concentration was elevated to 2000 mg/l at
day 98, to 2500 mg/l at day 117 and to 3000 mg/l at day 140.
Thus, the FAN concentration in the reactors reached 160 mg/
l (Fig. 2a). Although this level doubles the minimum free
ammonia inhibition threshold indicated by Koster and Let-
tinga [5], detrimental effects were not observed on the
performances of reactors. In contrast, 90% COD removal
efficiency in R5 at day 98 increased up to 95% at day 150
and approached the performances of R2 and R4 (Fig. 3e).
By increasing the influent TAN concentration to
3500 mg/l at day 189, to 4000 mg/l at day 210 and to
4500 mg/l at day 230, FAN concentrations in the reactors
 B. Calli et al. / Process Biochemistry 40 (2005) 1285–1292
exceeded 160 and reached 240 mg/l (Fig. 2a). FAN con-
centrations attained in this period were reported as com-
pletely inhibitory and responsible for unbalancing in the
reactors [14]. In addition to high FAN levels, problems in
feeding pumps resulted in a shock organic load at day 210.
Consequently, the acetic acid concentration in the effluent of
R1 exceeded 500 mg/l at day 212 which was usually around
100 mg/l. Thus, COD removal efficiency in R1 decreased
from 91 to below 85%. The reductions were from 96 to 92%
in R2 and from 89 to 81% in R3. In spite of, shock organic
load and FAN concentrations of about 240 mg/l, R4 and R5
achieved 94 and 96% COD removal efficiencies, respec-
tively without any decline in performance (Fig. 3).
Until day 267, influent TAN concentration was kept at
4500 mg/l to provide an adaptation period for R1, R2 and R3
which were inhibited after the previous jump in FAN level.
In this 37-day period, COD removal efficiencies of about 92
and 83% were restored to 96 and 90% in R2 and R3,
respectively. At day 267, the influent TAN concentration
was increased to 5000 mg/l which resulted in 280 mg/l FAN
in the reactors. However, until day 275, the TAN concentra-
tion in the feed could not be kept at 5000 mg/l and fluctuated
around 4800 mg/l. Therefore, the increase in FAN was
tolerated until day 285 in R2 and until day 289 in R1 and
R3. At day 287, the COD removal efficiency in R2 suddenly
decreased from 95 to 86% within 3 days and the acetic acid
concentration in the effluent jumped to 130 mg/l at day 292
and to 250 mg/l at day 299 which was usually below 30 mg/l
until then. Reductions in COD removal efficiencies in R1
and R3 were not as severe as in R2 while R4 and R5 were
still very efficient in this period.
At day 303, the influent pH was adjusted to 7.8 which
was previously kept at 7.7. Subsequently, the FAN level in
the reactors reached 340 mg/l on average. In this period,
COD removal efficiency in R1 fluctuated between 81 and
89% due to the presence of acetic acid in the effluent at
concentrations as high as 600 mg/l. COD removal effi-
ciency in R2 exhibited a rise up to 92% at day 322 after the
unexpected inhibition at day 287, however it again
dropped to 85% at day 329 and nevermore exceeded
90%. Above 200 mg/l propionic acid and about 400 mg/
l acetic acid in the effluent of R3 were indications of
inhibition and thus COD removal efficiency decreased
from 91 to 84%. Although R1, R2, and R3 were more
or less affected from FAN concentration of about 340 mg/l
in this period, once more R4 and R5 had satisfactory COD
removal of above 93% (Fig. 3).
On increasing the influent pH to 7.9 at day 340 and to 8.0
at day 364, FAN concentrations in the reactors increased to
537 mg/l (Fig. 2b). Consequently, effluent COD concentra-
tions exceeded 900 and 600 mg/l in R1 and R2, respectively.
Moreover, parallel to the increase in acetic and propionic
acid concentrations of up to 470 and 340 mg/l respectively,
COD concentrations as high as 1085 mg/l were measured in
R3 at day 376. R4 and R5 tolerated even these increments in
FAN and achieved 93 and 95% COD removal efficiencies,
1289
respectively with very low total VFA concentrations in the
effluents of less than 50 mg/l (Fig. 3).
At day 383, the TAN concentration in the feed was
increased from 5000 to 6000 mg/l which resulted in about
620 mg/l FAN in the reactors (Fig. 2). Hence, in the effluent
of R1, both acetic and propionic acid concentrations
approximately doubled and reached to 400 mg/l at day
404, 21 days after the increase. A similar trend was observed
in R2. In R3, acetic and propionic acid concentrations in the
effluent decreased to 200 mg/l level and about 85% COD
removal rate was provided once more (Fig. 3).
Finally at day 406, the pH was increased to 8.1 to increase
the FAN concentration to 800 mg/l on average (Fig. 2). In
literature, usually severe or complete inhibitions were
reported at similar or lower FAN concentrations and most
of these studies were conducted under thermophilic condi-
tions. However, methanogenic activities were reported at
FAN concentration as high as 1100 mg/l in thermophilic
digestion of different animal manures [10,17].
After day 406, R1 and R2 exhibited constant COD
removal efficiencies of 82 and 87%, respectively until the
end of the experimental period. Acetic acid of about 400 mg/
l and propionic acid around 300 mg/l were measured in the
effluents of R1 while in R2, concentrations were almost half
of these. R3 exhibited a sudden drop in COD removal
efficiency from 85 to 78%. The most resistant reactor R5
was affected drastically at this FAN level and 96% removal
decreased to 85% progressively within 40 days associated
with the increase in effluent acetic acid concentration up to
370 mg/l. However, very low propionic acid concentrations
of less than 10 mg/l in the effluent of R5 indicated the
presence of propionate degraders that are resistant to these
extremely high FAN levels. In R4, reduction in methano-
genic activity was still inconsiderable, consequently 90%
COD removal efficiency was obtained with acetic and
propionic acid concentrations of less than 100 mg/l in the
effluent.
In addition to effluent COD and VFA parameters, VSS
concentrations in sludge samples taken from bottom sam-
pling ports of the reactors exhibited interrelated results
between inhibition and adaptation events. After start-up,
biomass increased to significant amounts in R4 and R5
which gradually adapted to elevated FAN concentrations
and achieved high COD removal efficiencies. Nevertheless,
even though no sludge wastage was observed, VSS con-
centrations in R1 and R3 remained almost constant. Con-
tinuously growing biomass in R2 started to decline after the
sudden drop in COD removal efficiency from 94 to 87% at
day 282 (Fig. 4).
Furthermore, to determine the quantitative variations in
methanogenic archaea and acetogenic bacteria in the reac-
tors, fluorescent in situ hybridization experiments were
conducted by using archaea (Arch 915) and bacteria (Eub
338) specific oligonucleotide probes. The 16S rRNA based
in situ hybridizations provided valuable information about
the abundance of active methanogenic archaea and aceto-
1290 B. Calli et al. / Process Biochemistry 40 (2005) 1285–1292

Fig. 4. VSS concentrations in the bottom sampling port of reactors.

genic bacteria and gave an opportunity to investigate the
inhibitory effect of ammonia on syntrophic relation. Even
under high free ammonia levels, there was a superior
correlation between the reactor performances and abun-
dance of Methanosarcina-like methanogenic archaea in
multicellular clusters in all reactors (Fig. 5b and d). On
the other hand, as FAN exceeded 200 mg/l, acetogenic
bacteria (Eub 338) prevailing in R1, R2 and R3 gradually
lost their activity and obviously decreased in numbers
parallel to propionic acid accumulation (Fig. 5a and c).
Very low butyric acid concentrations in the effluents indi-
cated that butyrate degraders were the resistant member
of acetogenic bacteria in the reactors. In that case, the
reason for the decrease in Eub338 signals in R1, R2 and
R3 may certainly be associated with inhibition of sensitive
propionate degraders above 200 mg/l FAN. In contrast,
according to FISH results, the presence of resistant propio-
nate degraders was indicated in R4 and R5 which was also
supported by effluent propionic acid concentrations of less
than 50 mg/l.
It was difficult to identify the adaptation phenomena as
modification of the appropriate enzymes or as growing and
domination of resistant microorganisms using conventional
analysis. Therefore, investigations should also be conducted
on microbial diversity at genus and species levels. In this
manner, recent molecular microbiology techniques should
be used for identification of anaerobic microorganisms in
their natural habitats, since methanogens and acetogens are
especially difficult to study by cultivation-based techniques.
In a recent study, identification of microorganisms in sludge

Fig. 5. Fluorescent in situ hybridization photomicrographs (1000); sludge samples (R1) hybridized with Fluos labeled Eub 338 (a and c) and Cy3 labelled
Arch 915 (b and d) probes.
 B. Calli et al. / Process Biochemistry 40 (2005) 1285–1292
samples is still going on in detail and results will be
discussed in a forthcoming paper.
4. Conclusions
As FAN concentrations were increased gradually to
800 mg/l throughout the 450 days experimental period,
COD removal efficiencies in the range of 78–96% were
observed in five identical UASB reactors seeded with dif-
ferent sludges. High removal efficiencies indicated that seed
sludges taken from anaerobic reactors treating low ammonia
containing wastewaters may be successfully adapted to
elevated free ammonia concentrations. Variations in the
efficiency of the reactors may be explained by the diver-
gence in the amount of free ammonia resistant microorgan-
isms in seed sludges.
According to 16S rRNA-based FISH results, it was
indicated that, in R1, R2 and R3 propionate degrading
acetogenic bacteria were significantly inhibited above
200 mg/l FAN. Quite high propionic acid concentrations
in the effluents of these reactors confirmed this consequence.
However, low propionic acid concentrations of less than
10 mg/l in the effluent of R5 indicated the existance of
propionate degraders resistant to extremely high free ammo-
nia levels. On the other hand, there was no problem in the
conversion of butyric acid at any free ammonia level in all
reactors revealing the resistance of butyrate degraders.
Granulated sludges known to be resistant to environmen-
tal factors and toxic substances did not show the same
resistance to high free ammonia concentrations in this
long-term study. Besides, the lowest removal efficiencies
were observed in R1 and R3 which were seeded with
granular sludges. This revealed that the microbial content
is more significant than physical structure of the sludge
against ammonia inhibition. Thus, to elucidate the inhibitory
effects and adaptation phenomena accurately, microbial
identification and quantification studies should certainly
be conducted.
Acknowledgements
Baris Calli and Orhan Yenigun would like to acknowl-
edge the financial support of Bogazici University Research
Fund, project number 01Y101D.
Appendix A. Nomenclature
COD chemical oxygen demand (mg/l)
FAN free ammonia nitrogen (mg/l)
OLR organic loading rate (kg COD m3/day)
Ri reactor i
TAN total ammonia nitrogen (mg/l)
TSS total suspended solids (mg/l)
1291
VFAs volatile fatty acids (mg/l)
VSS volatile suspended solids (mg/l)
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