Hindawi Publishing Corporation
Archaea
Volume 2016, Article ID 4089684, 10 pages
http://dx.doi.org/10.1155/2016/4089684

Research Article
Archaea and Bacteria Acclimate to High Total Ammonia in
a Methanogenic Reactor Treating Swine Waste
Sofia Esquivel-Elizondo,1,2 Prathap Parameswaran,3 Anca G. Delgado,1 Juan Maldonado,1
Bruce E. Rittmann,1,2 and Rosa Krajmalnik-Brown1,2
1

Swette Center for Environmental Biotechnology, The Biodesign Institute, Arizona State University, P.O. Box 875701,
Tempe, AZ 85287-5701, USA
2
School of Sustainable Engineering and the Built Environment, Arizona State University, Tempe, AZ, USA
3
Department of Civil Engineering, Kansas State University, 2118 Fiedler Hall, Manhattan, KS 66506, USA
Correspondence should be addressed to Rosa Krajmalnik-Brown; dr.rosy@asu.edu
Received 10 June 2016; Accepted 11 August 2016
Academic Editor: Jessica A. Smith
Copyright © 2016 Sofia Esquivel-Elizondo et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Inhibition by ammonium at concentrations above 1000 mgN/L is known to harm the methanogenesis phase of anaerobic digestion.
We anaerobically digested swine waste and achieved steady state COD-removal efficiency of around 52% with no fatty-acid or
H2 accumulation. As the anaerobic microbial community adapted to the gradual increase of total ammonia-N (NH3 -N) from
890 ± 295 to 2040 ± 30 mg/L, the Bacterial and Archaeal communities became less diverse. Phylotypes most closely related
to hydrogenotrophic Methanoculleus (36.4%) and Methanobrevibacter (11.6%), along with acetoclastic Methanosaeta (29.3%),
became the most abundant Archaeal sequences during acclimation. This was accompanied by a sharp increase in the relative
abundances of phylotypes most closely related to acetogens and fatty-acid producers (Clostridium, Coprococcus, and Sphaerochaeta)
and syntrophic fatty-acid Bacteria (Syntrophomonas, Clostridium, Clostridiaceae species, and Cloacamonaceae species) that have
metabolic capabilities for butyrate and propionate fermentation, as well as for reverse acetogenesis. Our results provide evidence
countering a prevailing theory that acetoclastic methanogens are selectively inhibited when the total ammonia-N concentration is
greater than ∼1000 mgN/L. Instead, acetoclastic and hydrogenotrophic methanogens coexisted in the presence of total ammonia-N
of ∼2000 mgN/L by establishing syntrophic relationships with fatty-acid fermenters, as well as homoacetogens able to carry out
forward and reverse acetogenesis.

1. Introduction
Animal wastes contribute more than half of the biomassbased wastes generated in the United States [1, 2]. The organic
carbon in animal wastes could be a major source of renewable
energy if it were captured as methane gas. Many animal
wastes, including swine waste, also are rich in organic nitrogen (N) due to the high protein content in the animals’ diet.
Anaerobic hydrolysis and fermentation convert the organic N
into ammonia-N (NH3 -N). While typical NH3 -N (i.e., unionized NH3 and NH4 + ) concentrations in anaerobic digesters
treating domestic wastewater sludge are 650–1100 mg L−1 [3],
concentrations in swine manure are as high as 8000 mg L−1
[4–6]. A challenge arises for treating these wastes (and
ultimately capturing the organic carbon as energy source) as

NH3 -N above 1000 mg L−1 is toxic to many groups of microorganisms [7, 8], including methanogenic Archaea [9, 10].
In anaerobic systems without inhibition by NH3 -N,
organic acids produced from acidogenesis are fermented to
acetate and H2 , and the typical distribution of the electron
flow to methane is 67% through acetate and 33% through H2
[11, 12]. Correspondingly, acetoclastic methanogens usually
predominate in anaerobic digesters with <1000 mg NH3 N L−1 [13, 14]. In contrast, the vast majority of studies on
methanogenesis from swine waste report that the dominant methanogenesis pathway switches from acetoclastic to
hydrogenotrophic. Several studies [15–19] reported that the
methane production in anaerobic reactors treating wastes
with high NH3 -N occurs mainly via hydrogenotrophic

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Table 1: Stoichiometry and thermodynamics of syntrophic acetate, propionate, and butyrate fermentation.

Acetate fermentation
Propionate fermentation
Butyrate fermentation
Hydrogen oxidation
(1a) + (2)
(1b) + (2)
(1c) + (2)

(1a) CH3 COO− + H+ + 2H2 O → 2CO2 + 4H2
(1b) C3 H5 OO− + 2H2 O → CH3 COO− + CO2 + 3H2
(1c) C4 H7 OO− + 2H2 O → 2CH3 COO− + H+ + 2H2
(2) 4H2 + CO2 → CH4 + 2H2 O
(3) CH3 COO− + H+ → CO2 + CH4
(4) C3 H5 OO− + (1/2)H2 O → CH3 OO− + (3/4)CH4 + (1/4)CO2
(5) C4 H7 OO− + 2H2 + CO2 → 2CH3 COO− + H+ + CH4

Δ𝐺∘󸀠 = +55.0
Δ𝐺∘󸀠 = +76.0
Δ𝐺∘󸀠 = +48.3
Δ𝐺∘󸀠 = −130.8
Δ𝐺∘󸀠 = −75.8
Δ𝐺∘󸀠 = −28.0
Δ𝐺∘󸀠 = −42.6

The standard free enthalpies of formation (Δ𝐺∘󸀠 ) are reported in kJ reaction−1 at 1 M, pH 7, and 25∘ C.

methanogenesis, since acetoclastic methanogens are inhibited and washed out. The loss of acetoclastic methanogens in
the bioreactors raises questions about the fate of acetate generated by fermentation. It was previously postulated that the
loss of acetoclastic methanogens was compensated by syntrophic “acetate oxidation” to CO2 and H2 (more accurately
termed reverse acetogenesis) coupled with hydrogenotrophic
methanogenesis [20]. Specifically, acetate generated by fermentation is converted into H2 and CO2 by reverse acetogenesis, and the H2 and CO2 are utilized by hydrogenotrophic
methanogenesis. The reactions involved in the syntrophy
of reverse acetogenesis coupled with H2 conversion into
methane with their corresponding Δ𝐺∘󸀠 are illustrated in
Table 1, equations (1a) and (2), respectively. Reverse acetogenesis might not be the only mechanism that allows acetate
conversion into methane when treating high-ammonium
wastes. Methanogenesis is possible if some acetoclastic
methanogens are able to adapt to high-ammonium concentrations and avoid being washed out. In fact, Westerholm
et al. detected acetoclastic methanogens in methanogenic
reactors operating at increasing NH3 -N concentrations (from
800 to 6900 mg L−1 ) [21]. Thus, an acclimation period may
be crucial for developing a microbial community that has
acetoclastic methanogens capable of tolerating high NH3 -N.
Another key aspect of microbial community acclimation
is the scavenging of H2 produced in fermentation and reverse
acetogenesis. It is well known that fermentation of propionate
(equation (1b) in Table 1) and butyrate (equation (1c)) is
coupled with hydrogenotrophic methanogenesis [22–24].
The highly endergonic nature of these fatty-acid fermentation
instances means that they can occur only at very low partial
pressures of H2 (<10−4 atm), which requires a tight syntrophic
partnership with a H2 scavenger, such as a hydrogenotrophic
methanogen, to maintain a negative Δ𝐺∘󸀠 for fermentation to
proceed [25].
The microbial ecology of anaerobic reactors treating
high NH3 -N wastes has received limited attention [15, 26–
28], and the possible syntrophies among different Archaea,
fermenting Bacteria, and homoacetogens are yet poorly
understood. High-throughput sequencing in combination
with statistical analysis can illuminate microbial dynamics
with high NH3 -N concentrations by identifying key Archaea
and Bacteria involved in syntrophic fatty-acid conversion into
methane. In this study, we used high-throughput sequencing,
parametric correlation, and qPCR (quantitative polymerase
chain reaction) to analyze shifts in the Archaeal and Bacterial

communities during the startup phase (first 105 days) of
an anaerobic digester successfully treating swine manure
to generate methane and without significant accumulation
of acetate or H2 . Contrary to previous explanations of the
effects of NH3 -N concentration higher than 1000 mg L−1 ,
acetoclastic methanogens played a major role in methane
production. Our results point to syntrophies that involved
acetoclastic methanogens, hydrogenotrophic methanogens,
homoacetogens, and other syntrophic acid-fermenting Bacteria developed during the startup of a methanogenic
bioreactor able to function well with NH3 -N greater than
∼2000 mg NH3 -N L−1 .

2. Materials and Methods
2.1. Anaerobic Bioreactor Setup and Operation. The bioreactor consisted of a 1-L glass vessel continuously stirred at
170 rpm and operated at 37∘ C and at a pH of 6.9–7.6. The
reactor was inoculated with 1 : 2 volume ratio of anaerobic
digested sludge to swine waste to ensure that the concentration of NH3 -N was below 1100 mg N L−1 . The anaerobic
digester sludge was obtained from the Northwest Wastewater
Reclamation Plant (Mesa, Arizona, USA) and the swine waste
from Hormel Foods (Snowflake, Arizona, USA).
The bioreactor was operated in batch mode for 35 days,
during which time methane production reached a plateau.
Afterwards, the operation was switched to semicontinuous
mode by daily removing and adding 33 mL of swine waste
feed with a sterile syringe under vigorous N2 sparging. The
hydraulic retention time (HRT), equal to the solids retention
time (SRT), was 35 days (denoted as “cycle” in the manuscript
figures). The bioreactor was operated semicontinuously for
105 days.
2.2. Chemical Analyses. An array of chemical analyses was
employed to characterize the input swine waste and to monitor the performance of the anaerobic reactor through liquid
samples taken from the reactor’s homogenized contents. Total
and soluble chemical oxygen demand (TCOD and SCOD),
soluble total nitrogen (TN), and NH3 -N were assayed with
HACH kits using spectrophotometer absorbance at wavelengths of 620, 410, and 655 nm, respectively. Soluble concentrations were measured after filtering the sample through a
0.45 𝜇m membrane filter.
Gas samples (200 𝜇L) were withdrawn from the bioreactor headspace using a 500 𝜇L gas-tight syringe (SGE,

Archaea
Switzerland) to quantify methane (CH4 ) and hydrogen (H2 )
concentrations using a gas chromatograph (GC 2010, Shimadzu) equipped with a thermal conductivity detector and
a packed column (ShinCarbon ST 100/120 mesh, Restek
Corporation). N2 was the carrier gas supplied at a constant
pressure of 405 kPa and a constant flow rate of 10 mL min−1 ,
and the temperature conditions for injection, column, and
detector were 110, 140, and 160∘ C, respectively.
2.3. DNA Extraction. Four biomass samples were obtained
from the inoculum and the effluent of the anaerobic bioreactor at the end of the batch operation and at the end of the first
and second cycles of the semicontinuous operation, when
the system was at steady state based on the COD removed
as CH4 . Effluent samples were pelleted using an Eppendorf
microcentrifuge 5810R (Hauppauge, NY) at 13,200 rpm. DNA
was extracted from 0.25 g (wet weight) of two pellets per sampling point using the MOBIO PowerSoil DNA extraction kit
(Carlsbad, CA). DNA from duplicate pellets was merged for
sequencing.
2.4. High-Throughput Microbial Community Analysis. To
determine the structure of the Bacterial community during
startup of the semicontinuous reactor, we sequenced DNA
using the Illumina MiSeq platform at University of Minnesota
Genomics Center (http://www.health.umn.edu/research/resources-researchers/genomics-center). Bacterial primers
used were V4F (5󸀠 -GTGCCAGCMGCCGCGGTAA-3󸀠 ) and
V6R (5󸀠 -ACAGCCATGCANCACCT-3󸀠 ), which amplify the
V4–V6 hypervariable regions of the 16S rRNA gene. The
reads were paired-end, and each end of the DNA fragment
consisted of 300 bp (2 × 300 bp). Before processing the reads
using the QIIME 1.8.0 pipeline [29], we paired forward and
reverse sequences using PANDASeq [30]. The average length
of reads after overlap was 551 bp.
The Archaeal community was sequenced at MR DNA
(http://www.mrdnalab.com/, Shallowater, TX, USA) on an
Illumina MiSeq following the manufacturer’s guidelines. 16S
rRNA primers 349F and 806R, with barcode on the forward
primer, were used to amplify the V3 and V4 hypervariable
region of this highly conserved gene [31]. The reads were
paired-end, and each end of the DNA fragment consisted of
300 bp (2 × 300 bp). The average length of reads after overlap
was 449 bp. Bacterial and Archaeal raw sequences were
submitted to NCBI Sequence Read Archive and are available
under the following accession numbers: SAMN04481086–
SAMN04481094.
Sequences with at least one of the following characteristics were omitted for the downstream analysis: being shorter
than 200 bps, with quality score of 25 or below, with any
primer or barcode mismatches, and with more than 6
homopolymers. From the sequences that passed the quality
filtering, OTUs were picked based on 97% sequence similarity
using the UCLUST algorithm [32]. The most abundant
sequence of each cluster was picked as the representative
sequence. Sequences were aligned using the PyNAST method
[33] and filtered to remove gaps and Chimeras (using
ChimeraSlayer [34]). The UCLUST algorithm [32] was used
to assign taxonomy to the most abundant sequence of each

3
OTU by comparing the most abundant sequence of each
OUT to the Greengenes database [35]. OTU tables (1 each
for Bacterial and Archaeal sequences) were generated from
the representative sequences excluding Chimeras. OTUs with
single sequences (singletons) were removed from the OTU
tables. To avoid biases that occur when sampling various
species in a community, OTU tables were subsampled (rarefied) using the pseudorandom number generator (PRNG)
NumPy, an implementation of the Mersenne PRNG [36].
Final OTUs numbers were 3275 for Bacteria and 776 for
Archaea. The numbers of high-quality reads per sample in
the Bacteria and Archaea analysis were 25,000 and 90,000
sequences, respectively, on average.
2.5. Sample Diversity. We calculated sample species diversity
(alpha diversity) by estimating PD- (Phylogenetic Distance-)
whole-tree and observed-species metrics using the QIIME
1.8.0 pipeline [29]. For this, we performed multiple subsamplings (rarefactions) of the OTU table (Bacteria and Archaea
separately) at a depth of 100 sequences in 10 replicates, and
we analyzed rarefaction measures in which the sequence
numbers per sample were equal (18,000 sequences per sample
for Bacteria and 32,000 for Archaea).
2.6. Quantitative Real-Time PCR (qPCR). We performed
qPCR in 20 𝜇L reactions, each containing 6 𝜇L PCR grade
water, 0.04 𝜇L TAQMAN probe (200 nM), 1 𝜇L each of
forward and reverse primers (500 nM), 10 𝜇L TAQ PCR
SuperMix (1X) or SYBR green mix (1X), and 2 𝜇L template
normalized DNA to 10 ng/𝜇L (130 nM). Using TAQMAN
assays, we used the qPCR primers and conditions described
previously [37, 38] for the amplification of the 16S rRNA gene
of Archaea, the methanogenic orders (Methanomicrobiales,
Methanobacteriales, and Methanococcales), and the families
Methanosaetaceae and Methanosarcinaceae. We also used
qPCR to target the 16S rRNA gene of Bacteria [39] and the
highly conserved formyl tetrahydrofolate synthase (FTHFS)
gene in homoacetogens by performing SYBR green assays,
using methods described previously [38].
2.7. Statistical Analysis. Using the Statistical Package for the
Social Sciences (SPSS) software, we performed Pearson’s
parametric correlation among variables of interest: methane
production, total N and NH3 -N concentrations, and key fermenters, syntrophs, and hydrogenotrophic and acetoclastic
methanogens identified through sequencing analysis. These
variables were picked based on the hypothesis that NH3 N would have an effect on microbial community structure,
specifically on methanogens, and correction for multiple
comparisons was not performed. 𝑃 < 0.05 was accepted as
significant.

3. Results and Discussion
3.1. COD Was Converted into Methane during Bioreactor
Operation despite NH3 -N > 2000 mg/L. We monitored total
and soluble COD, NH3 -N and total N, and methane and
biogas production rates at regular intervals during the startup
phase of the methanogenic reactor treating swine waste.

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COD concentration (g L−1 )

90

within the samples. PD-whole-tree, which is based on the
phylogenetic tree, uses the branch lengths as a measure
of diversity; the observed-species metric counts all unique
OTUs in the sample [40]. Both metrics had similar decreasing
trends for Archaea and Bacteria over time as the NH3 -N
concentration rose from 684 mg NH3 -N L−1 at the startup of
the reactor to 890 ± 295 mg NH3 -N L−1 in batch operation
and to 2040 ± 30 mg NH3 -N L−1 for continuous operation.
These significant decreases suggest selective enrichment of
microorganisms tolerant to NH3 -N.

80
70
60
50
40
30
20
10
0

Batch
TCOD in
TCOD out
SCOD in

Cycle 1

Cycle 2

Cycle 3

SCOD out
CH4

Figure 1: Total COD (TCOD) and soluble COD (SCOD) in the
influent and effluent of the reactor and the COD content of the total
methane (CH4 ) produced during each operating phase (batch and
3 cycles of semicontinuous operation). The data are averages with
standard deviations of three or more measurements.

The results are summarized in Figures 1 and 2 and S1, in
Supplementary Material available online at http://dx.doi.org/
10.1155/2016/4089684; Table S1 documents good COD massbalance closure at four sampling times. Based on COD
removed as CH4 , the performance of the reactor approached
a pseudo-steady state in cycle 1 of semicontinuous operation,
with approximately 52% conversion. CH4 was 70% ± 16%
of the biogas, and H2 concentrations were below detectable
levels (<0.5% v/v) throughout the experiment. During semicontinuous operation, effluent soluble COD (which is composed of relatively small, biodegradable molecules [39])
represented only 4.5% ± 0.3% of the influent TCOD or ≤2.6 ±
0.6 g COD L−1 . This low SCOD concentration implies that
short-chain fatty acids (including propionate, butyrate, and
acetate), which mainly comprise the SCOD, did not accumulate because they were consumed by microbial metabolism
leading to methane production and biomass synthesis.
Figure 2(a) shows NH3 -N increased from 890 ±
295 mg NH3 -N L−1 during batch operation to 2040 ±
30 mg NH3 -N L−1 during semicontinuous operation. The
concentration of soluble total N (the sum of NH3 -N and
organic N) paralleled that of NH3 -N and was about 50%
higher than total NH3 -N. This increasing release of organic
N and NH3 -N indicates that hydrolysis and fermentation
of the protein fraction of the animal wastes increased after
startup and stabilized in cycle 2. Figure 2(b) shows that
methanogenesis continued to increase into cycle 3, even
though hydrolysis and fermentation of protein stabilized.
Higher methane generation was possible in cycle 3 because
the input TCOD increased with the batch of swine waste used
in that cycle; the input N did not increase in parallel with
input TCOD because the feed collected swine manure was
not uniform throughout the experimental period.
3.2. The Diversity of Archaea and Bacteria Decreased with
Increasing NH3 -N Concentration. Table 2 summarizes the
coefficients of the two metrics used to analyze diversity

3.3. Hydrogenotrophic and Acetoclastic Methanogens Were
Abundant at ∼2000 mg NH3 -N L−1 . We analyzed the Archaeal microbial community during reactor startup using
high-throughput sequencing in order to evaluate how well
acetoclastic methanogens (grouped under Methanosarcinales) or hydrogenotrophic methanogens (grouped under
Methanobacteriales, Methanomicrobiales, Methanococcales,
and the E2 order) were tolerant to NH3 -N concentrations of
∼2000 mg L−1 . Figure 3 compares the Archaeal communities
in the inoculum with the communities after batch and
semicontinuous operation. Phylotypes within the phylum
Euryarchaeota (i.e., Methanomicrobiales, Methanobacteriales, E2 group, and Methanosarcinales) and those of the order
pGrfc26 (within Crenarchaeota) [41] increased during the
semicontinuous operation at 2040 ± 30 mg NH3 -N L−1 , while
the unidentified phylotypes decreased from 38.6 to 1.5%.
These results confirm enrichment during the gradual acclimation to high and increasing total-ammonium concentrations.
The relative abundance of hydrogenotrophic and acetoclastic methanogens increased over time. This agrees with
increasing methane production, low effluent SCOD (including acetate in the measurement), and no detection of H2 during semicontinuous operation. Figure 3(b) shows 8 different
genera of hydrogenotrophic methanogens identified, including Methanoculleus, Methanogenium, and Methanobrevibacter. Although the genus Methanosaeta was the sole acetoclastic methanogen identified by high-throughput sequencing,
its relative abundance increased from 14% in batch operation
to 25% during semicontinuous operation. This increase in
relative abundance could be due to an increase in the absolute
abundance of Methanosaeta or a decrease in the abundance
of other phenotypes. qPCR results (summarized in Figure
S2) are consistent with Figure 3 and suggest that Methanomicrobiales were the most abundant methanogens followed by
Methanosaetaceae and lastly by Methanobacteriales.
It is likely that the first 35 d of batch operation, with
<1100 mg N-NH3 L−1 , provided an acclimation period for
acetoclastic methanogens, and ∼2000 mg NH3 -N L−1 was
not high enough to inhibit Methanosaeta, which were
selected by the third cycle, relative to other Archaea. This
corresponds to the finding by Schnürer and Nordberg that
it took 3000 mg NH3 -N L−1 to inhibit NH3 -N-acclimated
Methanosaeta spp. [18] and that this family was not detected
in anaerobic digesters treating chicken wastes with above
3400 mg NH3 -N L−1 [42]. However, a recent study reported
that NH3 -N-acclimated Methanosaetaceae spp. were the

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Table 2: Diversity of each sample at different time points during the startup phase of the methanogenic reactor treating swine waste. At the
highest NH3 -N concentration, samples species diversity both for Archaea and for Bacteria was lowest.
Sample

NH3 -N (mg/L)

Inoculum
Batch
∗
Semicontinuous

Bacteria
Observed-species
1512 ± 10
1419 ± 7
1173 ± 12

PD-whole-tree
24 ± 0.2
25 ± 0.4
21 ± 0.1

Archaea
Observed-species
375 ± 6
474 ± 10
361 ± 2

The indices corresponding to semicontinuous operation are averages of the indices for samples taken on days 70 and 105.

4000
3500
3000
2500
2000
1500
1000
500
0

35
Production rate (mL L−1 )

Concentration (mg L−1 )

∗

N/A
<1100
∼2000

PD-whole-tree
67 ± 0.3
62 ± 0.3
52 ± 0.4

Batch

Cycle 1

Cycle 2

30
25
20
15
10
5
0

Cycle 3

Total NH3 -N
Total N

Batch

Cycle 1

Cycle 2

Cycle 3

Methane
Biogas
(b)

(a)

Figure 2: (a) Total nitrogen and NH3 -N concentrations and (b) methane and total biogas production rates during batch and semicontinuous
operation. The data are averages with standard deviations of three or more measurements during each operating phase.

100

80

80

60

60
(%)

(%)

100

40

40

20

20

0

Inoculum

Batch

Other
[Methanomassiliicoccaceae]
U_pGrfC26
Methanosaetaceae
(a)

Semicontinuous
Methanobacteriaceae
WSA2
U_Methanomicrobiales
Methanomicrobiaceae

0

Inoculum

Batch

Other
vadinCA11
U_[Methanomassiliicoccaceae]
U_pGrfC26
Methanosaeta

Semicontinuous
Methanosphaera
Methanobrevibacter
U_WSA2
Methanoculleus

(b)

Figure 3: Archaeal distributions. (a) Phylotypes at the family level. (b) Phylotypes at the genera level. Shades of green are for Methanomicrobiales (hydrogenotrophic methanogens), blue for Methanobacteriales (hydrogenotrophic methanogens), red for Methanosarcinales
(acetoclastic methanogens), and black for pGrfC26. The total NH3 -N concentration at the time of the batch sampling was 1140 mg/L.
Semicontinuous operation is an average of two samples at ∼2000 mg NH3 -N/L. “U ” stands for unidentified microorganism within the
taxonomic classification.

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100

80

80

60

60
(%)

(%)

100

40

40

20

20

0

Inoculum

Batch

0

Semicontinuous

Batch

U_ML615J-28 (Tenericutes)
W22
W5
Syntrophus
Treponema
U_Porphyromonadaceae
U_Marinilabiaceae
U_Bacteroidaceae

Marinilabiaceae
Bacteroidaceae
Spirochaetaceae
Pseudomonadaceae
[Tissierellaceae]
Syntrophomonadaceae
Ruminococcaceae
Lachnospiraceae
Clostridiaceae

Other
[Cloacamonaceae]
Syntrophorhabdaceae
Syntrophaceae
Anaerolinaceae
Comamonadaceae
Spirochaetaceae
Rikenellaceae
Porphyromonadaceae

Inoculum

(a)

Semicontinuous
U_Bacteroidales
Sphaerochaeta
U_Pseudomonadaceae
Syntrophomonas
U_Ruminococcaceae
Coprococcus
Clostridium
U_Clostridiaceae

(b)

most abundant acetoclastic methanogens identified in
laboratory-scale anaerobic digesters operated at increasing
NH3 -N concentrations of up to 4000 mg L−1 [43].
3.4. Acetogens Played a Key Role in Methane Production at
∼2000 mg NH3 -N L−1 . The most abundant Bacterial phylotypes at the family and genus levels are summarized in
Figure 4. At the genus level, phylotypes representative of producers of key short-chain fatty-acid (e.g., acetate, propionate,
and butyrate) were enriched during exposure to high
NH3 -N. These Bacteria are summarized in panel (b).
The most abundant phylotypes were (i) Coprococcus, a
butyrate- and acetate-producer within the Lachnospiraceae
family [44], (ii) Sphaerochaeta (within the Spirochaetaceae
family), an acetate-, formate-, and ethanol-producer [45],
(iii) Treponema, acetogenic microorganisms within the
Spirochaetaceae family, and (iv) unidentified phylotypes in
the Bacteroidales and Clostridiales orders (represented in
purple and blue, resp.), known to harbor microorganisms
capable of fermenting carbohydrates and proteins to shortchain fatty-acids [46–48].
To elaborate on the acetogens, we used qPCR to quantify the highly conserved formyl tetrahydrofolate synthetase
(FTHFS) gene of homoacetogens [49, 50] and reverseacetogens (sometimes called “syntrophic acetate oxidizers”)
[6, 21]. Figure 5 shows a steady increase of the FTHFS
gene; quantified FTHFS genes increased about two orders
of magnitude from the beginning of the experiment to day
105 despite relative constant numbers of Bacterial 16S rRNA

Gene copies mL−1 reactor

Figure 4: Bacterial distributions. (a) Phylotypes at the family level. (b) Most abundant phylotypes at the genera level. Similar colors (purple,
yellow, black, blue, green, gray, and pink) indicate that the families are in the same order. The total NH3 -N concentration at the time of the
batch sampling was 1140 mg/L. Semicontinuous operation is an average of two samples at ∼2000 mg NH3 -N/L. “U ” stands for unidentified
microorganism within the taxonomic classification.

1.E + 10
1.E + 09
1.E + 08
1.E + 07
1.E + 06
1.E + 05
1.E + 04
1.E + 03
1.E + 02

0

35

70

105

Time (days)
General BAC
FTHFS

Figure 5: Gene copies for General Bacteria (BAC) and the FTHFS
gene (marker of homoacetogens) over the duration of operation of
the semicontinuous reactor.

genes. This indicates that there was not only an increase
in homoacetogenic Bacteria but also a relative increase in
their proportion of the Bacterial community, revealing an
enrichment of homoacetogens with increasing NH3 -N concentrations.
Low SCOD in the effluent, no detection of H2 , and the
high amount of acetoclastic and hydrogenotrophic methanogens mean that acetate and H2 were efficiently scavenged
to produce CH4 . The presence of homoacetogens means
that the sink for acetate during semicontinuous operation
could have been either one of the methanogenic pathways or
both. Homoacetogens could either have been doing forward

Archaea
acetogenesis, in which case acetoclastic methanogens scavenge acetate, or have been doing reverse acetogenesis, in
which hydrogenotrophic methanogens feed on H2 and CO2
generated from acetate.
Syntrophic acetate-utilizers were represented by phylotypes associated with the genus Clostridium within the
Clostridiaceae family. Their relative abundance increased
from 0.72% in the inoculum to 3.5 and 5.6% during batch
and semicontinuous operation, respectively. Some strains
(including C. ultunense [51] and strains similar to C.
botulinum, C. sticklandii, and C. beijerinckii [52]) have been
reported to perform reverse acetogenesis in methanogenic
communities [51, 52]. With high NH3 -N concentration, these
acetate-utilizers have been commonly found in syntrophy
with hydrogenotrophic methanogens such as Methanoculleus
[13, 53], the most abundant methanogen identified in our
reactor. Thus, it is possible that several Clostridium spp. in our
reactor contributed to methane production through reverse
acetogenesis coupled with hydrogenotrophic methanogenesis.
3.5. Syntrophic Fatty-Acid Fermenters Thrived at
∼2000 mg NH3 -N L−1 . In addition to syntrophic acetateutilizers among the Clostridia, propionate- and butyratefermenters that grow in syntrophy with hydrogenotrophic
methanogens also were detected at relative abundances
between 1.2 and 4.5% during semicontinuous operation. The
detected phylotypes at the genus level were Syntrophomonas
and W22 and W5. Syntrophomonas (within the Syntrophomonadaceae) ferment butyrate to acetate and H2 in
syntrophic association with hydrogenotrophic methanogens
and sulfate-reducers [22, 54]. Cloacamonas, a representative
genus of Cloacamonaceae, obtains its energy from the
fermentation of amino acids and can ferment propionate to
acetate, H2 , and CO2 in syntrophy with H2 and acetate
consumers [55, 56]. This syntroph has been named
Candidatus Cloacamonas acidaminovorans, and although it
has not been cultivated, its genome has been reconstructed
by metagenomics [55]. Comparing the sequences (300 bp)
associated with the W22 and W5 genera to available
sequences (NCBI, BLAST) reveals that these genera share
up to 96% similarity with Candidatus C. acidaminovorans.
Therefore, it is possible that Cloacamonaceae and Syntrophomonadaceae contributed to methane production
with 2040 ± 30 mg NH3 -N L−1 by fermenting butyrate
and propionate to H2 , CO2 , and acetate in syntrophy with
hydrogenotrophic and acetoclastic methanogens.
3.6. Correlation Analysis Supports Syntrophies between
Acetoclastic Methanogens with Acetogens and Hydrogenotrophic Methanogens with Syntrophic Fatty-Acid Fermenters.
In order to understand the effect of NH3 -N and total N
concentrations on methane production and the microbial
community structure, we calculated Pearson’s R coefficient
among methane production rates, NH3 -N and total N
concentrations, and fermenters, syntrophs, and hydrogenotrophic and acetoclastic methanogens identified at four
time points during operation of the anaerobic reactor treating
swine waste. The results of the parametric correlation analysis

7
are summarized in Figure 6. Hydrogenotrophic methanogens
(Methanoculleus, Methanogenium, Methanobrevibacter, and
an unidentified genus within Methanomicrobiales) and
acetoclastic Methanosaeta were positively correlated
with total N and NH3 -N concentrations (in some cases,
correlations were significant at the 0.05 level). These positive
correlations suggest that hydrogenotrophic and acetoclastic
methanogens thrived with increasing NH3 -N concentration
up to 2040 ± 30 mg NH3 -N L−1 when the pH was 6.9–7.6
and the HRT was 35 d. Moreover, acetogens/fermenters
Coprococcus, Sphaerochaeta, and an unidentified genus
within Porphyromonadaceae were positively correlated with
NH3 -N and almost all methanogens and, consequently,
correlation with methane production was positive. These
positive correlations underscore the important role of
acetogens for methane production at high NH3 -N.
Unidentified Clostridiaceae, which comprises several
short-chain fatty-acid producers, and Clostridium had positive correlations with acetoclastic Methanosaeta, and this supports acetate generation by acetogens and homoacetogens.
However, unidentified Clostridiaceae also showed a positive
correlation with unidentified hydrogenotrophic Methanomicrobiales and with hydrogenotrophs Methanogenium and
Methanoculleus. This supports that homoacetogens among
the Clostridiaceae were possibly carrying out reverse acetogenesis. Thus, parametric analysis supports an important role
for homoacetogens, but it cannot determine whether they
were performing forward or reverse acetogenesis.

4. Conclusions
Successful operation of an anaerobic reactor treating swine
manure proved that Bacterial and Archaeal communities
could acclimate to a steady increase in total NH3 -N concentration up to 2040 ± 30 mg NH3 -N L−1 . Both communities
became less diverse over time. NH3 -N tolerant phylotypes
that were enriched include (1) acetoclastic methanogens
(Methanosaeta); (2) Clostridia known to do forward and
reverse acetogenesis (Clostridium and Clostridiaceae spp.);
(3) fatty-acid producers (Coprococcus and Sphaerochaeta); (4)
hydrogenotrophic methanogens (Methanoculleus, Methanobrevibacter, and Methanogenium); and (5) syntrophic fattyacid fermenters (Syntrophomonas, Clostridium, Clostridiaceae spp., and possibly Cloacamonaceae species). Our
results suggest that the gradual increase in the NH3 -N
concentration led to a microbial community acclimated to
the high total NH3 concentrations associated with anaerobic
digestion of animal wastes. As summarized in Figure 7, acetoclastic and hydrogenotrophic methanogens could coexist
in the presence of NH3 -N concentrations ∼2000 mg L−1 by
establishing syntrophic relationships with propionate and
butyrate-fermenters, as well as homoacetogens able to carry
out forward and reverse acetogenesis.

Disclosure
Any opinions, findings, and conclusions or recommendations
expressed in this material are those of the authors and do not
necessarily reflect those of the NSF.

8

Archaea

TAN
Total N
Methane
Coprococcus
U_Porphyromonadaceae
Sphaerochaeta
Syntrophomonas
W5_Cloacamonaceae
W22_Cloacamonaceae
Clostridium
U_Clostridiaceae
U_Bacteroidaceae
U_pGrfC26
Methanobrevibacter
U_Methanomicrobiales
Methanoculleus
Methanogenium
Methanosaeta

TAN
1.00
0.76
0.90
0.99
0.59
0.62
−0.69
−0.67
−1.00
0.94
0.73
0.27
−0.36
0.53
0.78
1.00∗
0.72
0.94

Total_N
1.00 Methane
Coprococcus
1.00
0.97
0.67
0.83
1.00 U_Porphyromonadaceae
Sphaerochaeta
1.00
0.97
0.89
0.47
Syntrophomonas
1.00
0.98
0.90
0.51
1.00∗
W5_Cloacamonaceae
−0.06
−0.30
−0.78
0.18
0.14
1.00
−0.99
−0.93
−0.56
−1.00 −1.00 ∗
−0.08
1.00 W22_Cloacamonaceae
−0.81
−0.93
−0.98
−0.64
−0.67
0.64
0.72
1.00 Clostridium
0.48
0.68
0.97
0.26
0.30
−0.90
−0.36
−0.91
1.00 U_Clostridiaceae
0.96
0.64
0.98
0.99
−0.01
−1.00
−0.78
0.45
1.00 U_Bacteroidaceae
1.00 ∗
−0.42
−0.19
0.39
−0.63
−0.60
−0.88
0.54
−0.20
0.59
−0.46
1.00
−0.88
−0.74
−0.24
−0.97
−0.96
−0.42
0.94
0.43
−0.01
−0.90
0.80
−0.15
0.09
0.63
−0.38
−0.34
−0.98
0.28
−0.46
0.79
−0.19
0.96
−0.39
0.98
0.70
0.96
0.97
−0.09
−0.99
−0.83
0.52
1.00 ∗∗
1.00 ∗
0.77
0.90
0.99
0.60
0.63
−0.68
−0.68 −1.00 ∗
0.93
0.74
0.25
0.96
0.63
0.98
0.99
0.00
−1.00
−0.77
0.43
1.00
−0.48
1.00 ∗
−0.09
0.94
0.88
0.83
0.85
−0.39
−0.89
−0.96
0.75
0.93
1.00

Pearson’s R coefficient
−1

0

1

Figure 6: Parametric correlation (Pearson’s R coefficient) of methane production, total NH3 -N (TAN) concentration, and key Bacteria and
Archaea identified during operation of the anaerobic reactor treating swine waste. Phylotypes in purple, orange, blue, and red represent
fermenters, syntrophs, and hydrogenotrophic and acetoclastic methanogens, respectively. Phylotypes that are most similar to fermenters and
syntrophs are indicated in a combination of purple and orange colors. Note. ∗ Correlation is significant at the 0.05 level (2-tailed). ∗∗ Correlation
is significant at the 0.01 level (2-tailed). The statistical analysis was not corrected for multiple comparisons.

Complex organic
substrate

Acetogens

Fermenters
Short-chain
fatty acids
Syntrophic fatty-acid
fermenters

Fermenters
Syntrophic
fatty-acid
fermenters

H2 + CO2

Acetate

Homoacetogens

Acetoclastic
methanogens

Fermenters
H2 + CO2

Hydrogenotrophic
methanogens

Methane
Hydrogenotrophic
methanogens

Figure 7: Proposed key anaerobic food-web reactions occurring at ammonia-N concentrations of ∼2000 NH3 -N mg L−1 .

Competing Interests

Acknowledgments

The authors declare that there is no conflict of interests
regarding the publication of this paper.

This work was supported in part by the National Science
Foundation CAREER Program [Grant no. 1053939].

Authors’ Contributions

References

Sofia Esquivel-Elizondo and Prathap Parameswaran contributed equally to this work.

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