ORIGINAL RESEARCH
published: 10 December 2018
doi: 10.3389/fmicb.2018.03039
Metabolism and Occurrence of
Methanogenic and Sulfate-Reducing
Syntrophic Acetate Oxidizing
Communities in Haloalkaline
Environments
Peer H. A. Timmers 1,2* , Charlotte D. Vavourakis 3 , Robbert Kleerebezem 4 ,
Jaap S. Sinninghe Damsté 5,6 , Gerard Muyzer 3 , Alfons J. M. Stams 1,7 , Dimity Y. Sorokin 4,8
and Caroline M. Plugge 1,2
1
Edited by:
Gloria Paz Levicán,
Universidad de Santiago de Chile,
Chile
Reviewed by:
Ronald Oremland,
United States Geological Survey,
United States
Stefano Campanaro,
Università degli Studi di Padova, Italy
*Correspondence:
Peer H. A. Timmers
peer.timmers@kwrwater.nl
Specialty section:
This article was submitted to
Extreme Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 20 September 2018
Accepted: 26 November 2018
Published: 10 December 2018
Citation:
Timmers PHA, Vavourakis CD,
Kleerebezem R, Damsté JSS,
Muyzer G, Stams AJM, Sorokin DY
and Plugge CM (2018) Metabolism
and Occurrence of Methanogenic
and Sulfate-Reducing Syntrophic
Acetate Oxidizing Communities
in Haloalkaline Environments.
Front. Microbiol. 9:3039.
doi: 10.3389/fmicb.2018.03039
Laboratory of Microbiology, Wageningen University & Research, Wageningen, Netherlands, 2 European Centre
of Excellence for Sustainable Water Technology, Wetsus, Leeuwarden, Netherlands, 3 Microbial Systems Ecology,
Department of Freshwater and Marine Ecology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam,
Amsterdam, Netherlands, 4 Department of Biotechnology, Delft University of Technology, Delft, Netherlands, 5 Department
of Marine Microbiology and Biogeochemistry, NIOZ Netherlands Institute for Sea Research, Utrecht University, Utrecht,
Netherlands, 6 Department of Earth Sciences, Faculty of Geosciences, Utrecht University, Utrecht, Netherlands, 7 Centre
of Biological Engineering, University of Minho, Braga, Portugal, 8 Winogradsky Institute of Microbiology, Research Centre
of Biotechnology, Russian Academy of Sciences, Moscow, Russia
Anaerobic syntrophic acetate oxidation (SAO) is a thermodynamically unfavorable
process involving a syntrophic acetate oxidizing bacterium (SAOB) that forms
interspecies electron carriers (IECs). These IECs are consumed by syntrophic partners,
typically hydrogenotrophic methanogenic archaea or sulfate reducing bacteria. In this
work, the metabolism and occurrence of SAOB at extremely haloalkaline conditions
were investigated, using highly enriched methanogenic (M-SAO) and sulfate-reducing
(S-SAO) cultures from south-western Siberian hypersaline soda lakes. Activity tests
with the M-SAO and S-SAO cultures and thermodynamic calculations indicated that
H2 and formate are important IECs in both SAO cultures. Metagenomic analysis of the
M-SAO cultures showed that the dominant SAOB was ‘Candidatus Syntrophonatronum
acetioxidans,’ and a near-complete draft genome of this SAOB was reconstructed.
‘Ca. S. acetioxidans’ has all genes necessary for operating the Wood–Ljungdahl
pathway, which is likely employed for acetate oxidation. It also encodes several
genes essential to thrive at haloalkaline conditions; including a Na+ -dependent
ATP synthase and marker genes for ‘salt-out‘ strategies for osmotic homeostasis
at high soda conditions. Membrane lipid analysis of the M-SAO culture showed
the presence of unusual bacterial diether membrane lipids which are presumably
beneficial at extreme haloalkaline conditions. To determine the importance of SAO
in haloalkaline environments, previously obtained 16S rRNA gene sequencing data
and metagenomic data of five different hypersaline soda lake sediment samples
were investigated, including the soda lakes where the enrichment cultures originated
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Haloalkaliphilic Syntrophic Actetate Oxidation
from. The draft genome of ‘Ca. S. acetioxidans’ showed highest identity with two
metagenome-assembled genomes (MAGs) of putative SAOBs that belonged to the
highly abundant and diverse Syntrophomonadaceae family present in the soda lake
sediments. The 16S rRNA gene amplicon datasets of the soda lake sediments showed
a high similarity of reads to ‘Ca. S. acetioxidans’ with abundance as high as 1.3%
of all reads, whereas aceticlastic methanogens and acetate oxidizing sulfate-reducers
were not abundant (≤0.1%) or could not be detected. These combined results indicate
that SAO is the primary anaerobic acetate oxidizing pathway at extreme haloalkaline
conditions performed by haloalkaliphilic syntrophic consortia.
Keywords: syntrophic acetate oxidation, haloalkaliphiles, soda lakes, syntrophy, SAOB, syntrophic acetate
oxidizing bacteria, genome-centric metagenomics
INTRODUCTION
hypersaline soda lakes and the dominant SAOB, ‘Candidatus
Syntrophonatronum acetioxidans,’ represents a novel genus
within the family Syntrophomonadaceae (Sorokin et al.,
2014a, 2015a, 2016). The SAO pathways used by these first
haloalkaliphilic enrichment cultures, the mechanisms of energy
conservation, and the IEC that is transferred between the
partner organisms are, however, unknown. In this study, we
investigated the occurrence and metabolism of SAO using
these enrichment cultures of ‘Ca. S. acetioxidans’ with either
the hydrogenotrophic methanogenic partner Methanocalculus
natronophilus strain AMF5 (M-SAO) (Sorokin et al., 2015a, 2016)
or the sulfate-reducing partner Desulfonatronovibrio magnus
(S-SAO) (Sorokin et al., 2011). Acetate oxidation, IEC formation,
and rates of methane or sulfide formation, respectively,
were monitored in the presence and absence of inhibitors of
methanogenesis or sulfate-reduction and in presence of possible
IECs (i.e., formate or H2 ). A draft genome of the SAOB ‘Ca. S.
acetioxidans’ was obtained from the metagenome of the M-SAO
enrichment culture to identify the genes putatively involved in
acetate-oxidizing pathways and the adaptation mechanisms to
haloalkaliphilic conditions. The composition of membrane lipids
was investigated for possible adaptations to these conditions. The
occurrence and ecology of SAOBs and aceticlastic methanogens
and acetate-degrading sulfate reducers was investigated using
16S rRNA gene amplicon and metagenome sequencing datasets
from five different soda lake sediment samples of south-western
Siberia that were published recently (Vavourakis et al., 2018). The
sediment of one of these lakes, Bitter-1, was also the inoculum
of the studied SAO enrichment cultures. These results provide
more insights into the importance of SAO in these extreme
environments.
Syntrophic acetate oxidation (SAO) is an anaerobic process
where two microorganisms are responsible for the degradation
of acetate. In this process, syntrophic acetate oxidizing bacteria
(SAOB) oxidize acetate and produce H2 and CO2 or formate.
Hydrogen and formate can serve as interspecies electron
carriers (IECs) that are utilized by syntrophic partners, which
in most cases are hydrogenotrophic methanogens or sulfatereducing bacteria (SRB). Only a few bacterial species able
to perform SAO have been described, such as “strain AOR”
(Lee and Zinder, 1988), Clostridium ultunense (Schnürer
et al., 1996), Thermoacetogenium phaeum (Hattori et al.,
2005), Tepidanaerobacter acetatoxydans (Westerholm et al.,
2011), Pseudothermotoga lettingae (Balk et al., 2002) and
Syntrophaceticus schinkii (Westerholm et al., 2010). Acetate is an
important intermediate in the anaerobic degradation of organic
matter and up to 80% of produced methane can derive from
acetate (Mountfort and Asher, 1978; Lovley and Klug, 1982). In
highly reduced environments, acetate is degraded by aceticlastic
methanogens or by sulfate-reducing bacteria (SRB). However,
aceticlastic methanogenesis is inhibited at extreme conditions,
such as high ammonia, high fatty acid concentrations and high
temperatures and as a result, SAO becomes the dominant acetate
utilizing process (Shigematsu et al., 2004; Karakashev et al.,
2006; Nozhevnikova et al., 2007; Noll et al., 2010; Westerholm
et al., 2012). Methanogenic digesters of protein rich organic
matter exhibit high concentrations of ammonium, which strongly
inhibits aceticlastic methanogenesis (Sprott and Patel, 1986;
Steinhaus et al., 2007; Manzoor et al., 2016) and results in SAO
to become the dominant acetate degradation pathway (Schnürer
et al., 1994, 1999; Schnürer and Nordberg, 2008; Westerholm
et al., 2012; Jiang et al., 2018). SAO has also been reported to
be an important anaerobic process at high temperature (55◦ C)
oil fields (Mayumi et al., 2011, 2013; Dolfing, 2014) and acetate
utilization shifted from aceticlastic methanogenesis to SAO at
higher temperatures in rice paddy field soils (Rui et al., 2011).
Extreme conditions also exist in hypersaline soda lakes,
which are a specific type of salt lakes characterized by double
extremes; high pH (9.5–11) and sodium carbonate/bicarbonate
concentrations up to saturation (0.5–4 M Na+ ) (Sorokin et al.,
2014b). Recently, SAO communities have been enriched from
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MATERIALS AND METHODS
Inoculum
The enrichment cultures used were described previously
and consist of ‘Ca. S. acetioxidans’ (Sorokin et al., 2014a)
together with the haloalkaliphilic hydrogenotrophic methanogen
Methanocalculus natronophilus strain AMF5 (M-SAO) (Sorokin
et al., 2015a, 2016) or the hydrogenotrophic sulfate-reducer
Desulfonatronovibrio magnus (S-SAO) (Sorokin et al., 2011).
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Haloalkaliphilic Syntrophic Actetate Oxidation
All cultures derived from Bitter-1 soda lake sediment (southwestern Siberia).
by measuring optical density at 600 nm and H2 , formate and
methane or sulfide formation over time.
Media and Cultivation
Analytical Measurements
The sodium carbonate-bicarbonate alkaline media (1 M Na+
and pH 9.5) used in these experiments was made as described
previously (Sorokin et al., 2014a). SAO enrichment cultures and
pure cultures of hydrogenotrophic partners were pre-grown in
media with either 60 mM acetate or 80 mM formate and 2 mM
acetate (all sodium salts), respectively. For methanogenic SAO
and pure cultures, 0.1 mM coenzyme M was supplemented. For
sulfate-reducing SAO or pure cultures, 20 mM sodium sulfate was
added as electron acceptor. All incubations were done at 30◦ C, as
described previously (Sorokin et al., 2014a).
When pre-grown methanogenic SAO cultures had consumed
around 30 mM acetate, the gas phase of the cultures was
exchanged with 100% (v/v) N2 . Afterwards, 30 ml of pre-grown
cultures was added to autoclaved, crimp-capped, 100% (v/v) N2 containing 50 ml serum vials using a nitrogen-flushed syringe.
Incubations were done in triplicate with either (1) 45 mM
acetate – static incubation, (2) 45 mM acetate – shaking at
130 rpm, (3) 45 mM acetate with 100% (v/v) H2 , (4) 45 mM
acetate and 7 mM or 80 mM formate, (5) 45 mM acetate and
10 mM bromoethanesulfonate (BES).
When pre-grown sulfate-reducing SAO cultures had
consumed around 20 mM acetate, the cells were collected by
centrifugation (1 h at 4000 × g) and resuspended in fresh media
followed by gas exchanging with 100% (v/v) N2 to remove
produced sulfide. Afterwards, 30 ml of pre-grown culture was
added to autoclaved, crimp-capped, 100% (v/v) N2 -containing
50 ml serum vials using a nitrogen-flushed syringe. Incubations
were done in triplicate with either (1) acetate and sulfate –
static incubation, (2) acetate and sulfate – shaking, (3) acetate
and sulfate with 100% (v/v) H2 , (4) acetate, sulfate and 80 mM
formate, and (5) acetate and 5 mM molybdate (MoO2−
4 ). Acetate
was added after 65 h of incubation to a total concentration of
around 25 mM (the concentration of acetate was 5 mM before
additional acetate supplementation). Acetate was amended
later after it was confirmed that the cultures were still active
after washing. There was less acetate amended than to the
methanogenic cultures to prevent overproduction of sulfide that
is both inhibitory for the cultures and more difficult to measure
at high concentrations.
All cultures were incubated statically at 30◦ C, except for
condition 2 that was shaken at 130 rpm. For all cultures, H2 ,
organic acids (mainly formate and acetate), sulfate and sulfide
were monitored during incubation. Methanogenic cultures were
incubated for a total of 286 h while sulfate-reducing cultures were
incubated for 584 h.
Additionally, the production of formate from H2 and vice
versa during growth of the syntrophic partners in pure culture
was investigated. For this work, pure cultures of Methanocalculus
natronophilus strain AMF5 and Desulfonatronovibrio magnus
were pre-grown with 100% (v/v) H2 . After full growth, cultures
were gas exchanged with 100% (v/v) N2 and 10% (v/v) of the
cultures was transferred to new media with either 100% (v/v) H2
or 100 mM formate. Growth of these pure cultures was monitored
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Headspace Gasses
Headspace gas samples (0.2 ml) from the incubations were taken
at 20◦ C using a sterile N2 -flushed syringe and analyzed using a
Compact GC (Global Analyser Solutions, Breda, Netherlands)
equipped with a Carboxen 1010 pre-column, followed by two
lines: a Molsieve 5A column (pressure: 200 kPa, split flow:
20 ml min−1 , oven temperature: 80◦ C) and a RT-Q-bond column
(pressure: 150 kPa, split flow: 10 ml min−1 ) with a PDD detector
at 110◦ C. For samples with H2 and methane concentrations
above 1%, measurements were done on another Compact GC
12 4.0 (Global Analyser Solutions, Breda, Netherlands) with a
Molsieve 5A column (operated at 100◦ C) coupled to a Carboxen
1010 pre-column and a Rt-Q-BOND column (operated at 80◦ C)
with a thermal conductivity detector. Quantification of CH4 and
H2 was done using standards with known concentration.
Organic Acids
Liquid samples were taken using a sterile N2 flushed syringe
and needle and were centrifuged for 10 min at 14000 g at
4◦ C and stored at −20◦ C until further processing. Samples
were again centrifuged for 10 min at 14000 g at 4◦ C and
organic acids in the supernatant were analyzed using a Dionex
Ultimate 3000RS (Thermo Fisher Scientific, Sunnyvale, CA,
United States) equipped with a Phenomenex Rezex Organic Acid
H+ column (300 mm × 7.8 mm) (Phenomenex, Torrance, CA,
United States). The system was operated at a column temperature
of 80◦ C and a flowrate of 0.5 mL min−1 . Eluent consisted of
2.5 mM sulfuric acid. Detection was done using a UV detector
at 210 nm.
Sulfide
For sulfide analysis, liquid samples were directly fixed 1:1 in zinc
acetate (5% w/v). Samples were vortexed thoroughly and further
diluted when necessary in MQ water. Sulfide measurements were
done as described previously (Timmers et al., 2015).
Thermodynamic Analysis
The Gibbs free energy changes of the different redox reactions
that sustain microbial growth in the enrichment culture
incubations were calculated. Gibbs free energy changes under
standard conditions were calculated according to the tabulated
Gibbs free energy of formation values (Thauer et al., 1977).
Actual Gibbs free energy changes were calculated from the in situ
concentrations of the reactants in the different incubations.
To account for the elevated sodium bicarbonate/carbonate
concentration in the media, activity electrolyte correction
factors were estimated according to Buffle, 1988 (estimated
f -values are 0.5 and 0.04 for monovalent and bivalent ions,
respectively). Equilibrium was assumed in the gaseous carbon
dioxide, bicarbonate, carbonate system, as well as the gas-liquid
partitioning of carbon dioxide, methane, and molecular H2 .
Initially, thermodynamic equilibrium (1G = 0) was furthermore
assumed in the formate to bicarbonate/H2 conversion in order
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Haloalkaliphilic Syntrophic Actetate Oxidation
Prodigal (Hyatt et al., 2010), tRNA prediction with tRNAscanSE (Lowe and Eddy, 1997), rRNA prediction using rna_hmm3.
Protein homology searches were performed against the COG
(Tatusov et al., 2000) and TIGRFAMs (Haft et al., 2001) databases
with predictions of hmmer3 (Eddy, 2011). Best-hits of the CDS
and rRNA genes were obtained against the NCBI non-redundant
BLAST database (USEARCH, e-value = 0.00001) and SILVA
(MEGABLAST, e-value = 0.00001), respectively.
Archaeal and bacterial contigs were separated based on
the taxonomic annotation of the encoded CDS. Contigs that
contained genes from both kingdoms were manually inspected
and disregarded when they contained less than three genes, were
considerably mixed in phylogeny or were from phage/viral origin.
The contigs were further binned with MaxBin (version 2.2.1: (Wu
et al., 2014)) using 40 and 107 universal marker genes for Archaea
and Bacteria, respectively. MaxBin can infer contig abundance
after mapping reads to the contig with Bowtie2 (version 2.2.3)
(Langmead and Salzberg, 2012).
The resulting bins were evaluated using CheckM (Parks et al.,
2015), abundance information and inspected manually based
on the annotated CDS. Three Euryarchaeota-, three Firmicutesand two Deltaproteobacteria-related bins of reasonable quality
(completeness 83–98%, contamination 2–23%) were obtained
with estimated coverage >10×. Several very low-abundance bins
of mixed phylogeny were also obtained, but excluded from
further analysis.
The eight bins of interest were further optimized in iterative
steps until CheckM results stagnated after the third reassembly:
we checked manually the bins for contamination and spurious
contigs, re-assembled with MEGAHIT (–kmin 21, –k-max 121,
–k-step 10, –min-contig-len 1000) the subsets of mapped reads
to the bins from the previous round and their respective mates,
we annotated the contigs and binned archaeal and bacterial
contigs separately with MaxBin. After the three iterative binning
rounds, 16S rRNA genes were blasted against NCBI-nr (blastn,
e-value ≤ 0.00001) and manually placed in the correct bins
guided by taxonomic gene contig annotations. VizBin [default
settings; (Laczny et al., 2015)] was used for further inspection
and refinement of the selected bins based on PCA plots
(Supplementary Figure S1). Finally, taxonomic assignments
of the three Firmicutes-related MAGs were double-checked
with maximum-likelihood phylogenetic trees constructed with
16S ribosomal proteins as described previously (Hug et al.,
2016; Vavourakis et al., 2018), including all 36 available
closely related NCBI reference (draft) genomes (Supplementary
Figures S2, S3).
Characteristics and taxonomic assignments of the final eight
metagenome-assembled genomes (MAGs) are summarized in
Supplementary Tables S1, S2. The dominant populations in the
enrichment culture belonged to the SAOB (MSAO_Bac1 with
100% 16S rRNA gene identity to ‘Ca. S. acetioxidans’ clone
AAS1) and its syntrophic methanogenic partner (MSAO_Arc1
with 100% 16S rRNA gene identity to Methanocalculus sp. strain
AMF5).
The draft genome of ‘Ca. S. acetioxidans,’ MSAO_Bac1
(Table 1), was re-annotated against KEGG [Automatic
Annotation Server (KAAS)] (Moriya et al., 2007) and RAST
to estimate the actual formate concentrations in the system.
Henry coefficients that are not corrected for the elevated salt
concentration, give unrealistic values for the Gibbs energy
change for both H2 producing and consuming reactions. To
account for the decrease in solubility of H2 at 1 M sodium
bicarbonate/carbonate, an activity correction for gaseous H2 was
introduced and roughly estimated to amount to 0.2, according to
Engel et al. (1996).
DNA Isolation and Sequencing of
Methanogenic SAO Enrichment Cultures
Methanogenic SAO cultures were used for metagenome
sequencing. Cultures were pre-grown on 60 mM acetate.
When 30 mM acetate was utilized, cells were centrifuged (1 h,
4,700 g) and washed three times in 1x PBS with 1 M NaCl
(to remove Na+ -carbonates). Cells were resuspended in 1x
TE buffer and 0.4 mg L−1 of polyadenylic acid was added
to coat surfaces for prevention of sorption of nucleic acids.
Then, lysozyme solution (Masterpure Gram-positive DNA
purification kit, Epicentre, Madison, WI, United States) was
added and incubated for 30 min at 37◦ C. Next, proteinase K in
Gram-positive lysis solution (Masterpure Gram-positive DNA
purification kit) was added and samples were incubated for
15 min at 67◦ C and vortexed every 5 min. Samples were cooled
down to 37◦ C and placed on ice for 5 min. Then, 1 volume of
phenol:chloroform:isoamyl alcohol (24:24:1, v/v/v) was added to
the samples and mixed by inversion. Samples were centrifuged
at 12000 × g for 2 min at room temperature. The aqueous
upper layer was transferred to a new tube and nucleic acids were
precipitated by adding 1 volume of isopropanol and 0.1 volume
of 3 M sodium acetate (pH 5.2). Samples were kept on ice for
20 min and centrifuged at 12,000 × g for 20 min at 4◦ C. The
supernatant was removed and the pellet was rinsed with 70%
cold ethanol. Tubes were inverted and air dried. Afterwards,
1x TE buffer with RNAse (Masterpure Gram-positive DNA
purification kit) was added to the samples and incubated at 37◦ C
for 30 min and nucleic acids were again precipitated by adding
1 volume of isopropanol and 0.1 volume of 3 M sodium acetate
(pH 5.2). Samples were kept on ice for 20 min and centrifuged
at 12,000 g for 20 min at 4◦ C. The supernatant was removed
and the pellet was rinsed with 70% cold ethanol. Tubes were
inverted and air dried. The pellet was dissolved in 20 µl DNAse
free water. The DNA quality and quantity were checked using
the Nanodrop 2000 and the Qubit fluorometer (Thermo Fisher
Scientific, Waltham, MA, United States). Samples were sent
for sequencing using the Illumina HiSeq 4000 platform (GATC
Biotech, Konstanz, Germany).
Metagenome Analysis of Methanogenic
SAO Enrichment Cultures
Raw sequence reads were quality and length (minimum 21 b)
trimmed using the software program Sickle (version 1.33) (Joshi
and Fass, 2011). Trimmed reads were assembled into contigs
>1 kb with MEGAHIT (Li et al., 2015), using the default sensitive
mode for generic metagenomes. ORF calling was done with
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CheckM results to select only one representative MAG for each
species and re-calculated the phylogeny of the 16 ribosomal
proteins. The final phylogenetic tree, results of the recruitment
experiment and genome annotation [Ghost Koala; (Kanehisa
et al., 2016)] summaries were visualized using iTOL v4 (Letunic
and Bork, 2007).
TABLE 1 | Details on the draft genome of ‘Ca. Syntrophonatronum acetioxidans
MSAO_Bac1’ (accession number pending) reconstructed from the methanogenic
SAO enrichment culture.
Recovered genome size
1.97 Mb
Estimated sequence coverage
665×
G+C content
44.3%
Anvi’o/CheckM completeness
96/90%
Number of tRNA sequences
39
Number of 5S rRNA genes
1
Number of 16S rRNA genes
1
Number of 23S rRNA genes
1
Number of contigs
300
Number of coding sequences
1,876
Coding density
91.7%
Anvi’o redundancy/CheckM contamination
8/12%
CheckM strain heterogeneity
41%
Besthit NCBI-nr 16S rRNA gene
“Ca. Syntrophonatronum
acetioxidans clone AAS1,” partial
sequence
Identity
100%
e-value
0
Besthit NCBI-nr gene contigs
Dethiobacter alkaliphilus
Of total hits
25%
Lipid Analysis of Methanogenic SAO
Enrichment Cultures
Lipids were extracted with a Bligh Dyer method and the extract
was acid hydrolyzed. The resulting core lipids were methylated
and silylated and analyzed by gas chromatography and gas
chromatography-mass spectrometry. The Bligh Dyer extract
was also analyzed directly for intact polar lipids using liquid
chromatography-mass spectrometry. All methods have been
previously described in detail (Sinninghe Damsté et al., 2011).
RESULTS AND DISCUSSION
Activity of Methanogenic and
Sulfate-Reducing SAO Enrichment
Cultures
The methanogenic SAO (M-SAO) and sulfate reducing SAO
(S-SAO) enrichment cultures degraded acetate with coinciding
H2 production and subsequent methane or sulfide production,
respectively. Hydrogen accumulated up to 10.7 (±0.6) and 12.3
(±8.6) Pa in static and shaken M-SAO cultures (Figure 1A and
Supplementary Figure S4A, respectively) and to 4.3 (±0.7) and
5.1 (±1.0) Pa in static and shaken S-SAO cultures (Figure 2A
and Supplementary Figure S4B, respectively). The lower H2
concentration in the S-SAO cultures suggests that SRB are
capable of achieving a lower H2 partial pressure as compared to
methanogens. SAO was apparently energetically feasible at H2
concentrations higher than the values calculated for neutrophilic
SAOBs (Dolfing, 2014). The actual Gibbs free energy change of
acetate oxidation is energetically unfavorable if one considers the
H2 solubility in pure water (Table 2, 1G1 ). However, when a
H2 solubility of 20% of the solubility in pure water is assumed,
due to the in situ sodium bicarbonate/carbonate concentrations,
acetate oxidation becomes favorable (Table 2, 1G1∗ ). Using
this assumption, the Gibbs free energy changes of conversion
for both partner organisms are similarly negative at the rather
stable H2 partial pressures in these cultures, indicating that the
syntrophic partners equally share the energy gained from the
total reaction (Table 2). Formate was never detected (Figures 1A,
2A and Supplementary Figures S4A,B). Assuming that formate
conversion to H2 /CO2 is at thermodynamic equilibrium, the
calculated formate concentrations from the maximum H2
concentrations in these incubations are in the range of 10 µM,
which was always below the detection limit of 50 µM. Therefore,
a role of formate cannot be excluded.
There was no significant difference in acetate consumption
and H2 , methane or sulfide formation when cultures were shaken
or statically incubated (Figures 1A, 2A, and Supplementary
Figures S4A,B). This indicates that IEC transfer is not influenced
(Rapid Annotation using Subsystems Technology) (Aziz et al.,
2008). Manual blast runs of translated gene sequences were
done using the NCBI’s BLASTP service. Gene domain analysis
was done using NCBI’s CDD (Marchler-Bauer et al., 2009) and
EMBL’s Interpro (Jones et al., 2014) services. The location of
proteins (extra-, intracellular or transmembrane) were checked
using PROTTER (Omasits et al., 2014), the TMHMM server
v. 2.0 (Sonnhammer et al., 1998) and the SignalP 4.1 Server
(Petersen et al., 2011). Genome completeness and redundancy
were estimated also based on universal copy genes (Rinke et al.,
2013) using the Anvi’o platform (v4; Eren et al., 2015).
For comparative SAO analysis, all genomes of SAOBs so
far described and some acetogens were compared to MSAO
Bac1, including Acetobacterium woodii DSM 1030 (NC_016894)
(Poehlein et al., 2012), Clostridium ultunense Esp (HG764817)
(Manzoor et al., 2013b), Tepidanaerobacter acetatoxydans Re1
(NC_019954) (Manzoor et al., 2013a), Syntrophaceticus schinkii
strain Sp3 (NZ_CDRZ01000250) (Manzoor et al., 2015, 2016),
Pseudothermotoga lettingae (NC_009828) (Zhaxybayeva et al.,
2009), and Thermacetogenium phaeum DSM 12270 strain PB
(bioproject PRJNA168373) (Oehler et al., 2012).
To estimate the relative abundance of MSAO_Bac1 in native
soda lake sediments, a recruitment experiment was performed
using 10 million reads subsamples from previously described
metagenomes obtained from soda lake sediments in Kulunda
Steppe in the summer of 2010 and 2011 (Vavourakis et al., 2018).
Maximum likelihood phylogeny was calculated based on 16
ribosomal proteins predicted in selected Syntrophomonadaceae
reference genomes available at the time of analysis (NCBI) and
in MSAO_Bac1 as described previously (Vavourakis et al., 2018).
Species delineation was determined with Average Nucleotide
Identities [ANI, (Goris et al., 2007)]. We further used ANI and
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FIGURE 1 | Activity test results with pre-grown syntrophic acetate oxidizing cultures with a methanogenic partner (M-SAO) showing acetate (blue line), H2 (black
line), formate (gray line) and methane (red line) evolution in cultures supplemented with (A) acetate, (B) acetate and formate – 7 mM, (C) acetate and BES,
(D) acetate and formate – 80 mM, (E) acetate and 100% H2 . Standard deviations represent biological triplicate incubations.
equilibrium is 35 µM when BES was added and 26 µM when
molybdate was added, which are below the detection limit of
formate.
When formate was supplied together with acetate, formate was
consumed in both M-SAO and S-SAO cultures with concomitant
increase in H2 and subsequent methane or sulfide production,
respectively (Figures 1C, 2C). This indicates that formate was
first converted to H2 before being consumed by the syntrophic
partner. In the M-SAO cultures when only 7 mM formate was
supplied, formate was consumed rapidly which resulted in a H2
peak of 40.6 (±10.7) Pa and stoichiometric methane formation
of 1.8 (±0.2) mmol per liter media (Figure 1C). When 80 mM
formate was supplied to both cultures, formate was consumed
and no acetate consumption occurred if formate was present
(Figures 1D, 2C). Only when all formate was consumed after
by complete mixing. Microscopic investigations showed no
physical association of partner organisms (Sorokin et al., 2014a,
2016). Aggregation or close association is not essential when H2
and/or formate act as IEC (Stams and Plugge, 2009).
When the methanogenic inhibitor 2-bromoethanesulfonate
(BES) was supplied to M-SAO cultures, only 1.6 mM (±1.7) of
acetate was consumed and H2 accumulated up to 47.9 (±3.3)
Pa (Figure 1B). In the S-SAO cultures, the sulfate-reducing
inhibitor molybdate (MoO2−
4 ) inhibited acetate oxidation and
sulfide production and H2 accumulated until between 12.1 (±3.6)
and 15.3 (±0.4) Pa (Figure 2B). Acetate oxidation at these
H2 concentrations was indeed energetically less favorable for
S-SAO and even unfavorable for M-SAO cultures (Table 2).
The calculated concentration of H2 -derived formate from
the maximum measured H2 concentration at thermodynamic
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FIGURE 2 | Activity test results with pre-grown syntrophic acetate oxidizing cultures with a sulfate-reducing partner (S-SAO) showing acetate (blue line), H2 (black
line), formate (gray line) and sulfide (red line) evolution in cultures supplemented with (A) acetate, (B) acetate and molybdate, (C) acetate and formate – 80 mM, (D)
acetate and 100% H2 showing acetate, H2 and sulfide and (E) acetate and 100% H2 showing only acetate and formate evolution. Standard deviations represent
biological triplicate incubations.
TABLE 2 | Thermodynamic calculations according to the measured parameters in the incubations that performed syntrophic acetate oxidation coupled to
methanogenesis (M-SAO) or sulfate reduction (S-SAO) with and without inhibitors bromoethanesulfonate (BES) or Molybdate (MoO2−
4 ), respectively.
M-SAO cultures
Reaction
Acetate oxidation
+
CH3 COO− + 4 H2 O → 2 HCO−
3 + 4 H2 + H [H2 ] = 11.5 Pa
Hydrogenotrophic methanogenesis
+
4 H2 + HCO−
3 + H → CH4 + 3 H2 O [H2 ] = 11.5 Pa
Total
CH3 COO− + H2 O → HCO−
3 + CH4 [H2 ] = 11.5 Pa
Acetate oxidation + BES
+
CH3 COO− + 4 H2 O → 2 HCO−
3 + 4 H2 + H [H2 ] = 47.9 Pa
1G1 (kJ mol−1 )
1G1∗ (kJ mol−1 )
+2.2
−13.7
−31.2
−15.3
−29
−29
+16.3
+0.4
S-SAO cultures
Acetate oxidation
+
CH3 COO− + 4 H2 O → 2 HCO−
3 + 4 H2 + H [H2 ] = 4.7 Pa
−6.2
−22.3
Hydrogenotrophic sulfate-reduction
+
−
4 H2 + SO2−
4 + H → HS + 4 H2 O [H2 ] = 4.7 Pa
−33.3
−17.3
Total
−
−
CH3 COO− + SO2−
4 → 2 HCO3 + HS [H2 ] = 4.7 Pa
−39.5
−39.6
Acetate oxidation + MoO2−
4
+
CH3 COO− + 4 H2 O → 2 HCO−
3 + 4 H2 + H [H2 ] = 13.7 Pa
+4.4
−11.6
Gibbs free energy calculations were conducted assuming a Henry coefficient of 1.7 10−4 mol/L/Atm as defined for pure water (1G1 ) and a five time lower value to account
for the decreased solubility in a 1 M sodium bicarbonate/carbonate medium (1G1∗ ).
1G1 is calculated using media conditions, assuming bicarbonate/carbonate/and gaseous carbon dioxide in equilibrium, and formate in equilibrium with bicarbonate and
gaseous H2 . The resulting average concentrations are with activity correction according to Buffle (1988) (with small variations between experiments): acetate 60 mM, pH
2−
1∗
9.5, PCO2 0.01 bar, HCO−
3 0.65 M, CO3 0.17 M, formate 0.1 mM. Average H2 concentrations were estimated from the stable concentrations in the incubations. 1G
is calculated from 1G1 with an activity correction of 0.2 for gaseous H2 solubility, according to Engel et al. (1996).
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115.5 h, acetate oxidation started and 12.7 mM (±1.4) acetate
was consumed after 522 h (Figure 1D, methane and H2 data not
shown). In S-SAO cultures, formate consumption resulted in an
increase in H2 to a total of 1.3 (±0.1) kPa and sulfide to a total of
23.3 mM (±0.9) (Figure 2C). After 300 h, residual formate was
slowly consumed and no acetate consumption was noted during
the whole incubation time, probably because not all formate was
consumed, as opposed to the M-SAO cultures.
When 100% H2 was added together with acetate, H2 was
consumed to produce methane or sulfide in M-SAO or S-SAO
cultures, respectively. Formate was produced in both cultures and
its presence inhibited acetate consumption (Figures 1E, 2D,E).
The M-SAO cultures only started to consume acetate after 146 h
of incubation in parallel with H2 consumption. This inhibition
of acetate consumption corresponded to the presence of formate
during the first 146 h of incubation. When formate levels
decreased, acetate was consumed again (Figure 1E). In S-SAO
cultures, H2 consumption corresponded to sulfide production
(Figure 2D). Acetate consumption was also inhibited when
formate was produced after 297 h (Figure 2E). Mass transfer
limitation of externally supplied gaseous H2 in these static
incubations probably explains the different inhibitory effects of
H2 and formate.
Hydrogen-formate interconversion occurred in both M-SAO
and S-SAO cultures. Pure cultures of the methanogenic
partner M. natronophilus and of the sulfate reducing partner
D. magnus growing on H2 also produced formate and
vice versa (Supplementary Figure S5). This indicates that
M. natronophilus and D. magnus might be responsible for H2 formate interconversion in the SAO cultures. Interestingly, SAO
rates were four times higher with M. natronophilus as partner
than with D. magnus as partner (acetate consumption of 0.04 mM
h−1 vs. 0.01 mM h−1 , respectively) although the Gibbs free
energy change for S-SAO was higher (Table 2). Pure cultures
of M. natronophilus showed much faster methane production
and growth on H2 than on formate, whereas D. magnus showed
the opposite for sulfide production and growth (Supplementary
Figure S6). The assumption that H2 is probably the main IEC in
SAO would explain the faster SAO rates with a M. natronophilus
as partner. Overall, the data provides no answer to the question if
formate and/or H2 is the actual IEC, but do suggest that both are
largely equivalent and interchangeable.
Hydrogenases and Formate Dehydrogenases
The partial genome of ‘Ca. S. acetioxidans’ encodes two [NiFe]
hydrogenases. One is a homolog to the Escherichia coli Hya
hydrogenase which consists of a large and small cytochrome c3containing subunit and a cytochrome b subunit. Only the small
cytochrome c3 subunit has a twin-arginine translocation (TAT)
signal and is anchored in the membrane. The cytochrome b was
predicted to be transmembrane (k121-2698, [NiFe] hydrogenase
1). The TAT system can transport the complete hydrogenase
dimer across the membrane when the TAT signal peptide is
located only on one of the subunits via a so-called hitchhiker
mechanism (Rodrigue et al., 1999). The active site on the
large subunit is therefore probably extracellularly oriented and
involved in H2 oxidation or production and electron transfer
from or to menaquinone via cytochrome b. The second, [NiFe]
hydrogenase 2, (k121-3777) consists only of a large and small
cytochrome c3-containing subunit without signal peptides to
translocate it over the cell membrane. The small subunit is
anchored in the membrane, probably intracellularly. Such ‘group
5’ [NiFe] hydrogenases are involved in H2 oxidation at very low
H2 concentrations (Peters et al., 2015). Most SAOB described
so far encode for [FeFe] hydrogenases that are involved in
H2 production (Manzoor et al., 2018). A cytoplasmic [NiFe]
hydrogenases was also found to be expressed by S. schinkii during
SAO, and was thought to be involved in intracellular H2 sensing
for subsequent energy-transducing reactions since the gene was
located next to a response regulator receiver gene (Manzoor et al.,
2016). In ‘Ca. S. acetioxidans,’ the [NiFe] hydrogenases 2 genes
surround a methylenetetraydrofolate reductase gene (MetF) and
are probably directly involved in acetate metabolism (see section
“Energy Conservation”), either in regulation of gene expression
of other hydrogenases in response to SAO, or to produce H2
during SAO.
The partial genome of ‘Ca. S. acetioxidans’ also contains
several formate dehydrogenases (FDHs). Three of them contain
4Fe-4S ferredoxin-containing beta subunits (k121-3286-cds8,
k121-4883-cds7 and k121-6301-cds1). Most FDHs seem to have
the active site intracellularly and formate and H2 can therefore
be interconverted in the cytoplasm. One FDH alpha subunit
has a TAT signal, indicating that it is exported and thus might
be involved in extracellular formate conversion (k121-1471cds2). The genome does not encode formate transporters and it
therefore seems most plausible that the SAOB produces H2 as
an end product during SAO. Yet, it could potentially convert
H2 to formate outside of the cell, using extracellular [NiFe]
hydrogenases and formate dehydrogenases.
Genetic Potential of ‘Ca. S. Acetioxidans’
The metagenome of the M-SAO enrichment culture
consisted of eight metagenome-assembled genomes (MAGs)
(Supplementary Figure S1 and Supplementary Tables S1, S2).
The dominant populations in the enrichment culture belonged
to the SAOB (MSAO_Bac1), with 100% 16S rRNA gene
identity to ‘Ca. S. acetioxidans’ clone AAS1, and its syntrophic
methanogenic partner (MSAO_Arc1), with 100% 16S rRNA
gene identity to Methanocalculus natronophilus strain (AMF5).
Details on the draft genome of ‘Ca. S. acetioxidans’ are given in
Table 1. According to the genome completeness and redundancy
estimates obtained through Anvi’o, and the presence of rRNA
and tRNA genes, our MAG can be viewed as a high-quality draft
(Bowers et al., 2017).
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Acetate Activation and Uptake
‘Ca. S. acetioxidans’ does not contain the conventional acetate
kinase (ACK) gene for activating acetate to acetyl phosphate
(acetyl-P) that was found in genomes of the previously
characterized SAOBs Pseudothermotoga lettingae strain TMO
(Zhaxybayeva et al., 2009), Tepidanaerobacter acetatoxydans
strain Re1 (Müller et al., 2015), Syntrophaceticus schinkii strain
Sp3 (Manzoor et al., 2016), Thermacetogenium phaeum strain
DSM 12270 (Oehler et al., 2012), and Clostridium ultunense
strain Esp (Manzoor et al., 2013b). It also does not contain
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Haloalkaliphilic Syntrophic Actetate Oxidation
acetate concentrations, which corresponds to the high affinity
acetate activation via AMP-forming acetate-CoA ligase. It also
has genes for an AMP-forming phenylacetate-CoA ligase (k1216797/k121-3180) that can activate phenylacetate. This enzyme
also acts, albeit less specifically, on acetate, propionate and
butyrate (Martinez-Blanco et al., 1990) and could therefore also
be responsible for acetate activation during SAO. Since very little
energy can be conserved from SAO, it is surprising that ‘Ca. S.
acetioxidans’ only has a gene for the AMP-forming acetyl-CoA
synthetase because this enzyme requires two ATP molecules per
molecule of acetate whereas the ACK/PTA system requires only
one ATP (Berger et al., 2012). However, it is possible that the
generated pyrophosphate (PPi ) is not completely hydrolyzed by
pyrophosphatases and a part is used for transfer of phosphate
groups to other intermediates, thereby conserving some energy
from the reaction (Berger et al., 2012). Multiple genes were indeed
encoding H+ /Na+ translocating pyrophosphatases (Figures 3,
4 and Supplementary Data S1) that hydrolyse PPi for proton
or sodium translocation and thereby possibly creating a proton
or sodium motive force, respectively (Baykov et al., 2013). This
mechanism was indeed postulated to be an adaptation to low
energy supply (Luoto et al., 2013) and has been found in all other
SAOBs described so far (Manzoor et al., 2018).
The gene for an acetate-transporting permease (ActP) is often
found to cluster with acetyl-CoA synthetase (AMP-forming)
gene (Gimenez et al., 2003). However, ActP was not present
in the genome, nor was it present in any other SAOB genome
in our comparison. We found a putative Na+ /solute symporter
(k121-3905) encoded next to the gene for acetyl-CoA synthetase
(AMP-forming). The genome of another SAOB, S. schinkii Sp3,
also contained a gene related to Na+ /solute symporters and
this gene was previously identified to encode for an acetate
transporter in E. coli (Manzoor et al., 2016). Genome analysis of
T. acetatoxydans also showed presence of Na+ /solute symporters
(Müller et al., 2015).
The SAO Pathway
Most SAOBs described so far cluster with the physiological group
of homoacetogens, which possess the Wood–Ljungdahl (WL)
pathway and experimental evidence indicated that indeed this
pathway is used in reverse for SAO by at least T. phaeum and
C. ultunense (Schnürer et al., 1997; Hattori et al., 2005; Oehler
et al., 2012). The partial genome of ‘Ca. S. acetioxidans’ contains
all genes that encode for the (reverse) WL pathway to oxidize
acetyl-CoA (Figure 3) and it likely uses this route for acetyl-CoA
oxidation. An alternative route using the oxidative TCA cycle
was suggested for T. acetatoxydans (Müller et al., 2013). ‘Ca. S.
acetioxidans’ has an incomplete TCA cycle as it lacks the genes
for conversion of malate to oxaloacetate and citrate to isocitrate
(Supplementary Figure S7 and Supplementary Discussion).
Therefore, it seems unlikely that it oxidizes acetate through this
pathway.
FIGURE 3 | Summary of the Wood–Ljungdahl pathway and acetate
activation. Red lines and red names indicate absence of these conversions or
genes in the partial genome of ‘Ca. Syntrophonatronum acetioxidans.’
the gene for phosphotransacetylase (PTA), that further converts
acetyl-P to acetyl-CoA. The enzymes PTA and ACK are mainly
operational at high acetate concentrations. ‘Ca. S. acetioxidans’
does have the gene that encodes for acylphosphatase (acyP) that
could also produce acetyl-CoA from acetyl-P (Figure 3). For
acetate activation, it does have genes encoding AMP-forming
acetate-CoA ligase (k121-3905) that catalyzes the conversion of
acetate to acetyl-CoA via acetyl-AMP in Bacteria and Archaea
for anabolic acetate assimilation. It is reversible in vitro, but
the reaction is irreversible in vivo with presence of intracellular
pyrophosphatases (Wolfe, 2005). It, therefore, serves as the main
route for acetate assimilation at low acetate concentrations since
the enzyme was considered to have a high affinity for acetate
with a low activity (Starai et al., 2003; Berger et al., 2012). In
its natural environment, an acetate oxidizer must cope with low
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Energy Conservation During SAO
The reversal of the WL pathway generates several problems
for energy conservation. Firstly, it creates only one ATP
during formyl-tetrahydrofolate synthase activity, but acetate
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Haloalkaliphilic Syntrophic Actetate Oxidation
FIGURE 4 | Scheme showing transmembrane transporters, energy conserving enzymes and electron transport mechanisms found in the genome of ‘Ca.
Syntrophonatronum acetioxidans.’
This could imply that the Rnf complex has no significant
role in energy conservation during SAO, but this needs to be
investigated for ‘Ca. S. acetioxidans’. Lastly, two contigs of the
genome encoded for NADH:ubiquinone oxidoreductase subunits
NuoEFGI (Supplementary Data S1). These subunits form the
soluble fragment and catalyze the oxidation of NADH (Braun
et al., 1998). Since we did not find the hydrophobic membrane
fragment in the draft genome (NuoAHJKLMN subunits) (Leif
et al., 1995), it is not clear if the ‘Ca. S. acetioxidans’ genomes
encodes for the whole complex or if genes were missed by
incompleteness of the draft genome (Table 1). Still, since it
was proposed that acetogens can be bioenergetically classified
into Rnf and Ech-containing groups with either Na+ and H+ dependence (Hess et al., 2014), we propose to classify ‘Ca. S.
acetioxidans’ as a Rnf-containing Na+ -dependent acetogen.
The second challenge for energy conservation during
SAO is the production of NADH (E◦ = −320 mV) from
methyl-THF oxidation (E◦ = −200 mV). This conversion is
endergonic under standard conditions. Therefore, a bifurcation
mechanism that couples this to a favorable conversion is
expected. Interestingly, ‘Ca. S. acetioxidans’ contained two
copies of the gene coding for methylenetetrahydrofolate
(methylene-THF) reductase (metF). One was located in the
same contig with the genes for CODH/acetyl-CoA synthase and
methyltetrahydrofolate:corrinoid methyltransferase (acsE/metH)
(k121-702). Besides these genes, this acs operon also contains
genes for a MvhD/HdrABC-like complex. Strangely, the genes
coding for the HdrA subunit contains a TAT signal peptide
for export and is next to the TAT protein translocase system
(TatABC). The subunits HdrB and HdrC were encoded on a
different contig and are predicted to be cytoplasmic (k1214746). In the acetogen Moorella thermoacetica, metF and
HdrABC/MvhD form a transcript (Mock et al., 2014). There,
the genes encoding homologs of the soluble HdrABC complex,
similar to the one of Methanothermobacter marburgensis,
are downstream of methylene-THF reductase subunits MetV
activation costs two ATP. Energy must therefore come from
the generation of a sodium or proton motive force during
SAO. The partial genome of ‘Ca. S. acetioxidans’ encodes for
an F1 FO -type ATP synthase. Multiple sequence alignment
shows that the c-subunit of the F1 FO -ATP synthase of
‘Ca. S. acetioxidans’ (k121-2696) has conserved amino acids
involved in Na+ -binding that are present in Na+ -dependent
F1 FO -ATP synthases but not in H+ -dependent F1 FO -ATP
synthases (Mulkidjanian et al., 2008; Oehler et al., 2012; Schulz
et al., 2013) (Supplementary Figure S8). Na+ -dependent ATP
synthases provide an advantage at haloalkaline conditions where
extracellular Na+ concentrations are high and thus contribute
to the sodium motive force (Figure 4). To establish a sodium
motive force, the Na+ levels in the cell need to be kept lower
than externally. As mentioned above, the genome contains
H+ /Na+ translocating pyrophosphatases that produce a sodium
motive force during acetate activation. Secondly, the genome
contains genes encoding for Na+ efflux proteins and single- and
multisubunit Na+ /H+ antiporters (see section “Adaptations to
Haloalkaliphilic Conditions”).
The genome encoded for two transmembrane complexes
that combine production of a sodium motive force to NADH
recycling and subsequent H2 production. Three subunits
of the Rnf complex (Na+ -translocating ferredoxin: NAD+
oxidoreductase) were encoded in the genome; subunits C, D
and two genes for subunit G (Supplementary Data S1). Both
genes for subunit G contained a signal peptide for export and
RnfD was transmembrane. Subunit RnfC is directly involved in
NADH recycling that drives RnfD to pump Na+ or H+ ions.
These genes are homologs to the Na+ -pumping NADH:quinone
oxidoreductase (Na+ -NQR), but multiple sequence alignment
showed that they are more related to Rnf complex genes (data
not shown). T. phaeum contains only one gene with weak
similarity to the subunit RnfC (Oehler et al., 2012) and in
S. schinkii transcription levels of the Rnf complex were very
low when grown syntrophically on acetate (Manzoor et al., 2016).
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Haloalkaliphilic Syntrophic Actetate Oxidation
antiporters (Mnh, k121-5712) for regulation of pH and
electronegativity homeostasis. As opposed to the Nha antiporters,
Mnh antiporters have larger and more extensive proton gathering
funnels, which presumably makes them more suitable for H+
scavenging; essential at maintenance of pH homeostasis in
haloalkaliphiles (Horikoshi, 2011). Common compatible solutes
are L-proline, glycine betaine and ectoine. The partial genome
of ‘Ca. S. acetioxidans’ encodes most genes necessary for
biosynthesis of L-proline. We did not find the complete pathway
for choline biosynthesis or for glycine betaine production from
choline (Supplementary Data S1), and no genes for ectoine
production were present. Glycine, cysteine, and serine are
derivatives of 3-phosphoglycerate, which is an intermediate of the
glycolysis and pentose-phosphate pathway. The glycolysis, TCA
cycle and the non-oxidative branch of the pentose phosphate
pathway are all encoded in the genome (Supplementary
Figure S7 and Supplementary Discussion). Serine and glycine
can be produced from 3-phosphoglycerate (Supplementary
Data S1). Glycine can also be produced from serine using
serine hydroxymethyl transferase (k121-2696-cds48) or from
glyoxylate using alanine-glyoxylate transaminase (k121-3258cds5). Glycine betaine (trimethylglycine) is produced from
glycine betaine aldehyde using betaine aldehyde dehydrogenase
(k121-1838-cds1). The glycine betaine aldehyde is normally
produced from choline, but no choline dehydrogenases or
choline monooxygenases were found in the genome of ‘Ca. S.
acetioxidans’. Glycine betaine can, however, also be produced
from glycine using glycine methyltransferase (k121-2696-cds48)
to produce sarcosine, but no genes for sarcosine conversion
to glycine betaine were found. However, the genome encodes
a putative protein-S-isoprenylcysteine methyltransferase (k1211838-cds2) next to the betaine aldehyde dehydrogenase that
could act as a multifunctional enzyme to convert sarcosine
to N,N-dimethylglycine and subsequently to glycine betaine
by producing S-adenosyl L-homocysteine from S-adenosyl Lmethionine. Furthermore, uptake systems for glycine betaine,
choline, and L-proline are encoded in the genome (Figure 4
and Supplementary Data S1). The “salt-out” strategy with
osmotic solutes allows fast adaptation to rapid fluctuations in
salinity (Oren, 2013), which is probably key for survival of ‘Ca.
S. acetioxidans’ in soda lake environments that have a high
fluctuation of salinity due to dry and wet seasons.
Soda lakes have besides a high pH and high sodium carbonate
concentrations also very low concentrations of unbound divalent
ions (mainly Ca2+ and Mg2+ ). The partial genome of the
SAOB contains many ABC transporters and several divalent
ion uptake systems for magnesium, cobalt, nickel, calcium,
zinc, manganese, tungsten and iron (Supplementary Data S1).
It also contains a gene encoding an ammonium transporter
(AmtB, k121-1147-cds13) for ammonium uptake. At haloalkaline
conditions, ammonium occurs mainly as the free ammonia
which can diffuse through the membrane, but only at high
concentrations.
and MetF genes with a ferredoxin-coding gene in between
(Schuchmann and Müller, 2014). Partial purification of this
enzyme complex of M. thermoacetica showed that it is a
heterohexamer of MetFV, HdrABC, and MvhD that uses NADH
as electron donor. The complex, however, does not catalyze
NADH dependent methylene-THF reduction and does not
use ferredoxin as an electron acceptor. It still needs to be
investigated if this HdrABC performs electron bifurcation with
a second electron acceptor (Mock et al., 2014; Schuchmann
and Müller, 2014). In the SAOB T. phaeum, the acs operon also
contained Hdr-like genes (Oehler et al., 2012) and a Hdr/NADbinding oxidoreductase complex was expressed during SAO
in S. schinkii (Manzoor et al., 2016). In T. phaeum it was
assumed that a bifurcating hydrogenase was coupled (directly
or indirectly via menaquinone) to oxidation of methyl-THF
(Oehler et al., 2012; Manzoor et al., 2016). In Syntrophobacter
fumaroxidans, the Hdr/MvhD complex of that bacterium was
detected under sulfate-reducing conditions, whereas only the
subunits containing FAD/NAD binding domains were detected
under syntrophic conditions (Sedano-Nuñez et al., 2018). CO
oxidation to CO2 (−520 mV) by the CODH/ACS is coupled to
ferredoxin reduction (−450 mV) and is exergonic. Production of
NADH (−320 mV) during methyl-THF oxidation (−200 mV)
is endergonic. Therefore, we propose a flavin-based electron
bifurcating mechanism where the exergonic oxidation of
ferredoxin drives the endergonic reduction of NAD+ during
methyl-THF oxidation (Figure 4), similar as was found in
M. thermoacetica, but reversed. The other metF copy in the
genome of the SAOB (k121-3777) is located next to a [NiFe]
hydrogenase ([NiFe] hydrogenase 2) and possibly involved in H2
production during SAO (see section “Hydrogenases and Formate
Dehydrogenases”).
Adaptations to Haloalkaliphilic Conditions
At haloalkaline conditions, microorganisms must cope with two
extremes, namely high pH and high osmotic pressure. ‘Ca. S.
acetioxidans’ is an obligate haloalkaliphile as its optimal growth
condition is around pH 9.5–10 and 1 M Na+ in the form of
carbonate/bicarbonate. To be able to thrive at these conditions,
the organism needs some specific adaptations to maintain pH
homeostasis and osmotic balance. The two strategies for osmotic
adaptation are the “salt-in” and “salt-out” strategies. With the
“salt-in” strategy, extreme halophilic prokaryotes accumulate K+
to balance the high Na+ concentrations outside of the cell. We
found two genes encoding for K+ uptake proteins that belong
to the Trk K+ transport system (Figure 4 and Supplementary
Data S1). Halophilic microorganisms that accumulate KCl are
also characterized by an excess of acidic amino acids in their
proteins (Oren, 2013). The isoelectric point profile of the
predicted proteome of ‘Ca. S. acetioxidans’ does not show a
pronounced acidic proteome and thus probably employs mostly
a “salt-out” strategy (Supplementary Figure S9).
The “salt-out” strategy involves Na+ extrusion and organic
osmolytes (compatible solutes) production. As mentioned, the
partial genome encodes for multiple Na+ extrusion mechanisms,
such as Na+ efflux systems (NatB, k121-1613-cds22) and
single subunit (Nha, k121-1712-cds1) and multisubunit Na+ /H+
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Membrane Lipid Composition
The lipid composition of the M-SAO enrichment culture
showed the presence of two types of core membrane diether
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Haloalkaliphilic Syntrophic Actetate Oxidation
grow under less extreme condition is required to test this
hypothesis.
lipids – the archaeal lipid archaeol (37%) and bacterial diether
lipids (63%) (Supplementary Figure S10). Archaeol is derived
from M. natronophilus AMF5 as was confirmed by analysis
of a pure culture of M. natronophilus AMF5. The bacterial
diether lipids (63%) comprise the core lipids of the bacterial
fraction of the M-SAO enrichment culture, that was highly
enriched in ‘Ca. S. acetioxidans.’ These are composed of C31 C37 unsaturated dialkyl glycerol diethers. Their alkyl chains
are predominantly composed of C14 and C16 n-alkyl moieties
as demonstrated by hydrogenation and subsequent GC-MS
analysis: 1-n-tetradecyl-2 -n-hexadecyl glycerol diether and 1,2di-n-hexadecyl glycerol diether, identified by comparison to
literature data (Pancost et al., 2001), were the most prominent
hydrogenation products obtained. Fatty acids were only minor
fractions of lipids in comparison to the unsaturated diakyl
glycerol diethers, indicating that ‘Ca. S. acetioxidans’ is producing
a highly unusual lipid membrane that is predominantly
composed of diether lipids. Analysis of intact polar lipids revealed
that the head groups of these diethers was predominantly
phosphocholine. The occurrence of phosphocholine headgroups
in phospholipids in prokaryotes is not common, but it was
reported that around 10% of all bacterial genomes possess the
pathways to produce phosphocholine (Sohlenkamp et al., 2003).
Choline is also a precursor for the compatible solute glycine
betaine. It was hypothesized that choline could be released
from phosphocholine by phospholipase under hyperosmotic
conditions (Sohlenkamp et al., 2003). For the biosynthesis of
intact polar lipids, we indeed only found diacylglycerol kinase
(k121-2772-cds2) and phosphatidate cytidylyltransferase (k1213334-cds5) for conversion of 1,2 diacyl-sn-glycerol to CDP-diacyl
glycerol, but no genes for its conversion to phosphocholine
(Boumann et al., 2006). We did find a lysophospholipase
L1 in the draft genome (k121-3840-cds54). This enzyme
could catalyze the conversion of 2-lysophosphatidylcholine to
glycerophosphocholine which can subsequently be converted to
glycerophosphate and choline by a glycerophospho-diesterase
(k121-1838-cds3, k121-2762-cds8 or k121-4277-cds1). However,
the pathways of phosphocholine synthesis and degradation
in prokaryotes need further investigation (Sohlenkamp et al.,
2003), as well as the biosynthesis of bacterial dialkyl glycerol
diethers (Grossi et al., 2015). Alkyl glycerol ether lipids
are typically present in archaeal membranes and ensure
lower permeability of ions and higher stability than bacterial
membrane polar lipids containing esterified fatty acids (Koga,
2012; Grossi et al., 2015). Bacterial alkyl glycerol ether lipids
were found in (hyper)thermophilic bacteria and acidophilic
bacteria (Koga, 2012; Siliakus et al., 2017), but also in
mesophilic Planctomycetes (Sinninghe Damsté et al., 2005) and
sulfate-reducing bacteria (Grossi et al., 2015). The function
of this uncommon lipids in bacteria is not known, but
they probably give the cell membrane a higher degree of
stability and impermeability, as was shown for archaeal
dialkylglycerol ethers (Grossi et al., 2015). The dominance of
ether lipids with phosphocholine headgroups in the membrane
of ‘Ca. S. acetioxidans’ may reflect an adaptation to the
high pH and high salt concentrations, but knowledge on
the membrane lipid compositions of close relatives that
Frontiers in Microbiology | www.frontiersin.org
The Importance of SAO in Haloalkaline
Environments
The SAOB ‘Ca. S. acetioxidans’ belongs to the family
Syntrophomonadaceae (Sorokin et al., 2014a), mostly known for
its characterized syntrophic butyrate oxidizers (Müller et al.,
2010). Recently, it was shown that members of this family
are abundant in hypersaline soda lake sediments from the
Kulunda steppe (south-western Siberia) and 52 novel MAGs
were retrieved from five metagenomes (Vavourakis et al., 2018).
Based on the phylogeny of 16 conserved ribosomal proteins,
the ‘Ca. S. acetioxidans’ genome derived from our M-SAO
culture (MSAO_Bac1) was most closely related to two of those
MAGs with similar G+C content, namely Syntrophomonadaceae
CSSed11_10 (not shown) and T1Sed10_67 (Figure 5). The
two MAGs belonged likely to another species (ANI = 96.9%,
conDNA = 61%) within the genus ‘Ca. Syntrophonatronum’
(ANI = 83.25% with MSAO_Bac1). Since T1Sed10.67 encoded
for an AMP-forming acetyl-CoA synthetase and all genes of
the WL pathway, it has the genetic potential to perform acetate
oxidation, as was the case for ‘Ca. S. acetioxidans’ (Figure 5
and Supplementary Data S2). Other candidate (reversed)
acetogens able to produce H2 in syntrophic interactions
based on the predicted genome potential were present in the
sediment metagenomes, such as Dethiobacter alkaliphilus,
CSSed11.298R1 and CSSed11.131 (Figure 5). Some other
MAGs belonged to the genus ‘Ca. Syntropholuna’ (94% 16S
rRNA gene identity; Supplementary Data S3), from which a
syntrophic benzoate-degrading culture was recently obtained
(Sorokin et al., 2016). This culture was obtained in a study
where soda lake enrichment cultures with other fatty acids
and alcohols such as butyrate, propionate and ethanol also
mainly resulted in syntrophic cultures (Sorokin et al., 2016).
Previously obtained 16S rRNA gene profiles of sediments from
south-western Siberian hypersaline soda lakes (Vavourakis et al.,
2018) were also examined, including lake Bitter-1 from which
our enrichment cultures originated (Sorokin et al., 2014a, 2016).
Syntrophomonadaceae was among the most abundant family
of all Bacteria; their occurrence ranged from 6.8 to 24.5% at
100 g l−1 salinity (Figure 6). The highest identity (up to 99%)
with the full 16S rRNA gene sequence of ‘Ca. S. acetioxidans’
(Sorokin et al., 2014a) was with representative sequences
from the OTU assigned to ‘Candidatus Contubernalis’. ‘Ca.
Contubernalis alkalaceticum’ was the first cultivated syntrophic
SAOB obtained from a low-salt soda lake (Zhilina et al., 2005)
and ‘Ca. S. acetioxidans’ and ‘Ca. C. alkalaceticum’ form an
independent branch within the family of Syntrophomonadaceae
(Sorokin et al., 2014a). In line with the metagenomics data, the
abundance of this OTU was between 0.2 and 1.4% of all reads in
the four examined soda lakes. These combined results show that
syntrophic fatty acid oxidation might be an important anaerobic
carbon mineralization route in soda lake sediments.
Bacterial 16S rRNA gene community profiling of the
same hypersaline soda lake sediment samples showed that
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December 2018 | Volume 9 | Article 3039
Timmers et al.
Haloalkaliphilic Syntrophic Actetate Oxidation
FIGURE 5 | Maximum likelihood tree based on the phylogeny of 16 ribosomal proteins found in selected reference genomes from the family Syntrophomonadaceae
and the draft genome of “Ca. S. acetioxidans” (MSAO_Bac 1). The tree was rooted to the proteins found in Natranaerobius thermophilus. The estimated relative
abundance of each organism in five different hypersaline soda lake sediment samples is expressed as reads per Kb of genome sequence per Gb of mapped reads
(RPGK). Black dots at the nodes show the ≥90% confidence of 100x bootstraps. The reference genomes indicated as bin X Y are MAGs obtained from the same
metagenomes (CSSed10, CSSed11, T3Sed10, T1Sed10, and B1Sed10) for which the recruitment experiment was performed [33]. MAG IDs with an asterisk show
the presence of a partial 16S rRNA gene with 94% identity to ‘Ca. Syntropholuna alkaliphila’. Presence and absence of genes and pathways are indicated by the
symbols on the right hand side. Full = present, empty = partially present. Gene abbreviations for acetate activation: ack/pta = acetate kinase/phosphate
acetyltransferase, ACSS/hppA = acetyl-coA synthetase/H+ /Na+ translocating pyrophosphatases, paaK = phenylacetate-CoA ligase. WL-carbonyl and WL-methyl
are the carbonyl and methyl branch of the Wood–Ljungdahl pathway, respectively. PFOR = pyruvate ferredoxin oxidoreductase. Gene abbreviations encoding for
formate dehydrogenases: fdoGH, fdhAB. Gene abbreviations encoding for [NiFe] hydrogenase: B-cyt = b-type cytochrome subunit, hyaBA.
the potential acetate-oxidizing SRB clades were present only
in very low relative abundance (≤0.1%). Representatives of
the genera Desulfonatronospira and Desulfonatronobacter were
the most abundant, followed by Desulfonatronovibrio and
Desulfobacteriaceae sp. (Figure 6). Most cultured representatives
of these genera can use formate and H2 as electron donor but
need or prefer acetate as a carbon source and previous attempts to
isolate SRB with acetate as electron donor and sulfate as electron
acceptor have failed (Zhilina et al., 1997; Sorokin et al., 2008,
2011, 2012, 2015b; Sorokin and Chernyh, 2017). In fact, the
only acetate-oxidizing anaerobes cultivated from soda lakes so
far are sulfur-reducing bacteria belonging to the Chrysiogenetes
and Halanaerobiales (Sorokin et al., 2010) and the thiosulfate or
sulfite-reducing Desulfonatronobacter acetoxydans (Sorokin et al.,
2015b).
Archaeal 16S rRNA gene community profiling
showed that the most abundant methanogenic clades are
methylotrophic and hydrogenotrophic methanogens belonging
to Methanobacterium, Methanocalculus, Methanolobus, and
Frontiers in Microbiology | www.frontiersin.org
Methanosalsum (Figure 6). Thermoplasmata were abundant
and some MAGs were constructed from this order that
belonged to the methyl-reducing methanogens of the order
Methanomassiliicoccales (Vavourakis et al., 2018). Aceticlastic
methanogens from the genera Methanosaeta and Methanosarcina
were present at very low relative abundance (≤0.1%) and each in
only one of the soda lake sediment samples. In previous research,
aceticlastic methanogenesis did not occur in incubations with
several soda lake sediments (Nolla-Ardevol et al., 2012). Since
the minimum salinity of the soda lakes investigated here was
70 g l−1 (which corresponds to 1.1 M Na+ in defined media),
our findings are in line with other previous enrichment cultures
from several soda lakes that yielded aceticlastic Methanosaeta
sp. only at salinities below 0.6 M Na+ , whereas at higher
sodium concentrations, syntrophic communities with the
extremely salt tolerant hydrogenotrophic methanogenic partner
Methanocalculus sp. were considered responsible for acetate
oxidation (Sorokin et al., 2015a). Since most of the SRB and
methanogens in soda lake environments cannot use acetate as
13
December 2018 | Volume 9 | Article 3039
Timmers et al.
Haloalkaliphilic Syntrophic Actetate Oxidation
FIGURE 6 | Relative abundance (%) of OTUs of Firmicutes, Deltaproteobacteria, and Euryarchaeota in five different soda lake sediment samples with different
salinity. Only reads with higher relative abundance than 0.1% in at least one of the sediment samples are shown.
electron donor, it seems that acetate would mainly be oxidized
by syntrophic associations in soda lake environments with high
salinities.
substrate addition and metagenomics and metaproteomics will
aid to elucidate that this is true for acetate oxidation but also for
other fatty acids and alcohols.
CONCLUSION
DATA AVAILABILITY
Based on the results gathered from both the M-SAO and S-SAO
cultures, it can be concluded that H2 and/or formate are the main
electron carriers during SAO since; (1) H2 was produced in SAO
performing cultures at concentrations that were energetically
favorable; (2) inhibition of the sulfate reducing and methanogenic
partner resulted in H2 accumulation and unfavorable conditions
for acetate oxidation; (3) the draft genome of MSAO_Bac1
encoded for intra- and extracellular [NiFe] hydrogenases and
formate dehydrogenases and did not encode for formate
transporters, but formate and H2 were interconverted by the
syntrophic partner. The low solubility of H2 at soda lake
conditions explain why SAO is energetically feasible at the
measured H2 concentrations. The metagenomic and 16S rRNA
gene amplicon data of the five hypersaline soda lake sediment
samples, together with previous cultivation efforts, indicated
that syntrophy seems to be an important anaerobic process
for organic matter degradation at haloalkaline conditions. Most
haloalkaliphilic methanogens and SRB do not use acetate for
catabolic purposes, which implies that SAO might be the
dominant acetate-dependent catabolic process at haloalkaline
conditions. Further experiments involving cultivation, labeled
Datasets are in a publicly accessible repository. The raw
sequence reads of the metagenomes from the methanogenic
SAO enrichment culture have been deposited to the NCBI
Sequence Read Archive (SRP156567). The final MAGs described
in this paper have been deposited as individual Whole
Genome Shotgun projects at DDBJ/EMBL/GenBank (without
annotation). Accession numbers are given in Supplementary
Data S3 and Supplementary Table S3 (QZAD00000000QYZW00000000). All versions described in this paper are version
XXXX01000000.
Frontiers in Microbiology | www.frontiersin.org
AUTHOR CONTRIBUTIONS
PT designed the experiments, conducted most of the
experimental work, and wrote the article. CV analyzed the
metagenomic and 16S rRNA gene amplicon data, and helped
with analysis and interpretation of the work and writing of
the article. DS provided the cultures and the expertise to grow
them, and helped with design, analysis, and interpretation of the
experiments. RK performed the thermodynamic calculations,
14
December 2018 | Volume 9 | Article 3039
Timmers et al.
Haloalkaliphilic Syntrophic Actetate Oxidation
Organizations (0104-2018-0033), AS by the ERC Advanced Grant
Novel Anaerobes (No. 323009), and JD by the ERC Advanced
Grant Microlipids (No. 694569).
and helped with analysis and interpretation of the work. JD
performed the lipid analysis. GM, AS, and CP contributed with
experimental design and interpretation of the work. All authors
provided feedback and corrections on the manuscript, revised the
intellectual content, approved the final version to be published,
and agreed to be accountable for all aspects of the work.
ACKNOWLEDGMENTS
The authors want to thank Cristina M. Gagliano (Wetsus,
Leeuwarden, Netherlands) for help with genomic DNA isolation
at high salinity and W. Irene C. Rijpstra (NIOZ) for the
experimental lipid work.
FUNDING
This research was supported by the Soehngen Institute of
Anaerobic Microbiology (SIAM) Gravitation grant (024.002.002)
of the Netherlands Ministry of Education, Culture and Science
and the Netherlands Organisation for Scientific Research
(NWO). GM and CV were supported by the ERC Advanced
Grant PARASOL (No. 322551). DS also received support from
the Russian Foundation for Basic Research (16-04-00035) and the
Russian Academy of Sciences and Federal Agency of Scientific
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2018.03039/full#supplementary-material
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Conflict of Interest Statement: The authors declare that the research was
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