1. ARTICLE
Enrichment of Anaerobic Methanotrophs in
Sulfate-Reducing Membrane Bioreactors
Roel J.W. Meulepas,1,2 Christian G. Jagersma,3 Jarno Gieteling,1 Cees J.N. Buisman,1
Alfons J.M. Stams,3 Piet N.L. Lens1,2
1
Sub-department of Environmental Technology, Wageningen University, Bomenweg 2, 6703
HD Wageningen, The Netherlands
2
Environmental Resources, UNESCO-IHE, Westvest 7, 2611 AX Delft, The Netherlands;
telephone: þ31-15-215-1892; fax: þ31-15-212-2921; e-mail: r.meulepas@unesco-ihe.org
3
Laboratory of Microbiology, Wageningen University, Dreijenplein 10,
6703 HB Wageningen, The Netherlands
Received 19 February 2009; revision received 6 May 2009; accepted 11 May 2009
Published online 20 May 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22412
Introduction
ABSTRACT: Anaerobic oxidation of methane (AOM) in
marine sediments is coupled to sulfate reduction (SR). Anaerobic Methanotrophs
AOM is mediated by distinct groups of archaea, called
anaerobic methanotrophs (ANME). ANME co-exist with The anaerobic oxidation of methane (AOM) by microbes
sulfate-reducing bacteria, which are also involved in AOM was first discovered during geochemical studies, which
coupled SR. The microorganisms involved in AOM showed that AOM in marine sediments is coupled to
coupled to SR are extremely difficult to grow in vitro. Here, sulfate reduction (SR), according to Equation (1) (Barnes
a novel well-mixed submerged-membrane bioreactor
system is used to grow and enrich the microorganisms and Goldberg, 1976; Iversen and Jørgensen, 1985; Martens
mediating AOM coupled to SR. Four reactors were inocu- and Berner, 1974; Reeburgh, 1976, 1980). The AOM
lated with sediment sampled in the Eckernforde ¨ rates in marine sediments are low, between 0.001 and
Bay (Baltic Sea) and operated at a methane and sulfate 21 mmol gÀ1dry weight dayÀ1 (Kruger et al., 2005; Treude et al.,
¨
loading rate of 4.8 L LÀ1 dayÀ1 (196 mmol LÀ1 dayÀ1) and 2007).
3.0 mmol LÀ1 dayÀ1. Two bioreactors were controlled at
158C and two at 308C, one reactor at 308C contained also
anaerobic granular sludge. At 158C, the volumetric AOM CH4 þ SO42À ! HCO3À þ HSÀ þ H2 O
and SR rates doubled approximately every 3.8 months. After (1)
884 days, an enrichment culture was obtained with an AOM DG ¼ À16:6 kJ molÀ1
and SR rate of 1.0 mmol gvolatile suspended solidsÀ1 dayÀ1
(286 mmol gdry weightÀ1 dayÀ1). No increase in AOM and
SR was observed in the two bioreactors operated at 308C. AOM in marine sediments is mediated by uncultured
The microbial community of one of the 158C reactors was archaea, termed anaerobic methanotrophs (ANME). ANME
analyzed. ANME-2a became the dominant archaea. This are phylogenetically distantly related to cultivated metha-
study showed that sulfate reduction with methane as elec-
tron donor is possible in well-mixed bioreactors and that the nogenic members from the orders Methanosarcinales and
submerged-membrane bioreactor system is an excellent Methanomicrobiales (Hinrichs et al., 1999; Knittel et al.,
system to enrich slow-growing microorganisms, like metha- 2005; Niemann et al., 2006; Orphan et al., 2002). Three
notrophic archaea. groups of ANME have been distinguished so far, of which
Biotechnol. Bioeng. 2009;104: 458–470. ANME 1 and ANME 2 are the most abundant and
ß 2009 Wiley Periodicals, Inc. geographically widespread groups (Hinrichs et al., 1999;
KEYWORDS: anaerobic methane oxidation; sulfate reduc- Knittel et al., 2005; Niemann et al., 2006; Orphan et al.,
tion; enrichment; bioreactor 2002). Thus far, no gene analogs for enzymes involved in
dissimilatory SR have been found in archaea belonging to
the ANME groups (Thauer and Shima, 2008). It has been
suggested that the archeaon produces an electron carrier
compound from CH4 that is utilized by the sulfate-reducing
Correspondence to: R.J.W. Meulepas
Contract grant sponsor: Economie, Ecologie, Technologie (EET) partner (Alperin and Reeburgh, 1985; DeLong, 2000;
Contract grant number: EETK03044 Hoehler et al., 1994; Zehnder and Brock, 1980). This was
458 Biotechnology and Bioengineering, Vol. 104, No. 3, October 15, 2009 ß 2009 Wiley Periodicals, Inc.

2. supported by the finding that in Hydrate Ridge sediment, losses due to unwanted methanogenesis and acetogenesis
ANME live in consortia with sulfate-reducing bacteria can be avoided.
(SRB) (Boetius et al., 2000; Hinrichs et al., 2000). These To assess the potential of CH4 as electron donor for
archaea/SRB aggregates are not dominant in all AOM sites biological sulfate reduction in wastewater treatment, insight
though. In Black sea microbial mats, SRB occur in in the growth and conversion rates that can be achieved in
microcolonies surrounded by bulk ANME-1 cells clusters bioreactors is required. In addition, the obtainment of an
(Knittel et al., 2005; Michaelis et al., 2002). In samples from active methane-oxidizing sulfate-reducing enrichment will
Eel River Basin ANME-1 archaeal group frequently existed allow the physiological aspects of AOM coupled to SR to be
in monospecific aggregates or as single filaments, apparently studied.
without a bacterial partner (Orphan et al., 2002). And in
¨
Eckernforde Bay sediment, clusters of ANME-2a cells were
found without sulfate-reducing partners (Treude et al.,
2005). Current Research
In the present study, well-mixed ambient-pressure sub-
merged-membrane bioreactors (MBRs) were used to enrich
Sulfate Reduction in Biotechnology anaerobic methanotrophs. In these bioreactor systems,
the washout of cells and growth limitation, due to
Nauhaus et al. (2002, 2007) demonstrated in vitro AOM product depletion (SO42À and CH4) or product inhibition
coupled to SR and growth at a rate of 0.003 dayÀ1. (H2S toxicity), can be prevented. The MBRs were inoculated
Therefore, AOM coupled to SR might also be possible in ¨
with sediment from the Eckernforde Bay (Baltic Sea) and
bioreactors. Biological sulfate reduction in bioreactors is operated at 15 or 308C. One MBR at 308C was additionally
applied for the removal and recovery of metal and sulfur inoculated with methanogenic granular sludge. This was
compounds from waste or process streams produced in the done to assess if microorganisms from anaerobic granular
mining and metallurgical industry (Weijma et al., 2002). sludge could play a role in SR with CH4 as electron donor,
The produced sulfide and the dissolved metals form directly or indirectly by providing unknown compounds
insoluble metal sulfides, which are separated from the that may support growth (e.g. growth factors). Volumetric
water and reused in the metallurgical industry. Excess sulfide AOM and SR rates were followed in time and used to
can subsequently be biologically oxidized to elemental estimate the growth rate of the responsible organisms.
sulfur, which is a reusable product as well. This process The microorganisms responsible for the conversion were
allows complete sulfur and metal recovery from a waste identified by constructing a clone library of the enrichment
stream, however, the costs of the electron donor limit the and by monitoring the changes in microbial composition by
application of this process. Organic waste streams are cheap Denaturing Gradient Gel Electrophoresis (DGGE).
electron donors, but are not easily degradable and often
contain inert material. In addition, undesired byproducts
can be formed and the quantity and quality of these waste
streams is not constant. Fully degradable electron donors are
Materials and Methods
therefore a better option. Electron donors which have
Origin and Storage of the Inoculates
been extensively investigated as electron donor for SR in
bioreactors include hydrogen, synthesis gas, methanol, ¨
Sediment samples were taken in Eckernforde Bay (Baltic
ethanol, acetate, lactate, propionate, butyrate, sugar, and Sea) at station B (water depth 28 m, position 54831’15N
molasses (Liamleam and Annachhatre, 2007). According to 10801’28E) during a cruise of the German research vessel
van Houten (1996) hydrogen is the best electron donor at Littorina in June 2005. This sampling site has been described
large scale (5–10 kmol SO42À hÀ1 ), while ethanol is an by Treude et al. (2005); during the year, the temperature,
interesting electron donor at smaller and middle scale. salinity and sulfate concentration in the top 30 cm of the
However, natural gas (70–90% CH4) is two to four times sediment varied between 4 and 178C, 14 and 23% and 0 and
cheaper per amount of reducing capacity than hydrogen and ¨
22 mM, respectively. Eckernforde Bay sediment is a non-
ethanol (Mueller-Langer et al., 2007; www.ethanolmarket. seep sediment, AOM is fueled by CH4 produced by organic
com). Hydrogen was chosen as most suitable electron donor matter degradation. Sediment samples were taken with a
for a full-scale sulfate-reducing bioreactor in Budel (The small multicore sampler based on the construction
Netherlands; Weijma et al., 2002). However, if methane described by Barnett et al. (1984). The cores had a length
would be used as electron donor four times less gas needs to of 50 cm and reached 30–40 cm into the sediment bed.
be transferred from the gas to the liquid phase, as CH4 can Immediately after sampling, the content of the cores was
donate eight electrons and H2 only two. In addition, collected in a large bottle, which was made anaerobic by
the solubility of CH4 (1.44 mM in distillated water at replacing the headspace by anaerobic artificial seawater.
0.101 MPa CH4 and 208C) is higher than of hydrogen Back in the laboratory, the sediment was homogenized and
(0.817 mM at 0.101 MPa hydrogen and 208C). Another transferred into 1 L bottles in an anoxic glove chamber. The
advantage of methane as electron donor is that substrate 1 L bottles were closed with butyl rubber stoppers and the
Meulepas et al.: Enrichment of Anaerobic Methanotrophs 459
Biotechnology and Bioengineering

3. headspace was replaced by CH4 (0.15 MPa). The bottles were and sulfide solutions, the medium was boiled, cooled down
stored at 48C in the dark for 4 months until the experiments under a nitrogen (N2) atmosphere and transferred into a
were started. 10 L bottle with a CH4 headspace (kept at an overpressure of
Methanogenic granular sludge samples were obtained 10–20 kPa). The bottle was kept at 48C and connected to the
from two full-scale methanogenic mesophilic UASB influent pumps of the reactors.
reactors, one UASB reactor treating paper mill wastewater
(Eerbeek sludge; Eerbeek, The Netherlands, June 2005) and
one treating distillery wastewater (Nedalco sludge; Bergen
op Zoom, The Netherlands, July 2005), described in Experimental Set-Up of Membrane Bioreactors
detail by Roest et al. (2005) and Gonzalez et al. (2001), To enrich for anaerobic methanotrophs, 4 submerged-
respectively, and stored anaerobically at 48C in the dark. membrane bioreactors were built (Fig. 1). The reactor
system consisted of a cylindrical glass vessel (height:
520 mm, internal diameter: 70 mm, total volume: 2.0 L),
the vessel was equipped with sampling ports for the
Medium
headspace and the reactor suspension (mixture of liquid and
The basal medium consisted of: NaCl (19.8 g LÀ1), KCl suspended solids in the bioreactor). The glass reactor
(0.45 g LÀ1) MgCl2Á6H2O (4.25 g LÀ1), NH4Cl (0.25 g LÀ1), was covered with opaque plastic to prevent phototrophic
CaCl2Á2H2O (1.19 g LÀ1), MgSO4Á7H2O (5.10 g LÀ1), conversions. STEPDOS1 diaphragm metering pumps
KH2PO4 (0.34 g LÀ1), K2HPO4Á 3H2O (1.25 g LÀ1), a trace (KNF Flodos, Sursee, Switzerland) continuously supplied
element solution (1 mL LÀ1), a vitamin solution (1 mL LÀ1), the reactors with medium, the hydraulic retention time
a 0.5 g LÀ1 resazurin solution (1 mL LÀ1), a 0.1 M Na2S (HRT) was 7 days, which resulted in a sulfate loading
solution (1 mL LÀ1) and demineralized water. The trace of 3.0 mmol LÀ1 dayÀ1. Each reactor was equipped with
elements and vitamin solutions were made according to 4 polysulfone membranes (Triqua BV, Wageningen, The
Widdel and Bak (1992). Prior to the addition of the vitamins Netherlands), with a total effective surface of 0.028 m2, via
Figure 1. Schematic overview of a submerged-membrane bioreactor used for the enrichment experiments.
460 Biotechnology and Bioengineering, Vol. 104, No. 3, October 15, 2009

4. which the effluent was extracted by means of a peristaltic ¨
forde Bay sediment. One of the reactors at 308C (R2) was
pump (Watson Marlow 505S, Cornwall, UK). The mean additionally inoculated with 1.0 gdry weight Eerbeek sludge
pore size of 0.2 mm guaranteed complete cell retention. The and 1.0 gdry weight Nedalco sludge. During the first 330 days,
transmembrane pressure was monitored using a pressure 0.5 mM acetate was added to the medium, the volumetric
sensor (Sensortechnics, Puchheim, Germany). Due to acetate loading rate was 70 mmol LÀ1 dayÀ1. From
weekly manual back flushing, the transmembrane pressure day 330 onwards, CH4 was the sole electron donor and
remained below 20 kPa. The effluent pump was controlled carbon source. A fourth reactor was started 18 months later,
by a level switch (Electronics ATV, Wageningen, The ¨
it was inoculated with 20 gdry weight Eckernforde Bay
Netherlands), which kept the liquid volume at 1.0 L. Each sediment and operated at 158C with CH4 as sole electron
reactor was equipped with a water-jacket, through which donor and carbon source from the start onwards. Table I
water, cooled or heated in a thermostatic water bath (Julabo, shows the differences, in inoculation and operation of the
Seelbach, Germany) was recirculated to maintain a constant four bioreactors. The influent pumps, mass flow meters,
temperature of 15 (Æ1)8C or 30 (Æ1)8C in the reactor, pH-electrodes and gas flow meters were checked every
measured with a PT-100 electrode. The pH was monitored 2 months and recalibrated when needed. The sulfate
with a sulfide resistant Hamilton flushtrode pH-electrode and sulfide concentrations of the influent and effluent
(Reno, NV) connected to a pH monitor (Electronics ATV, (supernatant of the membranes), and the sulfide concen-
Wageningen, The Netherlands). The pH was maintained at tration in the wash bottle, were analyzed approximately
7.2 (Æ0.2) by means of the phosphate buffer in the medium every 3 weeks. Samples of the reactor suspension were taken
and manual addition of diluted hydrochloric acid (1 M). for activity assays, DNA isolation and quantification, and
CH4 gas (Praxair, Danbury, CT), with a purity of volatile suspended solids (VSS) and total suspended solids
99.9995%, was supplied via a gas sparger at the bottom of (TSS) analysis.
the reactor. This was done to supply methane to the micro-
organism, to promote reactor mixing, to strip off the sulfide
and to prevent fouling of the membrane surface (Chang
et al., 2002). The influent CH4 flow was measured
Activity Assays
and controlled at a gas loading rate of 4.8 L LÀ1 dayÀ1
(196 mmol LÀ1 dayÀ1) by a thermal mass flow controller CH4 oxidation rates were estimated from the 13C-
(MFC) type 5850E (Brooks, Veenendaal, The Netherlands). labeled CO2 (13CO2) production rate during batch incuba-
The gas with the hydrogen sulfide (H2S) and carbon dioxide tions with sampled reactor suspension and 13C-labeled CH4
(CO2) stripped from the liquid, left the reactor via two gas (13CH4). After determination of the exact weight and
cleaning bottles and a gas flow meter (Ritter, Bochum, volume of the 35-mL serum bottles, they were closed with
Germany). The first bottle (1 L) collected reactor liquid that butyl rubber stoppers and caps, and the gas phase was
was eventually transported with the gas out of the reactor. replaced eight times with nitrogen gas and made vacuum
The second bottle (1 L) was filled with a 0.5 M zinc chloride thereafter. Subsequently, 20 mL sampled reactor suspension
solution to selectively retain the H2S, and was placed on a was transferred into the bottles, using a syringe and a
magnetic stirrer. The sulfide concentration (including the hypodermic needle (internal diameter of 0.2 mm). To
precipitated zinc sulfide) in the bottle was measured once ensure homogeneous sampling of the reactor suspension,
every 2 weeks. The zinc chloride solution was replaced when the gas sparging rate in the reactors was temporally increased
the sulfide concentration reached 10–15 mM. The over- to 1 L minÀ1. After day 420, the sample withdrawn from R3
pressure in the headspace of the MBRs was 25 mbar. To was diluted with fresh medium, the dilution factor was the
provide additional mixing and to suspend the sediment/ last obtained rate divided by 50 mmol LÀ1 dayÀ1; the exact
biomass, the reactor suspension was recirculated from top to amounts of added reactor suspension and medium were
bottom at a rate of 0.3 L minÀ1. determined by weighing. Subsequently, the headspaces of
the bottles were made vacuum again and filled with pure
13
CH4 gas (Campro, Veenendaal, The Netherlands). The
Operation of the Membrane Bioreactors
bottles were incubated in an orbital shaker (rotating at
Initially, three reactors were started, two at 308C and one at 100 rpm) at the operation temperature of the source reactor.
158C, all three were inoculated with 10 gdry weight Eckern- Weekly, 100 mL headspace samples were taken for gas
Table I. Inoculation and operational conditions of the MBRs used in this study.
Inocula Inoculation date Duration run (days) Temperature (8C) 70 mmol LÀ1 dayÀ1 acetate
R1 ¨
10 gdry weight Eckernforde Bay sediment 15-8-2005 520 30 Till day 330
R2 ¨
10 gdry weight Eckernforde Bay sediment and 2 gdry weight 15-8-2005 520 30 Till day 330
Methanogenic biomass
R3 ¨
10 gdry weight Eckernforde Bay sediment 15-8-2005 884 15 Till day 330
R4 ¨
20 gdry weight Eckernforde Bay sediment 18-1-2007 355 15 None
Meulepas et al.: Enrichment of Anaerobic Methanotrophs 461
Biotechnology and Bioengineering

5. analysis (12CH4, 13CH4, 12CO2, and 13CO2). In addition, the Nanodrop spectrophotometer. The 16S rRNA gene was
headspace pressure, liquid and gas volume and pH were amplified from genomic DNA by PCR using the archaea-
measured. specific forward primer 4F (50 -TCCGGTTGATCCTGC-
To investigate the stoichiometry in batch, activity assays CRG-30 ) and the universal prokaryotic reverse primer 1492R
were done with reactor suspension sampled from R3 diluted (50 -CGGTTACCTTGTTACGACTT-30 ). 16S rRNA gene
with medium with a reduced sulfate concentration (2 mM). PCR was performed in a G-storm cycler (G-storm, Essex,
The headspace contained not-labeled CH4 or N2. Liquid UK) starting with 2 min at 948C, followed by 35 cycles of
samples were taken and used for sulfide and sulfate analyses, 948C for 30 s, 528C for 40 s, and 728C for 1.5 min. The final
after filtering over a 0.2 mm cellulose acetate membrane filter PCR extension step was at 728C for 5 min. PCR products
(Schleicher Schuell OE 66, Schleicher Schuell, Dassel, were ligated into pGEM-T (Promega Benelux BV, Leiden,
Germany). The Netherlands) and transformed into E. coli XL1-blue cells
(Stratagene, La Jolla, CA) as specified by the manufacturer.
For screening of the gene library by denaturing gradient gel
Analyses electrophoresis (DGGE), 10 mL of the overnight cultures of
the clones were mixed with 90 mL of TE and lysed for 10 min
Sulfide was measured photometrically using a standard kit
at 958C. Four hundred base pairs 16S rDNA gene fragments
(LCK 653) and a photo spectrometer (Xion 500) both
were amplified from 1 mL of the lysed clones using the
from Hach Lange (Dusseldorf, Germany). This method
primer pair A109T-F (ACT GCT CAG TAA CAC GT,
accounted for all dissolved sulfide species (H2S, HSÀ,
original Grosskopf et al. (1998) but with a third nucleotide
and S2À) and, if no filtration or centrifugation was applied,
changed into T) plus 515R (ATC GTA TTA CCG CGG CTG
precipitated sulfide (e.g., the ZnS in the wash bottle). Sulfate
CTG GCA, Lane, 1991) with a GC clamp (Muyzer et al.,
was measured on a DX-600 IC system (Dionex Corporation,
1993). The DNA clean and concentrator-5 kit (Zymo
Salt Lake City, UT) as described previously (Sipma et al.,
research, Orange, CA) was used for the purification and the
2004). Acetate was analyzed on a HP 5890A gas chromato-
DNA fragments were partially sequenced commercially
graph (Hewlett Packard, Palo Alto, CA) according to
(400–740 bp) by BaseClear (Leiden, The Netherlands).
Weijma et al. (2000).
The headspace composition was measured on a gas
chromatograph-mass spectrometer (GC-MS) from Inter-
Denaturing Gradient Gel Electrophoresis
science (Breda, The Netherlands). The system was com-
posed of a Trace GC equipped with a GS-GasPro column DGGE analysis was directly performed on extracted DNA
(30 m by 0.32 mm; JW Scientific, Folsom, CA), and a Ion- from the submerged membrane bioreactor. Four hundred
Trap MS. Helium was the carrier gas at a flow rate of base pair fragments of 16S rRNA genes were amplified by
1.7 mL minÀ1. The column temperature was 308C. The PCR using the universal archaeal primer pairs A109T-F plus
fractions of CH4 and CO2 in the headspace were derived 515R-GC clamp (as described above). DGGE was performed
from the peak areas in the gas chromatograph. The fractions by following a published protocol (Muyzer et al., 1998); the
of 13C-labeled CH4 (13CH4) and 13C-labeled CO2 (13CO2) temperature was 608C, the denaturant (urea and forma-
were derived from the mass spectrum as done by Shigematsu mide) gradient was 30–60%, the electrophoresis time was
et al. (2004), the method was checked using standards with 16 h, and the voltage was 85 V. Gels were stained with
known mixtures of 12CO2, 13CO2, 13CH4, and 12CH4. silver according to Sanguinetty et al. (1994) with minor
The pressure in the bottles and tubes was determined modifications. Selected DGGE bands were excised. The
using a portable membrane pressure unit, WAL 0–0.4 MPa DNA was extracted in 25 mL of TE buffer and incubated
absolute (WalMess- und Regelsysteme, Oldenburg, Ger- overnight at 378C. One microliter of DNA was reamplified
many). The pH was checked by means of pH paper with the same primers and sequenced commercially by
¨
(Macherey-Nagel, Duren, Germany). The VSS and TSS BaseClear.
content of the reactor suspension and the dry weight content
of the inocula were analyzed according to standard methods
(American Public Health Association, 1995). The VSS is Phylogenetic Analyses
obtained from the difference between dry weight (TSS) and
Partial sequences were processed using the DNASTAR
ash weight of the solids separated from the liquid by
Lasergene 6 package (Madison, WI) and verified by BLASTN
filtration.
(Altschul et al., 1997), possible chimerical sequences were
checked using the Pintail program (Ashelford et al., 2005).
The phylogenetic affiliation of the novel clones was deduced
DNA Isolation and Quantification
by means of BLASTN analyses (http://blast.ncbi.nlm.nih.
DNA was extracted from the reactor suspension using the gov). Identical migration patterns in DGGE were used to
FastDNA SPIN for Soil Kit (MP Biomedicals, Solon, OH). cluster the clones. Corrected sequences from representative
The extracted DNA was purified with the NucleoSpin PCR clones were deposited in GenBank (www.ncbi.nlm.nih.gov;
purification kit (Macherey-Nagel) and quantified with a accession numbers FJ210915 and FJ210925).
462 Biotechnology and Bioengineering, Vol. 104, No. 3, October 15, 2009

6. Calculation of Volumetric Activities kept below 7.6. Before day 590 and in the other three
reactors the pH remained between 7.2 and 7.5.
The volumetric sulfate removal, sulfide production and
acetate removal are calculated according to (for symbols and
abbreviations see Nomenclature):
Conversion Rates and Stoichiometry
ÀÂ Ã Â ÃÁ
SO42À influent À SO42À effluent Figure 2 presents the volumetric sulfide production, sulfate
Sulfate removal rate ¼
HRT removal, AOM and acetate consumption rates of the
4 MBRs in time. Three phases can be distinguished. During
Sulfide production rate the first phase (phase I), the SR rates were higher than the
ð½sulfideeffluent Š À ½sulfideinfluent ŠÞ AOM and acetate consumptions rates. Immediately after
¼ start-up, the sulfate removal and sulfide production rate
HRT
D½sulfidegaswash Š Â Vgaswash were relatively high, between 0.1 and 0.6 mmol LÀ1 dayÀ1,
þ but then over the course of a few weeks the SR rates dropped
Dt
and stabilized around 0.07 mmol LÀ1 dayÀ1. During phase I,
ð½Acetateinfluent Š À ½Acetateeffluent ŠÞ SRB are able to utilize substrates that were present in the
Acetate removal rate ¼
HRT inoculum or became available by decay of biomass. This
P13 endogenous activity dropped after the readily available
The absolute amount of CO2 (¼gaseous 13CO2,
13 13 endogenous organic compounds were depleted.
dissolved CO2, and C-labeled bicarbonate) in the activity
In the acetate-fed MBRs (R1, R2, and R3), a subsequent
assay bottles was plotted against time, the volumetric AOM
P phase can be distinguished (phase II) in which acetate is
rate was obtained from the D 13CO2/Dt over the period in
completely removed and during which sulfate removal,
which the increase was linear, at least four successive data
sulfide production and acetate consumption rates are almost
points were used.
equal, circa 0.07 mmol LÀ1 dayÀ1. AOM rates during phase
X
13 13 Vgas þ Vliquid II were at least five times lower. The dominant process in the
CO2 ¼f CO2 Â P reactors in this period was, therefore, sulfate reduction with
k  ð1 þ Kz =½H þ ŠÞ
acetate according to Equation (2).
P
ðD 13 CO2 =DtÞ
AOM rate ¼
Vinoculum CH3 COOÀ þ SO42À ! 2HCO3 þ HSÀ
À
(2)
DG ¼ À47 kJ molÀ1
Results Acetate was omitted from the feed of R1, R2, and R3 from
day 330 onwards, CH4 was thus the only available electron
Reactor Operation
donor and carbon source in this period (phase III). To R4,
The MBRs were kept anaerobic during operation and the no acetate has been added to the feed at all, therefore phase I
biomass was retained. To check if biomass was washed-out, is followed by phase III. In the reactors operated at 158C
100 mL effluent was monthly collected and centrifuged (R3 and R4), the sulfate removal, sulfide production and
at 32G. However, no pellet could visually be detected. AOM rates are coupled during phase III, according to
Moreover the transmembrane pressure in all four bior- Equation (1). Also in the activity assays done with
eactors remained between 15 and 20 kPa, which confirmed reactor suspension from R3, taken during phase III,
that the membranes were not leaking. During the 884 days of simultaneous CH4 and sulfate consumption was accom-
P
operation, the feeding, mixing, heating and/or cooling were panied by CO2 and sulfide production, according to
five times shortly interrupted (maximum 48 h). This was Equation (1) (Fig. 3a). In control incubations with nitrogen
because of power failure, equipment failure or the depletion gas instead of CH4 in the headspace, no conversion was
of CH4 gas or medium. During these interruptions, the observed (Fig. 3b). During the entire incubation, the AOM
redox potential of the reactor suspension always stayed rates obtained from activity assays, increased from 0.004 to
below À50 mV (at which the liquid would become pink 0.60 mmol LÀ1 dayÀ1 in 884 days for R3, and from 0.008 to
because of the present rezasurin). Also when the mem- 0.19 mmol LÀ1 dayÀ1 in 280 days for R4 (Fig. 2c and d).
branes, sparging stones or electrodes were cleaned or Figure 4a shows that the sulfate concentration in the
replaced, which was done under a nitrogen flow, the redox effluent of R3 decreased over time and the dissolved sulfide
potential stayed below that value. In all four reactors, concentration increased. A minimum sulfate concentration
the CH4 gas sparging and the recirculation of the reactor of 15.7 mM and a maximum sulfide concentration of
suspension were sufficient to keep all solids in suspension. 1.9 mM were reached.
The phosphate buffer in R3 was, after day 590, not sufficient In the reactors operated at 308C (R1 and R2), the sulfate
to cope with the increased alkaline production. By manual removal rate and sulfide production rate during phase III
dosing of hydrochloric acid two times a week the pH was were always below 0.01 mmol LÀ1 dayÀ1 (Fig. 2a and b).
Meulepas et al.: Enrichment of Anaerobic Methanotrophs 463
Biotechnology and Bioengineering

7. Figure 2. Volumetric conversion rates over time of four reactors inoculated with Eckernforde Bay sediment, R1 (a) and R2 (b) both operated at 308C, and R3 (c) and R4 (d),
¨
operated at 158C. R2 was additionally inoculated with anaerobic granular sludge. Symbols indicate: sulfide production rate (Â), sulfate removal rate (), AOM rate (*) and acetate
consumption (~). Three phases can be distinguished in R1, R2, and R3 and two in R4: during phase I endogenous organic matter from the inoculum was fueling sulfate reduction,
during phase II 0.07 mmol LÀ1 dayÀ1 (0.5 mM) acetate was added besides CH4, during phase III CH4 was the sole electron and carbon source.
There was no increase in AOM or sulfate reduction over a biomass. The decrease in solids can be explained by the
period of 640 days, after which the reactors where stopped. frequent sampling of reactor suspension for chemical
analyses and activity assays, in totally 2.3 L was sampled
during the 884-day incubation (dilution factor !3.3), and
by the decomposition of particulate organic matter present
Biomass Concentration and Composition of R3
in the inoculum (e.g., inactive and dead biomass). The
To assess which microorganisms are responsible for the products of particular organic mater decomposition can be
150-fold increase in AOM rate in R3, the biomass used as electron donor for SR, this resulted in the relative
concentration and composition were analyzed. Just after high SR rate during phase I (Fig. 2). Maximum 8 mmol
inoculation the TSS and VSS content in R3 were 8.4 and sulfate can be reduced from the 0.5 g VSS that was lost
1.1 g LÀ1, respectively. After 884 days the TSS and VSS during the experiment, when it is assumed that the average
content in R3 had decreased to 2.1 g LÀ1 and 0.59 g LÀ1 molecular structure of the particular organic matter
respectively. The DNA concentration in R3 also decreased is CH2O. These 8 mmol form only 3.2% of the total
over time (Fig. 4b), despite the exponential increase of the amount of sulfate that was reduced during the 884 days of
AOM and SR rate. The potential growth of microorganisms incubation. Therefore endogenous SR could not have
mediating AOM and SR did not result in a net increase in contributed significantly to SR in R3 during phase III.
464 Biotechnology and Bioengineering, Vol. 104, No. 3, October 15, 2009

8. P
Figure 3. Stochiometric sulfide production (Â), sulfate consumption (), CO2 production (*), and CH4 consumption (D) in 35-mL batch bottles containing 20 mL marine
medium with 2.5 mM sulfate and a headspace of 0.15 MPa CH4 (a) or 0.15 MPa nitrogen (b). The bottles were inoculated with 2.5 mL reactor suspension taken 760 days after start-up
from R3 and incubated shaken at 158C.
The biomass was mainly present as small flocks (up to Bay in early September: a temperature between 10 and 228C
0.1 mm in diameter). When the reactor suspension is left and a salinity between 14 and 19% (Treude et al.,
undisturbed, the flocks agglomerated to bigger flocks and 2005). However, in contrast to the in situ situation, the
settled at velocities between 16.8 and 3.4 m hÀ1. microorganisms in the bioreactors were continuously
Table II shows an overview of the clone library of the exposed to high shear forces, due to the liquid recirculation
archaea obtained from the biomass in R3, 809 days and gas sparging, and were suspended in the liquid
after inoculation. Ninety one percent of the obtained phase. Another difference was that gaseous and dissolved
archaeal clones had similarities with ANME-2a sequences compounds were continuously stripped out, due to the gas
found by BLASTN analysis (http://blast.ncbi.nlm.nih.gov). sparging, or washed out of the reactors system with
The DGGE scan of different samples of R3 in time the effluent. These issues are of importance if AOM is a
show the proliferation of two bands at the bottom of syntrophic conversion, in which an intermediate compound
the DGGE gel (Fig. 5), of which the sequences had high is transported between the partners (DeLong, 2000).
similarity with ANME-2a related clones (Band A). Ninety six Syntrophic partners could get separated due to the break
percent (300 bp) with clone SBAK-mid-10 (DQ522915) up of the sediment-biomass matrix under conditions of high
and Band B 92% (253 bp) with clone Hyd24-Arch25. shear forces. In addition, intermediate compounds could be
(AJ578107). transported away before reaching the syntrophic partner.
However, this study demonstrates that liquid recirculation,
gas sparging and a hydraulic retention time of 7 days did
not prevent the exponential development of the AOM
Discussion rate. Because these features help to prevent mass transfer
limitation, high volumetric conversion rates can be
Bioreactor System
obtained. This research opens possibilities for a biotechno-
In Figure 4c, the AOM, sulfate removal and sulfide logical sulfate reduction process with CH4 as electron donor.
production rates of R3 are shown on a logarithmic scale, The volumetric rate that was obtained in this study
during the 884 days of incubation the AOM rate increased (0.6 mmol LÀ1 dayÀ1), is still too low for application. In a
exponential, corresponding to a doubling time of 3.8 months full-scale sulfate-reducing bioreactor fed with hydrogen as
(R ¼ 0.99). In this way a very active enrichment was electron donor, a maximum volumetric sulfate reduction
obtained (1.0 mmol gÀ1 dayÀ1). The used submerged-
VSS rate of 175 mmol LÀ1 dayÀ1 has been reached (Weijma
membrane bioreactor system was therefore an excellent et al., 2002). However, the volumetric methane oxidation
system for growing the microorganisms mediating AOM and sulfate reduction rates can be increased further by
coupled to SR. The temperature, pH and salinity in this increasing the biomass concentration in the bioreactor,
reactor (158C, 7.5 and 30% respectively) were comparable MBR’s can operated at suspended solid concentrations up
¨
with conditions found at the sampling site in Eckernforde to 31 gdry weightÀ1 LÀ1 (Stephenson et al., 2000).
Meulepas et al.: Enrichment of Anaerobic Methanotrophs 465
Biotechnology and Bioengineering

9. study is that inactive and dead cells will not wash-out with
the effluent. The bands in the DGGE gel (Fig. 5) that were
not linked to ANME could be attributed to methanogens
that were already present in the original Eckernforde ¨
Bay inoculum. Despite the presence of these inactive
microorganisms, over 90% of the partial sequences (N ¼ 90,
400–750 bp) of the archaeal clones cluster in the ANME-2a
subgroup. Especially in samples taken from R3 in the period
that AOM coupled to SR was the only conversion taking
place (phase III) clearly showed the dominance of ANME-2a
clones in the archaeal clone library and in the sequences
from DGGE bands. Indicating that ANME-2a were involved
in the exponential increase in AOM coupled to SR. ANME-
¨
2a were also detected in the original Eckernforde Bay
sediment by Treude et al. (2005). ANME have been shown to
directly consume CH4 (Orphan et al., 2001) and to have
enzymes that can play a role in reversed methanogenesis
(Hallam et al., 2004). However, ANME have not been
shown to be capable of sulfate reduction nor to possess
enzymes involved in SR (Thauer and Shima, 2008).
Therefore, further research is required to unravel the
AOM pathway in the obtained enrichment. The bacterial
composition of the active biomass in the bioreactor is not
unraveled yet, but further research will focus on the
quantitative and phylogenetic aspects of these sulfate-
reducing bacteria.
SR and AOM did not increase in both reactors operated
at 308C, the presence of granular sludge in R2 did not
affect the outcome. The sulfate reduction in R2 during
phase I was slightly higher than in the other reactors
though, probably due to a higher fraction of readily
available organic matter in the sludge compared to the
sediment.
Doubling Time
Girguis et al. (2003, 2005), Nauhaus et al. (2007), and
¨
Kruger et al. (2008) also showed in vitro growth of anaerobic
Figure 4. The dissolved sulfide (Â) and sulfate () concentrations (a), the methanotrophs (Table III). The difference in reported
DNA concentration (b) and the volumetric sulfide production (Â), sulfate removal ()
and CH4 oxidation (*) rates on logarithmic scale (c) over time for a membrane doubling times can be related to the inocula that were
¨
bioreactor inoculated with 10 gdry weight Eckernforde Bay sediment, continuously fed used. However, there were also differences in incubation
with CH4 and sulfate and controlled at 158C (R3). conditions (e.g., CH4 partial pressure and temperature) and
techniques. One important difference is that the relative
short doubling times found by Girguis et al. (2005) were
obtained with sediment in which the AOM rates were low,
while Nauhaus et al. (2007) found much slower growth
using the active Hydrate Ridge sediment. For this study,
Responsible Microorganisms
both initial rates and doubling times were in between
The exponential increase in activity in R3 (Fig. 4c) indicates reported values, but growth did not slow down when rates
growth. However, VSS content and DNA concentration exceeded those of the Hydrate Ridge sediment. Further
(Fig. 4b) decreased over time. This decrease indicates that research should clarify which parameters are critical to
the original sediment contained many organisms not obtain optimal growth. An important difference in the
involved in the exponential increase in AOM coupled to approach of this research with those of others is that the aim
SR and that these organisms were slowly decaying. One was not to mimic the natural conditions, but to apply
aspect of the submerged-membrane bioreactor used in this conditions that allowed high conversion rates. The AOM
466 Biotechnology and Bioengineering, Vol. 104, No. 3, October 15, 2009

10. ¨
Table II. Phylogenetic summary based on clone library analysis of partial archaeal sequences from the MBR inoculated with Eckernforde Bay sediment and
operated at 158C for 809 days (R3).
Sequence with
Representative highest similarity in Genbank
clone Accession no. No. of clones (BLASTN) with accession no. Identity (%) Putative taxon
R3-1A3 FJ210916 27 Clone fos0642g6 (CR937012) 99 ANME2a
R3-1A2 FJ210915 23 Clone Hyd24-Arch25 (AJ578107) 99 ANME2a
R3-1A11 FJ210917 22 Clone GoM_GC232_4463_Arch65 (AM745238) 99 ANME2a
R3-1E5 FJ210918 8 Clone IV.4.Ar15 (AY367329) 99 ANME2a
R3-1D10 FJ210919 2 Clone SBAK-mid-74 (DQ640234) 99 ANME2a
R3-1B6 FJ210920 2 Clone WHA34-14 (AB426391) 95 Methanococcoides
R3-1G4 FJ210921 2 Methanomicrobiales archaeon ‘‘SBAK-CO2-reducing 99 Methanomicrobiales
Enrichment-4’’ (DQ280485)
R3-1A6 FJ210922 1 Clone MOB7-2 (DQ841237) 98 Methanosarcinales
R3-1E8 FJ210923 1 Uncultured euryarchaeote EHB95 (AF374283) 97 Methanosarcinales
R3-1F5 FJ210924 1 Clone SBAK-mid-25 (DQ522923) 96 Marine Benthic group-D
R3-1H9 FJ210925 1 Clone ss017b (AJ969786) 91 Thermoplasmatales—related group
Similarity to nearest neighbor in the GenBank nucleotide database as determined by BLAST results. A similarity of 100% indicates that the sequences were
indistinguishable.
activity of the enrichment obtained in this research is the operated at 308C (Fig. 2a and b). However there was some
highest reported so far (Table III). AOM activity at 308C (Fig. 2a and b). AOM by the original
¨
Eckernforde Bay sediment was only slightly lower at 288C
than at 208C (at which the highest rates were obtained)
Temperature (Treude et al., 2005). Despite the initial activity at 308C,
the responsible organisms were not able to grow at this
The AOM and SR rates increased in both reactors operated
temperate.
at 158C (Fig. 2c and d). In contrast, the AOM activity and
the SR during phase III, did not increase in the two reactors
AOM Activity Assays
P
The AOM rates are estimated from the 13CO2 production
in batch bottles to which only pure 13CH4 and P sampled
12
reactor suspension were added. Initially also CO2
was produced due to decomposition of particulate
organic matter. As the endogenous activity dropped, the
fraction 13CO2 became higher. The natural isotopic
signature of particulate organic matter is approximately
1.07% 13C, therefore the decomposition contributed to the
P13 P
CO2 formation. However, because the fraction 13CO2
of the total CO2 production was always at least 10 times
higher than the natural isotopic signature, this contribution
was neglected.
The AOM rate presented isP net 13CH4 oxidation rate,
the
thus the 13CH4 oxidation to 13CO2 minus the backward
P
reaction ( 13CO2 reduction). This CO2 reduction during
AOM might be similar to observed methane oxidation
during methanogenesis (Harder, 1997; Zehnder and
Brock, 1979). Treude et al. (2007) showed that in Black
sea sediments the CO2 reduction rate was about 10% of the
methane oxidation rate.
Figure 5. Analysis of changes in archaeal community over time, by 16S rRNA
gene-targeted PCR-denaturing gradient gel electrophoresis, in a MBR inoculated with ¨
Kruger et al. (2008) reported that the AOM rates are
¨
Eckernforde Bay sediment, fed with CH4 and sulfate and operated at 158C (R3) of a reduced by 30–80% if the fraction 13CH4 (of the total CH4)
¨
membrane bioreactor inoculated with Eckernforde Bay sediment, continuously and exceeds 25%. This was probably due to the inability of the
controlled at 158C (R3). The sequences obtained from bands A and B can be affiliated
with clones from the ANME-2a cluster. microbial community to use sufficient amounts of 13CH4 as
substitute for 12CH4 to sustain viability (Kruger et al., 2008).
¨
Meulepas et al.: Enrichment of Anaerobic Methanotrophs 467
Biotechnology and Bioengineering

11. Table III. Comparison of the doubling times and maximum AOM conversion rates found in different enrichment experiments with marine sediments.
Origin inoculum Monterey Bay Hydrate Ridge Golf of Mexico ¨
Eckernforde Bay
Incubation technique Continuous, prop flow Fed-batch, not mixed Batch, shaken Continuous, well-mixed
ones a week
CH4 partial pressure (during incubation) 1.5 mM (%0.1 MPa) 1.4 Mpa 1.5 Mpa 0.10 Mpa
Incubation temperature 58C n.r. 128C 158C
Involved microorganisms ANME-1, ANME-2, Consortia ANME-1 dominated ANME-2a, dominated
and SRB of ANME-2 and SRB
Estimated doubling time (months) 1.1 (ANME-2), 1.4 (ANME-1) 7.5 2 3.8
Maximum AOM rate (mmol g dry weightÀ1 dayÀ1) 0.1 230 13.5 286
Maximum AOM rate (mmol g VSSÀ1 dayÀ1) n.r. n.r. n.r. 1.0
References Girguis et al. (2005) Nauhaus et al. (2007) ¨
Kruger et al. (2008) This study
n.r., not reported.
In this study, the activity assays were performed with main electron donor for sulfate reduction. However, it
100% 13CH4 in the headspace, this was done to more directly cannot be excluded that some acetate was converted to CH4
and accurately quantify AOM. The reactors on the other and an equal amount of CH4 was used for sulfate reduction.
hand were fed with unlabeled CH4. An inhibitory effect of
13
CH4 will therefore result in a discrepancy between the
AOM rate obtained from the activity assays and the sulfate Nomenclature
removal and sulfide production achieved in the MBRs f fraction
(during phase III). However, the AOM rates are not HRT hydraulic retention time
systematically lower than sulfate removal and sulfide k Henry’s law constant for CO2 at sampling temperature (208C):
production rates (Fig. 2c). If the inhibitory effect of 0.0388 mol LÀ1
13
CH4 was due to a loss in viability of the cells, the effect can Kz dissociation constant of dissolved CO2 þ H2O: 4.5 Â 10À7
be expected to be less profound during the relative short P pressure
activity assays done in this research. t time
At the start of phase III, the coupling between sulfate TSS total suspended solids
removal, sulfide production and AOM is poor (Fig. 4c). This Vgas gas volume in serum bottle for activity assay
was related with a less accurate quantification of the sulfide Vgaswash liquid volume in gas wash bottle
production and sulfate removal, due to the small differences Vinoculum volume reactor suspension used for inoculation
between influent and effluent concentrations in this phase. Vliquid liquid volume in serum bottle for activity assay
The AOM rate in R3 just after start up was VSS volatile suspended solids
0.5 mmol gdry weightÀ1 dayÀ1. Treude et al. (2005) found AOM [X] molar concentration of compound X
activities between 0.1 and 0.3 mmol gdry weightÀ1 dayÀ1 with
sediment sampled 3 years earlier at the same site
¨
(station B in Eckernforde Bay). The difference between
the rates could be the result of growth prior to reactor
This work was part the Anaerobic Methane Oxidation for Sulfate
inoculation, as the wet sediment was stored for 69 days at Reduction project (AMethOx for SuRe, number EETK03044)
48C in an unshaken bottle with 100% CH4 in the headspace. supported by the Dutch ministries of Economical affairs, Education,
culture and science and Environment and special planning as
part their EET (Economie, Ecologie, Technologie) program. Anna
Lichtschlag and Tina Treude from the MPI-Bremen are acknowledged
Acetate as Co-Substrate ¨
for providing access to the Eckernforde Bay sediment. We thank the
¨
crew of the LITTORINA from the Leibniz-Institut fur Meereswis-
Acetate was initially fed to the reactors, in addition to CH4, senschaften for their excellent support with the sediment sampling.
in order to obtain and maintain sulfate-reducing conditions.
Another reason to add acetate was to supply a carbon source,
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