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 Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

A metaproteomic approach gives functional insights into

anaerobic digestion
F. Abram1, A.-M. Enright2, J. OReilly1, C.H. Botting3, G. Collins2 and V. OFlaherty1
1 Microbial Ecology Laboratory, Department of Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland
2 Microbial Ecophysiology Research Group, Department of Microbiology, School of Natural Sciences, National University of Ireland,
Galway, Ireland
3 Centre for Biomolecular Sciences, University of St Andrews, St Andrews, Fife, UK

Keywords
metabolic processes, microbial phylogenetics,
proteomics, sludge, wastewater treatment.
Correspondence
Vincent OFlaherty, Microbial Ecology
Laboratory, Department of Microbiology,
School of Natural Sciences, National University
of Ireland, University Road, Galway, Ireland.
E-mail: vincent.oflaherty@nuigalway.ie
2010 1925: received 26 October 2010,
revised 9 March 2011 and accepted 19 March
2011
doi:10.1111/j.1365-2672.2011.05011.x
Abstract
Aims: The objective of this work was to provide functional evidence of key
metabolic pathways important for anaerobic digestion processes through the
identification of highly expressed proteins in a mixed anaerobic microbial
consortium.
Methods and Results: The microbial communities from an anaerobic indus-
trial-like wastewater treatment bioreactor were characterized using phylogenetic
analyses and metaproteomics. Clone libraries indicated that the bacterial
community in the bioreactor was diverse while the archaeal population was
mainly composed of Methanocorpusculum-like (76%) micro-organisms. Three
hundred and eighty-eight reproducible protein spots were obtained on 2-D
gels, of which 70 were excised and 33 were identified. The putative functions
of the proteins detected in the anaerobic bioreactor were related to cellular
processes, including methanogenesis from CO2 and acetate, glycolysis and the
pentose phosphate pathway. Metaproteomics also indicated, by protein assign-
ment, the presence of specific micro-organisms in the bioreactor. However,
only a limited overlap was observed between the phylogenetic and metaproteo-
mic analyses.
Conclusions: This study provides some direct evidence of the microbial activi-
ties taking place during anaerobic digestion.
Significance and Impact of Study: This study demonstrates metaproteomics as a
useful tool to uncover key biochemical pathways underpinning specific anaerobic
bioprocesses.

wastes are discharged at ambient, or low, temperatures,

Introduction
Anaerobic digestion (AD) has become an essential and
well-established technology for the treatment of a wide
range of wastes and wastewaters. The natural process of
AD is driven by intricate, poorly characterized, microbial
interactions, which can be applied for the conversion of
complex unwanted biodegradable organic molecules into
a renewable source of energy (McCarty 2001). Because of
anaerobic microbial growth characteristics, reaction rates
and temperature optima, most full-scale, anaerobic, indus-
trial wastewater treatment systems are operated at temper-
atures higher than 18C (Lettinga et al. 2001; Hulshoff Pol
et al. 2004). However, the vast majority of industrial
which results in increased heating costs necessary to main-
tain mesophilic treatment. Therefore, low-temperature
anaerobic bioreactors represent an attractive and cost-
effective alternative. The success of laboratory-scale, low-
temperature anaerobic digestion (LTAD) has been well
documented for a wide range of food-processing, phenolic
and other industrial wastewaters (Kato et al. 1994, 1999;
Rebac et al. 1995, 1999; van Lier et al. 1997; Lettinga et al.
1999; Connaughton et al. 2006; Enright et al. 2007; Scully
et al. 2007; OReilly et al. 2009).
Despite the widespread application of AD, as well as
emerging LTAD technology development, the micro-
organisms involved have not yet been fully characterized.

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F. Abram et al.
Indeed, little is known about the functional activities of
many of the abundant groups found in anaerobic biofilms
(sludge granules) from AD bioreactors. Consequently, the
performance and optimization of AD processes remain
limited. Up to now, much effort has been made to eluci-
date the microbial community structure of consortia in
anaerobic bioreactors (Godon et al. 1997; Leclerc et al.
2001, 2004; Roest et al. 2005; Lee et al. 2009; McKeown
et al. 2009; OReilly et al. 2009). However, only limited
data are available on the links between diversity and
microbial functionality in anaerobic bioreactors.
In this study, the microbial communities of an anaerobic
bioreactor treating a synthetic, glucose-based wastewater at
15C for 300 days (OReilly et al. 2010) were characterized
using clone libraries and metaproteomics. The functions of
the proteins recovered from the bioreactors have been
attributed based on homologies to other proteins, and
their possible contributions to the biology of AD are
discussed. Protein assignment also indicated the presence
of specific micro-organisms in the bioreactor, and this was
compared with the results of the phylogenetic analysis.
Materials and Methods
Source of biomass
The anaerobic bioreactor, which was the source of the bio-
mass investigated in this study, was previously described
by OReilly et al. (2010). Briefly, the bioreactor had a
working volume of 35 l and was operated at 15C for
300 days to treat a synthetic, glucose-based wastewater,
which was buffered at pH 71 with NaHCO3. Samples of
sludge granules for phylogenetic and metaproteomic anal-
yses were obtained at the conclusion of the trial (day 300).
DNA extraction
Total DNA was extracted from granular biomass using an
automated nucleic acid extractor (Magtration 12 GC; PSS
Co., Chiba, Japan) following the manufacturers instruc-
tions. Prior to extraction, the biomass (05 g wet weight)
was finely crushed using a mortar and pestle and resus-
pended in 50 ml of deionized, distilled water. A 100-ll
aliquot of the biomass suspension was loaded per
extraction, and the extracted DNA was eluted in Tris
HCl buffer (pH 80) and stored at )20C. All extractions
were carried out in duplicate.
Generation of 16S rRNA gene clone libraries, sequencing
and phylogenetic analysis
Partial bacterial 16S rRNA genes were amplified with for-
ward primer 27F (5-AGAGTTTGATCCTGGCTCAG-3;
2011 The Authors
Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology
Metaproteomics of anaerobic sludge
Delong 1992) and reverse primer 1392R (5-ACGGGC-
GGTGTGTTRC-3; Lane et al. 1985). Partial archaeal 16S
rRNA genes were amplified with forward primer 21F
(5-TTCCGGTTGATCCYGCCGGA-3; Stackebrandt and
Goodfellow 1991) and reverse primer 958R (5-YCCGG-
CGTTGAMTCCAATT-3; Delong 1992). The concentra-
tions of the PCR components and the PCR conditions
were as outlined by Collins et al. (2003). Cloning
(TOPO XL; Invitrogen, Carlsbad, CA), restriction frag-
ment length polymorphism (RFLP) pattern analysis and
nucleotide sequencing were performed, as previously
described (Collins et al. 2003), by Gene Analysis Service,
Berlin, Germany. The sequence data were screened for
vector contamination using the National Center for
Biotechnology Information (NCBI) VecScreen, and
vector contamination was removed from contaminated
sequences. The presence of chimeric amplification prod-
ucts was also screened for using the Ribosomal Database
Project (rdp chimera_check software package (Maidak
et al. 2000) and none were present in the data generated
from this study. The resultant sequence data were then
compared to nucleotide databases using Blastn as previ-
ously described (Altschul et al. 1997). Sequences were
analysed using arb software (Ludwig et al. 2004) and
aligned with nearest relatives from the Blastn database
and a 16S rRNA ARB database (Hugenholtz 2002) down-
loaded from the Ribosomal Database Project II (Olsen
et al. 1992). Phylogenetic trees were constructed using the
neighbour-joining algorithm following the protocol of
Lueders and Friedrich (2000). All bacterial and archaeal
16S rRNA gene sequences from this study were deposited
in the GenBank database under accession numbers
GU322883-GU32288, GU322890-GU322899 and
GU946454-GU946455 for the bacterial sequences and
GU322876, GU322880-GU322881 and GU946456-
GU946457 for the archaeal sequences.
Two-dimensional gel electrophoresis (2-DGE)
Proteins were extracted from 50-ml granular sludge sam-
ples by sonication (Abram et al. 2009) and subsequently
separated by 2-DGE using the protocol of OFarrell
(1975), with modifications as previously described by
Abram et al. (2009). Briefly, the first dimension consisted
of isoelectric focusing using 7-cm IPG strips with linear
pH gradients (pH 47; Amersham, Buckinghamshire,
UK). The second dimension acrylamide (12% [w v]) gels
were run in pairs along with molecular weight markers
with a range of 10225 kDa (Broad Range Protein Molec-
ular Markers; Promega, Madison, WI). Gels were stained
overnight in GelCode Blue staining reagent (Pierce,
Rockford, IL) and then destained in deionized, distilled
water for several hours. Gel images were captured by
1551
Metaproteomics of anaerobic sludge
scanning with an Epson Perfection V350 photo scanner
(Epson, Long Beach, CA) at a resolution of 800 dpi. Six
gels were run corresponding to two duplicate indepen-
dent extractions and three technical replicates. Gel images
were processed and analysed with pdquest-Advanced
software, version 8.0.1 (BioRad, Hercules, CA). Spot
counts were obtained using the spot detection wizard
enabling the Gaussian model option as recommended by
the manufacturer.
Nanoflow liquid chromatography-electrospray ionization
tandem mass spectrometry (nLC-ESI-MS MS) and
protein identification
Proteins were excised from the gels and subjected to
in-gel digestion, using a ProGest Investigator in gel-
digestion robot (Genomic Solutions, Ann Arbor, MI) fol-
lowing standard protocols (Shevchenko et al. 1996).
Briefly, the gel pieces were destained by washing with ace-
tonitrile and subjected to reduction and alkylation prior to
tryptic digestion at 37C. The peptides were then extracted
with 10% (v v) formic acid. Peptide mixtures were subse-
quently separated using an UltiMate nanoLC (LC Packings,
Amsterdam, The Netherlands) equipped with a PepMap
C18 trap and column, developing a gradient from 5 to 50%
(v v) acetonitrile over either 30 min or 60 min, depending
on the molecular weight of the sample being analysed
(transition 40 kDa), followed by an extended washing per-
iod with 95% (v v) acetonitrile. The eluent was sprayed
into a Q-Star XL tandem mass spectrometer (Applied Bio-
systems, Foster City, CA) and analysed in information-
dependent acquisition mode. MS MS data for doubly and
triply charged precursor ions were converted to centroid
data, without smoothing, using the Analyst QS1.1 mas-
cot.dll data import filter with default settings. The MS MS
data file generated was analysed using the Mascot 2.2
search engine (Matrix Science, London, UK) against the
NCBInr database (2 Feb 2011, 12852469 sequences) and
Trembl (23 Feb 2011, 13499622 sequences), with no spe-
cies restriction. The identification of proteins was based on
the Mowse score from Mascot; a Mowse score of >55 was
statistically significant, with a 95% confidence level, when
the MS MS data were used to query both NCBInr and
Trembl. In addition, a minimum of two peptides had to be
detected for positive protein identification.
Results
Microbial community analyses of the low-temperature
bioreactor
PCRRFLP pattern analysis was carried out on 98 bacte-
rial clones and 79 archaeal clones. Unique electrophoretic
1552 Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology
F. Abram et al.
banding patterns were designated as operational
taxonomical units (OTUs), and only representative clones
of clonal OTUs exceeding 1% of respective clone libraries
were then sequenced. This implied that representatives of
only 50% of the bacterial clones and 90% of the archaeal
clones were selected for sequencing as the abundance of
the remaining clones was smaller than 1%. Phylogenetic
analyses distinctly grouped the bacterial clonal sequences
into the Planctomycetes (11%), the Verrucomicrobia (5%),
the Proteobacteria (2%), the Bacteroidetes (15%), the can-
didate division OP11 (2%), the Firmicutes (11%) and the
Thermotogae (4%; Fig. 1). The archaeal clonal sequences
could be grouped into the Methanomicrobiales (76%) with
only Methanocorpusculum-like clones, the Methanobacteriales
(115%) and the Crenarchaeota (25%; Fig. 2).
Metaproteomics of low-temperature anaerobic
granular sludge
The protein patterns of two independent, sludge samples
were analysed by 2-DGE. On average, 388 reproducible
protein spots (SD 179; n = 6) could be detected on the
2-D gels (Fig. 3). Seventy distinct spots were excised for
protein identification. Thirty-three of the 70 protein spots
were positively identified using nLC-ESI-MS MS
(Table 1, Fig. 3). A number of proteins, such as F420-
dependent N5, N10-methylenetetrahydromethanopterin
reductase from Methanothermobacter thermautotrophicus
and the c-subunit of the methyl-coenzyme M reductase
from Methanosaeta thermophila, migrated as several dis-
tinct spots as indicated in Table 1. Multiple spots detec-
tion of one gene product is a well-known characteristic of
the 2-DGE technique (OFarrell 1975; Gygi et al. 2000)
and could result from, for example, strain variations, dif-
ferential protein processing or post-translational modifi-
cations. It could also be attributed to artefactual
modifications imposed during the experimental analyses
of the proteins. However, the origin and biological signifi-
cance of the observed multiple spots detection is yet
unclear. In total, 18 different proteins were detected,
excluding redundant identifications but taking into
account proteins of the same suggested function attrib-
uted to different microbial species (Table 1). Some pro-
tein hits could not be unambiguously attributed to
specific microbial species as the peptides found matched
to regions of sequence homology across several species.
Microbial species resolution from metaproteomics
The metaproteomic approach applied here allowed for
the identification of proteins from specific microbial
species. The assignment of proteins to micro-organisms
led to six different situations detailed below. First, in the
2011 The Authors
F. Abram et al. Metaproteomics of anaerobic sludge

72 B17 GU946454 (3%)

52 Deepsea sediment clone BD216, AB015544
B6 GU322888 (2%)
36 Uncultured bacterium clone FGL3_B161, FJ437830
Uncultured Plantomycetes bacterium QEDQ3CE08, CU923200
Anaerobic digestor clone vadinBA30, U81656
B4 GU322886 (2%)
B7 GU3228899 (2%)
78
99
B5 GU322887 (2%)
Uncultured bacterium clone VHSB3_14, DQ394931
Planctomycetes
Planctomyces sp. str. 130, X81952
91 Planctomyces maris, S39798 X
62 Planctomyces limnophilus, S39795 X
Planctomyces sp. AGA/M18, X85249
Planctomyces brasiliensis, X85247
Pirellula sp. str. 302, X81942
59 Pirellula staleyi, M34126
99 Uncultured Verrucomicrobia bacterium QEDN7CE07, CU925564
92 Estuary sediment clone RB24, U62845
Uncultured Verrucomicrobia bacterium cloneC01_WMSP1, DQ450783 Verrucomicrobia
B1 GU322883 (2%)
B18 GU946455 (3%)
61 Uncultured delta proteobacterium clone MS491, GQ354944

18
Uncultured delta proteobacterium clone B10, 3
B3 GU322885 (2%)
Proteobacteria
B13 GU322895 (4%)
Uncultured eubacterium AA26, AF275921
Uncultured Cytophagales bacterium clone TDNP_Wbc97_200_1_88, FJ517044
B15 GU322897 (7%)
74 Uncultured bacterium clone MS4 31, GQ354928
43 Uncultured Cytophagales bacterium clone TDNP_USbc97_190_1_47, FJ516914
99 B8 GU322890 (2%)
63 Anaerobic digestor clone vadinHA17, U81712
40
Anaerobic digestor clone BC27, U81676 Bacteroidetes
Contaminated aquifer clone WCHB129, AF050544
Bacteroides putredinis, L16497
79 99 Rikenella microfusus, L16498
Bacteroides splanchnicus, L16496
B2 GU322884 (2%)
Uncultured bacterium clone BS17, EU358692
99 Uncultured Bacteroidetes/Chlorobi group bacterium clone D1\12_3, EU266837
Uncultured Anaerophaga sp. clone MDAF11, EU214540
63 Uncultured candidate division OP11clone pltbvmat84, AB294976
93
99 B9 GU322891 (2%)
80 Deepsea sediment clone BD74, AB015580 Candidate division OP11
98 Lake clone CL50013, AF316798
77 Deepsea sediment clone BD513, AB015569
Acetivibrio cellulolyticus, L35516
49 Clostridium aldrichii, X71846
Clostridium thermocellum, L09173
98 57 Uncultured Clostridium sp. clone 62K, AB198871
Clostridium cellulolyticum, X71847
Clostridium papyrosolvens, X71852
B12 GU322894 (2%)
99 Uncultured bacterium clone R1p16, AF482434
Ruminococcus callidus, L76596
92 Ruminococcus flavefaciens, X83430
Eubacterium siraeum, L34625
Fusobacterium prausnitzii, X85022
93 Carnobacterium funditum, S86170
99 Carnobacterium mobile, X54271
66 Carnobacterium alterfunditum, L08623
Carnococcus allantoicus, M98448
Lactosphaera pasteurii, X87150
B14 GU322896 (5%)
99 Uncultured bacterium clone A59, EU234092
Firmicutes
Bacillus sp. S110 (18)1 GU136579
32 B10 GU322892 (2%)
58 Uncultured bacterium clone IIIB9, AJ488100
Sphaerochaeta sp. TQ1, DQ833400
89 DCE dechlorinating consortium clone DCE25, AJ249227
96 Spirochaeta sp. Buddy, AF357916
51 Spirochaeta species, M87055
29 Spirochaeta bajacaliforniensis, M71239
41 Spirochaeta africana, X93928
Spirochaeta species, X79548
99 Uncultured bacterial clone, X89043
98 Thermophilic spirochete, M71240
B11 GU322893 (2%)
Uncultured Spirochaetes clone, QEDN2BE08, CU925939
99 Treponema denticola, M71236
Treponema phagedenis, M57739
Treponema pallidum, M34266 M
Spirochaeta thermophila, X62809
Uncultured bacterium, AB195923
97 Uncultured bacterium clone 28, FJ462028
89 B16 GU 322898 (4%) Thermotogae
Thermotoga elfii, X80790
Thermotoga subterranea, U22664
Thermotoga maritima, M21774
Ferroglobus
Euryarchaeota
Methanococcus

010

Figure 1 Phylogeny of bacterial sequences obtained from low-temperature anaerobic granular biomass (in bold type) together with the closest
relatives according to the NCBI and ARB (tree calculated with ARB, neighbour-joining method). Bootstrap replicates (out of a total of 1000
replicate samplings) that supported the branching order are shown at the relevant nodes in percentage. GenBank accession numbers are provided
for clonal and reference sequences, and percentage abundance of relevant clone libraries (in parenthesis) is also indicated. The scale bar
represents 10% sequence divergence.

cases of spots 1 and 33, a set of peptides that matched spot 2, a single spot led to the identification of a different
proteins of the same functions from different microbial set of peptides that matched proteins of similar function
species (spot 1) or genera (spot 33) was identified from (enolase) from a wide range of bacterial genera (Table 1).
analysis of a single spot (Table 1). Second, in the case of This situation also pertained to spots 3, 4 and 7; however,

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Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology 1553
Metaproteomics of anaerobic sludge F. Abram et al.

48 Anaerobic ciliate symbiont, M96976

24 Methanocorpusculum parvum, M59147
27 Methanocorpusculum bavaricum, AF042197
Methanocorpusculum sinense, AF095268
58 Methanocorpusclum parvum, AY260435
Methanocorpusculum labreanum, AF095267
A3 GU946457 (25%)
Methanomicrobiales
Uncultured archaeon gene for 16S rRNA, AB355102
Uncultured archaeon clone LTR-A3, FJ347529
A1 GU322876 (73%)
79 58 Uncultured archaeon clone LTR-A4, FJ347530

Methanoculleus
99

Methanosaeta Methanosarcinales
57
59 Uncultured archaeon clone OK3, GQ406364
68 A5 GU322880 (25%)
80 Archaeon enrichment culture clone C1-52C, GU470575
Methanobacteriales
Uncultured euryarchaeote clone F3, EU910627
89
Uncultured archaeon clone R2 Arc5, FJ005036
37 A6 GU322881 (9%)
Pyrococcus

Thermococcus
Euryarchaeota
62
Ferroglobus
13
Methanococcus

Sulfolobus tengchongensis, AY135637

56
55 Sulfolobus shibatae, M32504
Sulfolobus yangmingensis, AB010957
Uncultured Sulfolobus MS1, AF1690111
90 Metallosphaera hakonensis, D86414
Metallosphaera prunae, X90482
47 Sulfolobus sp H, D14052 Crenarchaeota
99 Sulfolobus metallicus, D85519
Thermofilum pendens, X14835
55 Thermofilaceae archaeon SRI-370, AF255605
95 Uncultured archaeonP15, EU662668
A2 GU946456 (25%)
Uncultured methanogenic archaeon, SMPFLSS56m_9, FJ982763
Uncultured crenarchaeote D39, EU910615
Thermodesulfobacteria
Thermodesulfobacterium

010

Figure 2 Phylogeny of archaeal sequences obtained from low-temperature anaerobic granular biomass (in bold type) together with the closest
relatives according to the NCBI and ARB (tree calculated with ARB, neighbour-joining method). Bootstrap replicates (out of a total of 1000
replicate samplings) that supported the branching order are shown at the relevant nodes in percentage. GenBank accession numbers are provided
for clonal and reference sequences, and percentage abundance of relevant clone libraries (in parenthesis) is also indicated. The scale bar
represents 10% sequence divergence.

for those spots, lower scoring peptide matches were
obtained for the protein homologues. Third, for spots 5,
6, 8, 26, 27 and 30, a single spot led to the identification
of a protein that could be assigned to one microbial spe-
cies (Table 1). Fourth, in the cases of spots 1317, 1822,
28 and 29, one protein assigned to one microbial species
migrated as multiple spots (Table 1). Fifth, for spots
912, a different set of peptides matched proteins of the
same function from diverse genera and was found to
migrate as different spots (Table 1). Finally, the last situa-
tion was observed for spots 2325, 31 and 32, where the
same set of peptides matched proteins of the same
function from several species or genera, and migrated as
multiple spots (Table 1). The assignment of multiple spe-
cies to one protein migrating as one or several spots
could be the result of two scenarios: either a mix of pro-
teins of similar functions from different micro-organisms
migrated to one spot or the spot contained one protein
that has homology to the proteins of all the different
microbial species and whose actual sequence may not be
available in the databases used here (NCBInr and Trembl).
The data presented here do not allow us to distinguish
between those two propositions.
Discussion
In this study, we applied metaproteomics and phylo-
genetic analysis to characterize the microbial communities
of an anaerobic EGSB bioreactor treating a synthetic,
glucose-based wastewater at 15C. Overall, 33 protein

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1554 Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology
F. Abram et al.
225
150
100
24
75
9 25
4
50 5 7
6
10 11
30
23 33
1 merase a-subunit of diverse strains belonging to both
26
12 3 2
35 8 Streptomyces and Mycobacterium genera (Table 1). Fourth,
13 29 28
27
22
15
25 14
16
31 32
17
20
15
10 (master)
4 5 6 7
Figure 3 Master 2-D gel (generated by pdquest-Advanced 8.0.1) of
proteins extracted from low-temperature anaerobic granular biomass.
Proteins excised and identified by mass spectrometry analysis are high-
lighted. Spot numbering corresponds to the numbering used in
Table 1. Molecular weight marker values indicated on the left of the
gel image are in kilodalton, and the 7-cm IPG strip used displayed a
pH gradient ranged from 4 to 7 (indicated below the gel picture).
spots excised from 2-D gels were successfully identified
corresponding to 18 distinct proteins excluding redun-
dant identification. Fourteen of the 18 proteins identified
could be classified into the broad functional category of
metabolism, with proteins mainly involved in glycolysis
and methanogenesis (Table 1; Fig. 4). The four remaining
enzymes were found to fall respectively under the cate-
gory of membrane protein, oxidation reduction, tran-
scription, and finally, protein degradation (Table 1). First,
the LamB porin family protein (Table 1), which facilitates
the diffusion of maltodextrins across the outer membrane
of gram negative bacteria (Schirmer et al. 1995), was
identified as a membrane protein from Pelobacter propio-
nicus. Maltodextrin is a glucose polymer that can be easily
converted to glucose, which can be subsequently used in
glycolysis or in the pentose phosphate pathway (Fig. 4).
Second, the oxygen-sensitive iron-containing alcohol
dehydrogenase (Table 1) from P. propionicus was also
detected. This protein can catalyse the interconversion
from alcohol to acetaldehyde. In many bacteria, pyruvate
produced via glycolysis can be converted to carbon diox-
ide and acetaldehyde, which in turn can be reduced to
ethanol by alcohol dehydrogenase (Fig. 4). This last step
is accompanied by the oxidation of NADH leading to the
necessary regeneration of NAD+. NAD+ can then act as
an electron acceptor for the continued oxidation of
glyceraldehyde-3-phosphate, a glycolytic intermediate, and
therefore enable glycolysis to take place in a continuous
2011 The Authors
Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology
Metaproteomics of anaerobic sludge
manner. Third, the RNA polymerase a-subunit, which
displays a putative function crucial for transcription
activation (Ishihama 1992), was identified. The set of
peptides identified for this protein matched RNA poly-
a proteasome a-subunit, responsible for the cleavage of a
21
broad range of proteins, was detected. The proteasome
18 19 subunit peptides were found to match the corresponding
M. thermophila protein as well as proteins of the same
function from various Methanosarcina species (Table 1).
Amongst the 14 proteins categorized as metabolism-
associated, two main enzymes of the glycolysis pathway
were detected in our protein samples, namely glyceralde-
hyde-3-phosphate dehydrogenase from Sanguibacter
keddieii, and enolase matching to various bacterial species
(Table 1). The presence of those glycolytic enzymes
coincides with the fact that the bioreactor was treating
wastewater containing glucose, which can be utilized via
glycolysis or the pentose phosphate pathway. The latter
pathway appeared active in the bioreactor as indicated by
the presence of transketolase from P. propionicus (Table 1;
Fig. 4), which is a key enzyme that constitutes a reversible
link between glycolysis and the pentose phosphate
pathway. The pentose phosphate pathway begins with one
intermediate of glycolysis, glucose, and ends with the for-
mation of two others, fructose-6-phosphate and D-glycer-
aldehyde-3-phosphate (Fig. 4). At the end of the glycolytic
pathway, pyruvate can be fermented to produce CO2 and
acetaldehyde, or formate and acetate. Acetaldehyde can be
further converted to ethanol via the activity of an alcohol
dehydrogenase (Table 1; Fig. 4), and CO2 can be used by
methanogens to produce methane. The data indicate that
methanogenesis from CO2 is taking place in the low-
temperature bioreactor with the identification of a key
enzyme of this pathway, the F420-dependent N5, N10-
methylenetetrahydromethanopterin reductase from Me.
thermautotrophicus. Our results also suggest that methano-
genesis from acetate is another active pathway based on
the detection of the b-subunit of the acetyl-CoA decarbon-
ylase protein from M. thermophila and Methanosarcina
barkeri (Table 1; Fig. 4). Methane production from both
CO2 and acetate correlates with the observation of a tem-
porally increasing ratio (2133 times) of hydrogeno-
trophic to acetoclastic methanogenic activity values during
the trial in the glucose-fed bioreactor (OReilly et al.
2009). Two other enzymes of methanogenesis were identi-
fied, namely, tetrahydromethanopterin S-methyltransferase
from M. thermophila leading to the production of methyl-
CoM and subunits of methyl-coenzyme M reductase from
M. thermophila, Me. thermautotrophicus and Methanother-
mobacter marburgensis catalysing the reduction of methyl-
coenzyme M to methane (Table 1; Fig. 4).
1555
 Table 1 Identification and putative function of proteins excised from 2-D gels from sludge from low-temperature anaerobic granular biomass*

1556
Spot Mascot No. matching % sequence
label Accession no. Protein name Species assignment score peptides (unique) covered Database source Suggested function

1 gi|269795306 Glyceraldehyde-3-phosphate Sanguibacter keddieii 304 1 > Ident 20 NCBInr Trembl Glycolysis
dehydrogenase 2 < Ident>Homol
6 < Homol
2 gi|229819515 Enolase Multiple bacterial 306 2 > Ident 13 NCBInr Trembl Glycolysis
species 1 < Ident>Homol
1 < Homol
3 gi|119715173 Enolase Nocardioides sp. 232 3 > Ident 9 NCBInr Trembl Glycolysis
Metaproteomics of anaerobic sludge

4 gi|29830076 Enolase Streptomyces avermitilis 247 2 > Ident 10 NCBInr Trembl Glycolysis
1 < Ident>Homol
5 Q3LFH7 Enolase Propionibacterium 385 3 > Ident 14 NCBInr Trembl Glycolysis
freudenreichii 2 < Ident>Homol
1 < Homol
6 D5UK74 Enolase Cellulomonas flavigena 238 2 > Ident 12 NCBInr Trembl Glycolysis
3 < Homol
7 A8U6I2 Enolase Carnobacterium sp. AT7 293 2 > Ident 15 NCBInr Trembl Glycolysis
1 < Ident>Homol
3 < Homol
8 gi|118579459 Transketolase central region Pelobacter propionicus 132 1 > Ident 13 NCBInr Trembl Pentose phosphate
1 < Ident>Homol pathway
1 < Homol
9, 10, gi|116753609 Acetyl-CoA Methanosaeta thermophila 175 1 > Ident 7 NCBInr Trembl Methanogenesis
11, 12 decarbonylase synthase Methanosarcina barkeri 2 < Ident>Homol From acetate
b subunit 4 < Homol
13, 14, 15, gi|1002717 Coenzyme F420-dependent Methanothermobacter 515 5 > Ident 36 NCBInr Trembl Methanogenesis
16, 17 N5,N10- thermautotrophicus 1 < Ident>Homol from CO2
methylenetetrahydromethanopterin 4 < Homol
reductase
18, 19, 20, gi|116753883 Methyl-coenzyme M reductase, M. thermophila 181 6 < Homol 16 NCBInr Trembl Methanogenesis
21, 22 c subunit
23, 24, 25 gi|304315293 Methyl-coenzyme M reductase, Methanothermobacter 217 2 > Ident 9 NCBInr Trembl Methanogenesis
a subunit marburgensis 1 < Ident>Homol
thermautotrophicus 2 < Homol
26 gi|517427 Methyl-coenzyme M reductase, Me. thermautotrophicus 406 4 > Ident 11 NCBInr Trembl Methanogenesis
b subunit
27 gi|116754675 Tetrahydromethanopterin M. thermophila 129 1 > Ident 2 NCBInr Trembl Methanogenesis
S- methyltransferase H subunit 1 < Ident>Homol
28, 29 gi|118579324 Iron containing alcohol P. propionicus 269 1 > Ident 21 NCBInr Trembl Oxidation reduction
dehydrogenase 3 < Ident>Homol
3 < Homol

Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology
F. Abram et al.

2011 The Authors

F. Abram et al. Metaproteomics of anaerobic sludge

No protein of unknown function was identified during

Protein degradation

homology match and Homol represents matches that are outliers from the distribution of random matches but do not reach the 95% confidence level. NCBInr and Trembl are cited together
Indicates that also other lower scoring peptides match to protein homologues from other species. Ident refers to the Mascot Identity threshold of 95% confidence for identical or extensive
Suggested function

Membrane protein
this study. A similar metaproteomic approach based on

Transcription
2-DGE applied to activated (aerobic) sludge from a muni-
cipal wastewater treatment plant resulted in the detection
of four proteins of unknown function from the 46 proteins
identified (Wilmes et al. 2008). Using 2-DGE as a protein
separation method is likely to result in biased analysis
towards the most abundant proteins, such as metabolic or
Database source

NCBInr Trembl

NCBInr Trembl

NCBInr Trembl
housekeeping proteins. In the study of Wilmes et al.
(2008), the metaproteomic work was supported by
metagenomic data, while in the case of our study no me-
*Spot numbers refer to those in Figure 3. Accession numbers starting with gi| belong to the NCBInr database while others belong to the Trembl database. tagenomic data were available from the bioreactor. More-
over, most of the micro-organisms composing the mixed
communities prevailing in our LTAD bioreactor are
% sequence

unlikely to have been previously sequenced. Therefore,

covered

identification of unknown proteins from microbial species,

whose genomes have not been sequenced and are hence
15

16

13

not in the NCBInr and Trembl databases, was not possible

without undertaking de novo analyses of individual
peptides (unique)

Ident>Homol

Ident>Homol

MS MS spectra.
No. matching

This study is, to our knowledge, the first metaproteo-

Homol

Homol

Homol
Ident

Ident

mic analysis conducted using granular biomass from an

anaerobic wastewater treatment bioreactor. Despite the
>
<
<
>
<
<
<
2
1
3
2
2
2
2

lack of corresponding metagenomic data, we could iden-

when the same protein appeared in both searches; under these instances, the best Mascot score is reported.

tify proteins of relevant functions and outline the micro-

bial activities taking place in the cold, anaerobic,
Mascot
score

237

183

135

bioreactor treating food-processing-like wastewater, with

glucose degradation via glycolysis and the pentose
phosphate pathway, and the production of methane via
Methanosarcina sp.

methanogenesis from both CO2 and acetate. The meta-

Mycobacterium sp.
Species assignment

Streptomyces and
M. thermophila

proteomic approach also indicated, by protein assign-

P. propionicus

ment, the presence of specific micro-organisms. The 33

protein spots successfully identified corresponded to 18
distinct proteins, from which six were assigned to the
Actinobacteria (Sanguibacter keddieii, Nocardioides sp.,
Cellulomonas flavigena, Streptomyces sp., Mycobacterium
sp. and Propionobacterium freudenreichii), one to the
RNA polymerase a subunit
LamB porin family protein

Firmicutes (Carnobacterium sp.), three to the Proteobacteria

Proteasome a subunit

(P. propionicus) and seven to the Archaea. Within the

Archaea, four proteins were assigned to the Methanosarci-
Protein name

nales (M. thermophila and Methanosarcina sp.) and three

to the Methanobacteriales (Methanothermobacter sp.). The
remaining protein (enolase) was assigned to a wide range
of bacteria (spot 2). The phylogenetic analyses indicated
that 11% of the bacterial clones belonged to the Firmi-
cutes and 2% to the Proteobacteria, and proteins could be
gi|302524022
Accession no.

assigned to micro-organisms of both phyla. While six

A0B8W6
A1ALD2

proteins were assigned to the Actinobacteria, no bacterial

Table 1 (continued)

clone was found to belong to this phylum. The other

bacterial phyla identified by phylogenetic analyses were
not represented in the metaproteomic investigations,
31, 32

despite the availability of complete genome sequences for

label
Spot

30

33

all of them apart from the candidate division OP11.

2011 The Authors

Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology 1557
Metaproteomics of anaerobic sludge F. Abram et al.

LamB porin
Intracellular Pel Extracellular
Glucose
Oxidative reaction maltodextrin maltodextrin
Xylulose-5-P of pentose
phosphate pathway
Ribulose-5-P Glucose-6-P

Ribose-5-P
Transketolase Fructose-6-P
Pel

Glyceraldehyde-3- Sedoheptulose-7-P Glyceraldehyde-3-P(2)

Glyceraldehyde-3-P-
dehydrogenase
San
Erythrose-4-P Fructose-6-P 1,3-Biphosphoglycerate (2)

Xylulose-5-P
Transketolase
Pel Fructose-6-P 2-Phosphoglycerate (2)

Enolase
Glyceraldehyde-3-P Noc/Str/Pro/Cel/Car
Phosphoenopyruvate (2)

Pyruvate (2)

Methanofuran CO2 + Acetaldehyde Formate + Acetate

Alcohol
dehydrogenase
Pel
Ethanol
Acetyl-CoA
CO2 Acetyl CoA
decarbonylase
Msa/Msr
5,10-methylene- 5-methyl-
tetrahydromethanopterin tetrahydrosarcinapterin

F420-dependent N5,N10 Coenzyme M

methylenetetrahydro-
methanopterin reductase
Mtt
Tetrahydromethanopterin
S-methyltransferase
Msa
5-methyl-tetrahydromethanopterin Methyl-CoM
Methyl coenzyme
Coenzyme M M reductase
Mtt/Msa

CoB-CoM heterodisulfide Methane

Coenzyme B

Figure 4 Proposed metabolic model for anaerobic digestion of synthetic glucose-based wastewater inferred from the metaproteomic data.
Enzymes identified in this study are in bold. Not all intermediate metabolites are shown. P stands for phosphate; Pel for Pelobacter; San for
Sanguibacter; Noc for Nocardioides; Str for Streptomyces; Pro for Propionibacterium; Cel for Cellulomonas; Car for Carnobacterium; Msa for
Methanosaeta; Msr for Methanosarcina; Mtt for Methanothermobacter.

2011 The Authors

1558 Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology
F. Abram et al.
Specifically, bacterial clones were grouped into the Plan-
ctomycetes, a phylum from which five complete genomes
are sequenced, the Verrucomicrobia, which to date
encompasses 28 full genomes, the Proteobacteria with 657
complete genomes, the Bacteroidetes with 59 sequenced
genomes, the Firmicutes with 337 available complete
genomes and finally the Thermotogae, with 11 genomes.
It should be noted however that the clones identified in
this study were only related to micro-organisms with
fully sequenced genomes. For the archaeal clones, 115%
belonged to the Methanobacteriales and proteins assigned
to micro-organisms of this order were identified. Sev-
enty-six per cent of archaeal clones were found to belong
to the Methanomicrobiales with exclusively Methanocor-
pusculum-like organisms, while no protein identified in
this study was assigned to members of this order, despite
the availability of the full genome sequence of Methano-
corpusculum labraneum. Two per cent of archaeal clones
were found to belong to the Crenarchaeota, while no pro-
tein could be assigned to micro-organisms of this order
despite the availability of 34 complete genomes amongst
which that of Thermofilum pendens (Fig. 2). While five
proteins were assigned to the Methanosarcinales, no
bacterial clone was found to belong to this phylum.
Overall, only a somehow limited overlap existed between
the phylogenetic and the metaproteomic analyses; the
reasons for this observation are yet unclear. We expect,
however, that an RNA-based phylogenetic approach
would better reflect the active component of the consor-
tium and might allow a stronger correlation with meta-
proteomics.
In conclusion, metaproteomics is demonstrated as a
powerful tool with the ability to uncover key biochemical
metabolic pathways occurring in specific bioreactor pro-
cesses. Metaproteomics can be applied to characterize the
communities underpinning LTAD, which will be a neces-
sary step to optimize the application of this emerging
technology. Future work could focus on the acquisition
of metagenomic data from AD systems, which would
considerably augment the usefulness of metaproteomic
data. Monitoring the performance and temporal protein
patterns of AD bioreactor communities in response to
environmental and operational parameters will assess fur-
ther the potential of the approach tested in this study. It
might also be possible to identify enzymatic markers, or
temporal shifts in the protein expression patterns of the
microbial communities, which could be used as predictive
indicators of process failure.
Acknowledgements
This publication has emanated from research conducted
with the financial support of Science Foundation Ireland
2011 The Authors
Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology
Metaproteomics of anaerobic sludge
(Principal Investigator Award). We also acknowledge the
support of the Wellcome Trust in funding the purchase
of the mass spectrometer at the Centre for BioMolecular
Sciences, University of St Andrews.
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