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A metaproteomic approach gives functional


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Article in Journal of Applied Microbiology March 2011


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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 Abstract
metabolic processes, microbial phylogenetics,
proteomics, sludge, wastewater treatment. Aims: The objective of this work was to provide functional evidence of key
metabolic pathways important for anaerobic digestion processes through the
Correspondence identification of highly expressed proteins in a mixed anaerobic microbial
Vincent OFlaherty, Microbial Ecology consortium.
Laboratory, Department of Microbiology,
Methods and Results: The microbial communities from an anaerobic indus-
School of Natural Sciences, National University
of Ireland, University Road, Galway, Ireland.
trial-like wastewater treatment bioreactor were characterized using phylogenetic
E-mail: vincent.oflaherty@nuigalway.ie analyses and metaproteomics. Clone libraries indicated that the bacterial
community in the bioreactor was diverse while the archaeal population was
2010 1925: received 26 October 2010, mainly composed of Methanocorpusculum-like (76%) micro-organisms. Three
revised 9 March 2011 and accepted 19 March hundred and eighty-eight reproducible protein spots were obtained on 2-D
2011 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
doi:10.1111/j.1365-2672.2011.05011.x
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
which results in increased heating costs necessary to main-
Anaerobic digestion (AD) has become an essential and tain mesophilic treatment. Therefore, low-temperature
well-established technology for the treatment of a wide anaerobic bioreactors represent an attractive and cost-
range of wastes and wastewaters. The natural process of effective alternative. The success of laboratory-scale, low-
AD is driven by intricate, poorly characterized, microbial temperature anaerobic digestion (LTAD) has been well
interactions, which can be applied for the conversion of documented for a wide range of food-processing, phenolic
complex unwanted biodegradable organic molecules into and other industrial wastewaters (Kato et al. 1994, 1999;
a renewable source of energy (McCarty 2001). Because of Rebac et al. 1995, 1999; van Lier et al. 1997; Lettinga et al.
anaerobic microbial growth characteristics, reaction rates 1999; Connaughton et al. 2006; Enright et al. 2007; Scully
and temperature optima, most full-scale, anaerobic, indus- et al. 2007; OReilly et al. 2009).
trial wastewater treatment systems are operated at temper- Despite the widespread application of AD, as well as
atures higher than 18C (Lettinga et al. 2001; Hulshoff Pol emerging LTAD technology development, the micro-
et al. 2004). However, the vast majority of industrial organisms involved have not yet been fully characterized.

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1550 Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology
F. Abram et al. Metaproteomics of anaerobic sludge

Indeed, little is known about the functional activities of Delong 1992) and reverse primer 1392R (5-ACGGGC-
many of the abundant groups found in anaerobic biofilms GGTGTGTTRC-3; Lane et al. 1985). Partial archaeal 16S
(sludge granules) from AD bioreactors. Consequently, the rRNA genes were amplified with forward primer 21F
performance and optimization of AD processes remain (5-TTCCGGTTGATCCYGCCGGA-3; Stackebrandt and
limited. Up to now, much effort has been made to eluci- Goodfellow 1991) and reverse primer 958R (5-YCCGG-
date the microbial community structure of consortia in CGTTGAMTCCAATT-3; Delong 1992). The concentra-
anaerobic bioreactors (Godon et al. 1997; Leclerc et al. tions of the PCR components and the PCR conditions
2001, 2004; Roest et al. 2005; Lee et al. 2009; McKeown were as outlined by Collins et al. (2003). Cloning
et al. 2009; OReilly et al. 2009). However, only limited (TOPO XL; Invitrogen, Carlsbad, CA), restriction frag-
data are available on the links between diversity and ment length polymorphism (RFLP) pattern analysis and
microbial functionality in anaerobic bioreactors. nucleotide sequencing were performed, as previously
In this study, the microbial communities of an anaerobic described (Collins et al. 2003), by Gene Analysis Service,
bioreactor treating a synthetic, glucose-based wastewater at Berlin, Germany. The sequence data were screened for
15C for 300 days (OReilly et al. 2010) were characterized vector contamination using the National Center for
using clone libraries and metaproteomics. The functions of Biotechnology Information (NCBI) VecScreen, and
the proteins recovered from the bioreactors have been vector contamination was removed from contaminated
attributed based on homologies to other proteins, and sequences. The presence of chimeric amplification prod-
their possible contributions to the biology of AD are ucts was also screened for using the Ribosomal Database
discussed. Protein assignment also indicated the presence Project (rdp chimera_check software package (Maidak
of specific micro-organisms in the bioreactor, and this was et al. 2000) and none were present in the data generated
compared with the results of the phylogenetic analysis. 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
Materials and Methods
analysed using arb software (Ludwig et al. 2004) and
aligned with nearest relatives from the Blastn database
Source of biomass
and a 16S rRNA ARB database (Hugenholtz 2002) down-
The anaerobic bioreactor, which was the source of the bio- loaded from the Ribosomal Database Project II (Olsen
mass investigated in this study, was previously described et al. 1992). Phylogenetic trees were constructed using the
by OReilly et al. (2010). Briefly, the bioreactor had a neighbour-joining algorithm following the protocol of
working volume of 35 l and was operated at 15C for Lueders and Friedrich (2000). All bacterial and archaeal
300 days to treat a synthetic, glucose-based wastewater, 16S rRNA gene sequences from this study were deposited
which was buffered at pH 71 with NaHCO3. Samples of in the GenBank database under accession numbers
sludge granules for phylogenetic and metaproteomic anal- GU322883-GU32288, GU322890-GU322899 and
yses were obtained at the conclusion of the trial (day 300). GU946454-GU946455 for the bacterial sequences and
GU322876, GU322880-GU322881 and GU946456-
GU946457 for the archaeal sequences.
DNA extraction
Total DNA was extracted from granular biomass using an
Two-dimensional gel electrophoresis (2-DGE)
automated nucleic acid extractor (Magtration 12 GC; PSS
Co., Chiba, Japan) following the manufacturers instruc- Proteins were extracted from 50-ml granular sludge sam-
tions. Prior to extraction, the biomass (05 g wet weight) ples by sonication (Abram et al. 2009) and subsequently
was finely crushed using a mortar and pestle and resus- separated by 2-DGE using the protocol of OFarrell
pended in 50 ml of deionized, distilled water. A 100-ll (1975), with modifications as previously described by
aliquot of the biomass suspension was loaded per Abram et al. (2009). Briefly, the first dimension consisted
extraction, and the extracted DNA was eluted in Tris of isoelectric focusing using 7-cm IPG strips with linear
HCl buffer (pH 80) and stored at )20C. All extractions pH gradients (pH 47; Amersham, Buckinghamshire,
were carried out in duplicate. 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-
Generation of 16S rRNA gene clone libraries, sequencing
ular Markers; Promega, Madison, WI). Gels were stained
and phylogenetic analysis
overnight in GelCode Blue staining reagent (Pierce,
Partial bacterial 16S rRNA genes were amplified with for- Rockford, IL) and then destained in deionized, distilled
ward primer 27F (5-AGAGTTTGATCCTGGCTCAG-3; water for several hours. Gel images were captured by

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Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology 1551
Metaproteomics of anaerobic sludge F. Abram et al.

scanning with an Epson Perfection V350 photo scanner banding patterns were designated as operational
(Epson, Long Beach, CA) at a resolution of 800 dpi. Six taxonomical units (OTUs), and only representative clones
gels were run corresponding to two duplicate indepen- of clonal OTUs exceeding 1% of respective clone libraries
dent extractions and three technical replicates. Gel images were then sequenced. This implied that representatives of
were processed and analysed with pdquest-Advanced only 50% of the bacterial clones and 90% of the archaeal
software, version 8.0.1 (BioRad, Hercules, CA). Spot clones were selected for sequencing as the abundance of
counts were obtained using the spot detection wizard the remaining clones was smaller than 1%. Phylogenetic
enabling the Gaussian model option as recommended by analyses distinctly grouped the bacterial clonal sequences
the manufacturer. into the Planctomycetes (11%), the Verrucomicrobia (5%),
the Proteobacteria (2%), the Bacteroidetes (15%), the can-
didate division OP11 (2%), the Firmicutes (11%) and the
Nanoflow liquid chromatography-electrospray ionization
Thermotogae (4%; Fig. 1). The archaeal clonal sequences
tandem mass spectrometry (nLC-ESI-MS MS) and
could be grouped into the Methanomicrobiales (76%) with
protein identification
only Methanocorpusculum-like clones, the Methanobacteriales
Proteins were excised from the gels and subjected to (115%) and the Crenarchaeota (25%; Fig. 2).
in-gel digestion, using a ProGest Investigator in gel-
digestion robot (Genomic Solutions, Ann Arbor, MI) fol-
Metaproteomics of low-temperature anaerobic
lowing standard protocols (Shevchenko et al. 1996).
granular sludge
Briefly, the gel pieces were destained by washing with ace-
tonitrile and subjected to reduction and alkylation prior to The protein patterns of two independent, sludge samples
tryptic digestion at 37C. The peptides were then extracted were analysed by 2-DGE. On average, 388 reproducible
with 10% (v v) formic acid. Peptide mixtures were subse- protein spots (SD 179; n = 6) could be detected on the
quently separated using an UltiMate nanoLC (LC Packings, 2-D gels (Fig. 3). Seventy distinct spots were excised for
Amsterdam, The Netherlands) equipped with a PepMap protein identification. Thirty-three of the 70 protein spots
C18 trap and column, developing a gradient from 5 to 50% were positively identified using nLC-ESI-MS MS
(v v) acetonitrile over either 30 min or 60 min, depending (Table 1, Fig. 3). A number of proteins, such as F420-
on the molecular weight of the sample being analysed dependent N5, N10-methylenetetrahydromethanopterin
(transition 40 kDa), followed by an extended washing per- reductase from Methanothermobacter thermautotrophicus
iod with 95% (v v) acetonitrile. The eluent was sprayed and the c-subunit of the methyl-coenzyme M reductase
into a Q-Star XL tandem mass spectrometer (Applied Bio- from Methanosaeta thermophila, migrated as several dis-
systems, Foster City, CA) and analysed in information- tinct spots as indicated in Table 1. Multiple spots detec-
dependent acquisition mode. MS MS data for doubly and tion of one gene product is a well-known characteristic of
triply charged precursor ions were converted to centroid the 2-DGE technique (OFarrell 1975; Gygi et al. 2000)
data, without smoothing, using the Analyst QS1.1 mas- and could result from, for example, strain variations, dif-
cot.dll data import filter with default settings. The MS MS ferential protein processing or post-translational modifi-
data file generated was analysed using the Mascot 2.2 cations. It could also be attributed to artefactual
search engine (Matrix Science, London, UK) against the modifications imposed during the experimental analyses
NCBInr database (2 Feb 2011, 12852469 sequences) and of the proteins. However, the origin and biological signifi-
Trembl (23 Feb 2011, 13499622 sequences), with no spe- cance of the observed multiple spots detection is yet
cies restriction. The identification of proteins was based on unclear. In total, 18 different proteins were detected,
the Mowse score from Mascot; a Mowse score of >55 was excluding redundant identifications but taking into
statistically significant, with a 95% confidence level, when account proteins of the same suggested function attrib-
the MS MS data were used to query both NCBInr and uted to different microbial species (Table 1). Some pro-
Trembl. In addition, a minimum of two peptides had to be tein hits could not be unambiguously attributed to
detected for positive protein identification. specific microbial species as the peptides found matched
to regions of sequence homology across several species.
Results
Microbial species resolution from metaproteomics
Microbial community analyses of the low-temperature
The metaproteomic approach applied here allowed for
bioreactor
the identification of proteins from specific microbial
PCRRFLP pattern analysis was carried out on 98 bacte- species. The assignment of proteins to micro-organisms
rial clones and 79 archaeal clones. Unique electrophoretic led to six different situations detailed below. First, in the

2011 The Authors


1552 Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology
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 could be the result of two scenarios: either a mix of pro-
obtained for the protein homologues. Third, for spots 5, teins of similar functions from different micro-organisms
6, 8, 26, 27 and 30, a single spot led to the identification migrated to one spot or the spot contained one protein
of a protein that could be assigned to one microbial spe- that has homology to the proteins of all the different
cies (Table 1). Fourth, in the cases of spots 1317, 1822, microbial species and whose actual sequence may not be
28 and 29, one protein assigned to one microbial species available in the databases used here (NCBInr and Trembl).
migrated as multiple spots (Table 1). Fifth, for spots The data presented here do not allow us to distinguish
912, a different set of peptides matched proteins of the between those two propositions.
same function from diverse genera and was found to
migrate as different spots (Table 1). Finally, the last situa-
Discussion
tion was observed for spots 2325, 31 and 32, where the
same set of peptides matched proteins of the same In this study, we applied metaproteomics and phylo-
function from several species or genera, and migrated as genetic analysis to characterize the microbial communities
multiple spots (Table 1). The assignment of multiple spe- of an anaerobic EGSB bioreactor treating a synthetic,
cies to one protein migrating as one or several spots 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. Metaproteomics of anaerobic sludge

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

2011 The Authors


Journal of Applied Microbiology 110, 15501560 2011 The Society for Applied Microbiology 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. Metaproteomics of anaerobic sludge

Specifically, bacterial clones were grouped into the Plan- (Principal Investigator Award). We also acknowledge the
ctomycetes, a phylum from which five complete genomes support of the Wellcome Trust in funding the purchase
are sequenced, the Verrucomicrobia, which to date of the mass spectrometer at the Centre for BioMolecular
encompasses 28 full genomes, the Proteobacteria with 657 Sciences, University of St Andrews.
complete genomes, the Bacteroidetes with 59 sequenced
genomes, the Firmicutes with 337 available complete
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