Natural enrichment resulted in a microbial community that degraded poplar under anaerobic conditions, as was evident from strong visual signs of decomposition of the bulk biomass, enzymatic attack of the cell walls, and the disappearance of cellulose and hemi-cellulose. Therefore, this community must contain the necessary functionality to break down the various polymers found in poplar hard wood, including the major polysaccharides xylan and glucomannan as part of the hemicellulose, and cellulose, in addition to less abundant polysaccharides. The complex network of these polysaccharides and their side chains, and the presence of lignin, all contribute to the recalcitrance of the poplar biomass. An overview of the poplar hard wood polysaccharides and the key enzymes that are theoretically required for the efficient decomposition of poplar hard wood are presented in . When comparing the enzymatic requirements for the breakdown of poplar hardwood with the presence of glycoside hydrolases among the dominant members of the biomass decomposing community (), most of the necessary activities were found among several of the dominant community members. However, there were some exceptions. For example, hydrolysis of (1–2)-α-D-(4-O
-methyl)glucuronosyl links in the main chain of hardwood xylans is thought to require enzymes of the GHase Families 67 and 115, both of which were only found among the contigs assigned to Bacteroides
. And although several other dominant community members encoded putative GHase 43 family members, required for the hydrolysis of terminal non-reducing β-D-galactose residues in (1–3)-β-D-galactopyranans, only Bacteroides
encoded putative GH43/GH95 and GH95 enzymes (with α-1,2-L-fucosidase and α-L-fucosidase activities). In general, the contigs assigned to Bacteroides
contained the largest number of unique putative glycoside hydrolases. We therefore hypothesize that the Bacteroides
-like community members play a major role in the breakdown of cellulose and hemicelluloses under these conditions. Members of this phylum also play an important role in other anaerobic microbiomes with high biomass turnover rates, including the cow rumen 
. Also, the two Clostridiales
appear, based on the presence of various putative GHases, to play a key role in the breakdown of these two recalcitrant plant cell wall polysaccharides. This is not unexpected, as many members of the Clostridiales
have been found in environments with high plant biomass turnover rates.
Because the cellulose and hemicellulose polymers are complexed with lignin, the anaerobic microbes must possess mechanisms to access these polysaccharides, most likely via mechanisms that allow for local depolymerization of lignin, as no major removal of lignin from the cell walls was observed. Despite the lack of enzymes involved in lignin decomposition, the presence of bacterial genes that show homology to fungal lignin oxidases might provide some insights in this process. Homologs to the LO3 family (FOLy database) were common among the six dominant members of the community studied. Most of the genes with similarity to this family are annotated as dihydrolipoamide dehydrogenase, cellobiose dehydrogenases or have more general annotations, as this is a large family of proteins only some of which are involved in lignin breakdown. However, putative cellobiose dehydrogenases can very well be involved in lignin breakdown, as they were found to be able to interact with lignin in three important ways: (1) to break beta-ethers; (2) to demethoxylate aromatic structures in lignins; (3) and to introduce hydroxyl groups in non-phenolic lignins 
. The presence of LO2 type peroxidases in the Clostridiales
genome bins might hint towards an anaerobic mechanism for lignin depolymerization, since members of the Clostridiales
require strict anaerobic conditions for their growth. In addition, genes with homology to the two Dyp-like peroxidase genes, involved in lignin degradation 
by Rhodococcus jostii
RHA1, were identified on low-coverage contigs and individual reads, suggesting that minor community members may also be important in local depolymerization of lignin, rendering the hemicellulose and cellulose available to depolymerization by the glycoside hydrolases that are synthesized by many members of the community.
In fungi, the general role of lignin degrading enzymes is perhaps becoming clearer. Vanden-Wymelenberg et al
. reported up-regulated Phanerochaete chrysosporium
genes in piles of decaying ball milled pine and the hardwood, aspen 
. The piles of hardwood (aspen) displayed high transcript levels for a glucose oxidase-like oxidoreductase, a catalase, and an alcohol oxidase 
. P. chrysosporium
oxidoreductase transcripts, found when grown on pine, included the copper radical oxidase CRO2 and cellobiose dehydrogenase. In the study presented here, the anaerobic bacterial consortium grown on poplar hardwood appears to produce oxidoreductases more consistent with those produced by P. chrysosporium
grown on pine, suggesting the bacterial homologs may have different specificity.
Although very little information is available on the anaerobic breakdown of lignin, some genetic clues on the evolution of anaerobic catabolism of aromatic compounds 
have been described, and anaerobic degradation of aromatic compounds has been reported for various bacteria, including Azoarcus
spp. CIB 
and Geobacter metallireducens
. Also, members of the genus Magnetospirillum
that degrade aromatic compounds anaerobically including toluene, phenol and benzoate, were previously isolated from denitrifying enrichment cultures 
. Although members of this genus have not previously been identified as dominant members of biomass decomposing communities, nor have they been known to harbor metabolic potential to break down lignocellulosic biomass, their unexpected dominant presence in the microbial community decaying poplar biomass seems to indicate that they might play a role in lignin depolymerization or the breakdown of aromatic compounds released from the wood. The toluene-degrading strain Magnetospirillum
TS-6 was found to contain genes that are homologous to those encoding benzylsuccinate synthase (Bss) and benzoyl-CoA reductase (Bcr), two key enzymes of anaerobic toluene and benzoate degradation respectively in known denitrifying bacteria 
. These two genes were also found on a large contig that based on sequence composition was assigned to Magnetospirillum
: a putative bssD
gene, encoding a benzylsuccinate synthase activating enzyme I, was found immediately downstream of a bssA
gene coding for a putative alpha subunit of benzylsuccinate synthase (PBDCA2_4994280). Since Magnetospirillum
does not seem to contain any key functions for lignin depolymerization, such as LO1 and LO2, our hypothesis is that the presence of Magnetospirillum
is key for the efficient degradation of toxic aromatic compounds that are released from the poplar wood during its decay. As such, Magnetospirillum
acts as the “liver" of the community, making sure that the community doesn't collapse due to the accumulation of toxic lignin-derived aromatic compounds.
Members of the genus Magnetospirillum
have been reported to play a significant role in oxic-anoxic transition zones of freshwater ecosystems, where opposing gradients exist of reduced iron and sulfide with oxygen, creating a suitable environment for microorganisms that derive energy from the oxidation of iron or sulfide 
. It cannot be totally excluded that opposing redox gradient also existed in our reactor system, and that this resulted in the enrichment of Magnetospirillum
The distribution and relative abundance of cellulases, hemicellulases, debranching enzymes and enzymes homologous to lignolytic enzymes revealed compelling patterns of enzyme frequencies among different types of communities. In this respect, the strong conservation in the relative distribution of candidate GHase between the top and bottom layers of the fungal garden, as shown in Figure S3
, is striking, as both layers have a clear distinction: the top layer, which retains the green, harvested state of plant leaves; and the bottom layer, which contains mature fungus and partially degraded plant material. Furthermore, comparison of the phylogenetic diversity between top and bottom layer indicated distinct differences, although both layers are dominated by phylotypes in the α-Proteobacteria
. The common presence of these dominant phylotypes might explain the conservation in functionality. It was also noted that no measurable lignin degradation occurred in the fungal garden. However, in order to explain the observed breakdown of hemicellulose and cellulose, some local degradation of lignin is required in order to access these polymers. The presence of bacterial genes with similarity to fungal peroxidases might provide a possibility for lignin breakdown.
In comparison, more light has been shed recently regarding the complete picture of fungal cellulose degradation by the discovery of the probable role of GH Family 61 oxidative enzymes 
. For example, Vanden-Wymelenberg et al.
found high levels of GH61 transcripts in decaying softwood piles inoculated with P. chrysosporium
. In the study presented here, the anaerobic bacterial consortium grown on the hardwood, poplar, appears to lack the ability to produce GH61 enzymes. Recently, the cellulose binding module (CBM) from Family 33, which is a structural analogue to GH61, was implicated in bacterial oxidative cellulose deconstruction 
. In this consortium, four homologs of CBM33 were found, all located on relatively small unbinned sequences: two on individual 454 reads, and 2 on contigs of 2–3× depth. All putative CBM33 genes showed high GC content (64–69%), so on that basis could potentially belong to the Magnetospirillum
, whose contigs varied considerably in coverage due to apparent sequencing bias in this high-GC genome. Furthermore, the bacterial consortium studied here encodes putative cellobiose dehydrogenases. This is significant, because it was suggested nearly 15 years ago that cellobiose dehydrogenase could be involved in cellulose depolymerization 
and today it appears that GH61 (or CBM33) and cellobiose dehydrogenase could work cooperatively to effect oxidative cellulose depolymerization.
From a community perspective, a striking observation is the near-complete lack of putative lignin degrading enzymes shared by all the gut communities examined (termite hindgut, cow rumen, wallaby, dog, human and mouse); whereas compost, soil, and fungal garden microbes all harbored some of these genes, as did the members of the poplar biomass decomposing community. For example, the termite hindgut community has very few candidate functions involved in the decomposition of lignin; only one candidate LDA6 with homology to a glucose oxidase and 20 putative LO3 candidates, all displaying homology to dihydrolipoyl and dihydrolipoamide dehydrogenases, could be identified (). The lack of enzymatic functions involved in the decomposition of lignin in the gut communities suggests that the hosts may provide adequate pretreatment of the biomass to allow for microbial decomposition, through chewing in all animals in addition to an alkaline pretreatment in the higher termite gut (pH 8.5–12, depending on species) 
, which might provide suitable solubilization of some lignins to permit the needed GHase accessibility to wall polysaccharides. While the relative availability of oxygen may also impact on the prevalence of lignin degradation pathways, it is worth noting that the termite gut has been found to have relatively high oxygen concentrations in some areas 
. Significant lignin degradation has been proposed to occur in the Asian longhorned beetle, which could prove an exception to the pattern seen here 
. This near-complete lack of putative lignin degrading enzymes was shared by all other gut communities examined (including bovine rumen, wallaby, dog, human and mouse). In the case of the termite gut, the relatively high pH (10–12) of the higher gut may provide suitable solubilization of some lignins to permit the needed GHase accessibility to wall polysaccharides.
In conclusion, natural communities subsisting on untreated plant biomass provide an ideal environment for the bioprospecting of enzymes involved in the depolymerization of plant cell-wall polysaccharides and lignin. Our results seem to indicate that these communities have a much broader metabolic potential than host-associated communities and thus could provide a richer resource for finding new catalytic functions involved in biomass decomposition. This is exemplified by the microbial community that under anaerobic conditions is decaying poplar woodchips, as a very broad representation of glycoside hydrolases and putative lignin decomposing enzymes was found. Furthermore we unexpectedly identified bacteria among the dominant community members, similar to Magnetospirillum, that seem to play a key role in the anaerobic breakdown of aromatic compounds. We hypothesize that these compounds are released from the lignin fraction in the poplar hardwood during the decay process, which would point to lignin-depolymerization under anaerobic conditions.