Tracking dynamics of plant biomass composting by changes in substrate structure, microbial community, and enzyme activity

Abstract

Background: Understanding the dynamics of the microbial communities that, along with their secreted enzymes, are involved in the natural process of biomass composting may hold the key to breaking the major bottleneck in biomass-to-biofuels conversion technology, which is the still-costly deconstruction of polymeric biomass carbohydrates to fermentable sugars.However, the complexity of both the structure of plant biomass and its counterpart microbial degradation communities makes it difficult to investigate the composting process.

Results: In this study, a composter was set up with a mix of yellow poplar (Liriodendron tulipifera) wood-chips and mown lawn grass clippings (85:15 in dry-weight) and used as a model system. The microbial rDNA abundance data obtained from analyzing weekly-withdrawn composted samples suggested population-shifts from bacteria-dominated to fungus-dominated communities. Further analyses by an array of optical microscopic, transcriptional and enzyme-activity techniques yielded correlated results, suggesting that such population shifts occurred along with early removal of hemicellulose followed by attack on the consequently uncovered cellulose as the composting progressed.

Conclusion: The observed shifts in dominance by representative microbial groups, along with the observed different patterns in the gene expression and enzymatic activities between cellulases, hemicellulases, and ligninases during the composting process, provide new perspectives for biomass-derived biotechnology such as consolidated bioprocessing (CBP) and solid-state fermentation for the production of cellulolytic enzymes and biofuels.

Figure 1

Compost setup and sampling. (A) The apparatus for composting of yellow poplar wood chips. (B) Representative samples collected showing morphological changes of composted poplar chips, i.e., size reduction, color darkening, and material softening. (C) Temperature and oxygen concentration measured, as described in Materials and Methods, during composting process. wk: week.

Figure 2

Cross-section micrographs of yellow poplar wood chips. (Top panel) Bright field microscopy shows composting effects on the cell wall structure over 24 weeks. (Bottom panel) fluorescence microcopy of the same field labeled by the CtCBM3-GFP probe that binds to cellulose specifically. Increasing fluorescence intensity indicates higher cellulose accessibility to the probe. CtCBM3-GFP: family 3a carbohydrate-binding module tagged by green-fluorescent-protein. wk: week.

Figure 3

Relative abundance of total genomic DNAs extracted from composted yellow poplar chips, and microbial rDNAs by PCR using primers in Table 2. Samples were collected in composting time at 3, 6, 9, 15, 18, 24, and 27 weeks. (A) Amount of total genomic DNAs above the baseline genomic DNAs in compost at 1 week which was 32.7 ± 2.6 μg/g FW compost (FW: fresh weight). (B) Relative level of bacterial, archaeal and fungal rDNAs. 16 s rDNA was used in bacteria and archaea, and 5.8 s and ITS2 rDNA were used in fungi. The bacterial rDNA abundance at 3 weeks was set as 1- fold; archaeal and fungal rDNA relative level was adjusted to bacterial rDNA abundance at 3 weeks. The insert in (B) shows the archaeal rDNA profiling with a fine scale for the relative levels of archaeal rDNA. Note the bacteria-dominant stage at 9 weeks, whereas the fungi-dominant and overall peak stage occurs at 18 weeks. (C) Relative levels of Trichoderma spp. ITS rDNA. Error bars indicate the standard errors of the mean (S.E. ± mean) for the three replicates.

Figure 4

Transcriptional level of representative cellulolytic functional genes in Trichoderma sp. by real-time RT-PCR during composting of yellow poplar chips. (A) Xylanases 1 and 2 (xyn1 and xyn2). (B) Cellobiohydrolase I (cbh1), endoglucanase I (egl1) and β-glucosidase 1 (bgl1) were used in a set of representative species of Trichoderma genus. The gene expression level at each sampling time point of composting was first normalized with the Trichoderma sp. ITS rRNA, and then compared to their respective expression levels at 3 weeks (each of which was set as 1 fold). The primers for these genes were described in Table 3. Note that identical scales in the X axis of panels A-B allows a direct visual comparison of the magnitude of changes in gene expression levels.

Figure 5

Transcriptional level of representative lignin degradation-related genes during the composting of yellow poplar chips. (A) Manganese peroxidase (MnP1 and MnP2). (B) Lignin peroxidase (LiP A/B, D, H and J). Gene sequences of fungus Phanerochaete chrysosporium were used to design primers for real-time RT-PCR. For each gene the expression level at each sampling time point of composting was compared to its expression level at 3 weeks (which was set as 1 fold).

Figure 6

Total cellulase and hemicellulase activities agaist model substrates measured in composted yellow poplar, as a function of composting time. Activities are normalized to solids content of the compost sample and are averaged values from three replicates. Asterisks indicate statistically significant differences from the control (* for p < 0.05; ** for p < 0.01). Fluorogenic model substrates were used for the cellulase assay: MUC, 4-methylumbelliferyl-β-D-cellobioside; MUG, 4-methylumbelliferyl-β-D-glucoside. Hemicellulase assays utilized the respective 4-methylumbelliferyl-β-D-glycosides of the monosaccharides D-xylose, D-mannose, D-arabinose, and D-galactose.