As described in the Materials and Methods section, the composting bin (Figure ) was set up indoors, and temperature and oxygen levels in the composting mass were regularly monitored. Figure shows a typical compost pattern [3
] with three stages. Initially a mesophilic phase I (20°-50°C) of 6 weeks was observed, followed by a brief thermophilic phase (one week above 50°C). Thereafter, the temperature returned to the mesophilic range until the composting finished.
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) (more ...)
Conventional composts usually contain highly nitrogenous materials, such as sewage and manures, in relatively large proportions, and thus tend to produce more heat and result in higher temperatures, up to 80°C [3
], in the thermophilic phase. In contrast, our biomass compost contained less nitrogen and generated less heat, resulting in lower composting temperature.
The ambient oxygen concentration at the center of composter basically had a negative correlation with the compost temperature (Figure ). When the temperature was at 50°-53°C during the thermophilic phase, the ambient oxygen concentration reached levels as low as 4%.
Microscopic imaging reveals successive stages of deconstruction of composted biomass
Biomass was imaged at successive stages of decay using optical microscopy. Apparent decay of the plant cell wall structure was observed after 6 weeks of composting (Figure ). In the bright field (Figure , upper panel), the overall structure of the plant cell walls has begun to collapse at 15 weeks, and substantial biomass loss was observed at 24 weeks. In addition, a green-fluorescence-protein tagged carbohydrate-binding module (CBM) was used as probe to localize cellulose, and labeled samples were then imaged with fluorescence microscopy. The family 3a CBM used in this study, termed Ct
CBM3, was originally isolated from Clostridium thermocellum
scaffoldin protein, and has strong binding affinity to crystalline celluloses [17
]. Figure (lower panel) shows that the Ct
CBM3-GFP (green fluorescent protein) binding to yellow poplar cross-sections increased after 15 to 24 weeks of composting, suggesting that more hemicellulosic material was degraded in the early stages of composting, leading to progressively greater cellulose exposure and increased access for the Ct
CBM3-GFP. Such observation is consistent with previous reports that removal of xylan enhances cellulose accessibility and digestibility [21
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 (more ...)
Compositional analysis for the composted materials
To assess the degradation effect of composting on the feedstocks, the compost samples collected at 1 and 27 weeks were used to measure the remaining amounts of cellulose, hemicellulose and lignin, along with other compositions, using the chemical analysis procedures described in Materials and Methods.
The results are shown in Table . Taking the data for week 1 samples as the "initial" numbers, we found that the cellulose content in compost samples changed from 39.2% to 19.5%, i.e. decreased by 50%. The contents of xylan and mannan, two major hemicellulose components, in compost samples changed from 13.9% to 7.1%, and from 2.3% to 1.4%, respectively. In other words, xylan and mannan decreased by 49% and 40%, respectively. Taken together, these data indicate that the rates of the decrease in amounts of cellulose, xylan and acetyl groups (which mainly link with or exist in xylan) are very similar, between 47% and 50%. This degradation effect is significant, and is comparable with the literature reported recalcitrance index (RI) value for hardwood biomass yellow poplar, which is 0.56 (means 44% of biomass is degradable; see the Discussion section).
Compositional data for the composted materials
Contrary to the trends seen for hemicellulose and cellulose contents, an increase in the content of lignin was observed (Table ). This is likely a result of the absolute amount of lignin staying the same while the absolute amounts of other components decrease, rather than of the generation of lignin in composting. The percent by weight of structural protein increased as well, the much larger proportional change (more than 10-fold) in this case likely reflecting actual increases in absolute amounts of composting organisms and enzymes.
Future study of chemical compositional analysis at more sampling time points will be helpful to provide deeper insights into the composting process.
rDNA shifts reflect environmental and microbial-population shifts
Samples from 3, 6, 9, 15, 18, 24, and 27 weeks of composting were collected for total genomic DNA extraction. Figure shows that the amounts of total genomic DNA increase steadily along the time course of composting with a peak at 18 weeks, followed by a decline in 24-27 weeks. This result appears to be correlated with the recorded drop in temperature during the late stages of composting (Figure ).
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 (more ...)
Like many other environmental samples, extracts from composted biomass materials may contain high concentrations of organic matter. For example, humic acids commonly persist in isolated genomic DNA, and can be inhibitory to PCR and thereby compromise the quantitation of rDNA abundance. To address this issue, a serial dilution of isolated genomic DNA was tested to optimize the template concentration and to eliminate the effect of inhibitors. Using primers listed in Table , we found that genomic DNA concentrations between 0.08 and 2.5 ng per well (20 μl reaction volume) resulted in a linear relationship between Ct (cycle threshold, which is the main output of real-time PCR data, defined as the number of cycles required for the fluorescent signal to cross the threshold, i.e. exceed the background level) and the log of DNA concentration. The PCR amplification efficiency values, calculated as 10(-1/slope)
(in which the slope refers to the above-constructed Ct vs. the log of DNA concentration standard curve), were calculated to be 1.72, 1.80, and 1.81 for the archaeal, bacterial, and fungal rDNA primers, respectively. Note that a PCR amplification efficiency value of 2 means 100% success in PCR amplification. The obtained amplification efficiency values are comparable to those in other reports using the same or similar universal primers [22
]. These values were used to calibrate the PCR-based measurement of rDNA abundance in this study.
Domain-level primers used to amplify short-subunit rDNA genes from DNA extracts of biomass compost
To assess the diversity of each group of microbes at each stage of the composting, real-time PCRs were conducted using 2.5 ng genomic DNA per reaction and universal primers for 16 s rDNA (bacteria and archaea) and 5.8 s and ITS2 rDNA (fungi) (Table ). The archaeal, bacterial, and fungal rDNA relative abundance was first calculated with the delta-delta Ct method, using the bacterial rDNA level at 3 weeks as the common calibrator, and then normalized to the yield of total genomic DNA in each sample. The results are shown in Figure . Interestingly, archaea and bacteria have similar patterns in rDNA abundance; both had a gradual increase with a peak at 15 weeks, followed by a decline until the end of composting. In contrast, fungi had a more distinctive pattern for rDNA abundance, which peaked at 18 weeks in a more abrupt rising and falling manner.
The observed higher proportion of fungi in the later stage of composting (Figure ) suggests that while bacteria may be more active when hemicelluloses are the easily accessible carbohydrates, fungi are more active when celluloses and lignins become exposed and accessible. The composting stages at week 9 and 18 therefore represent bacterial and fungal dominant phases, respectively (Figure ), and are candidate time points for sampling RNA for future metatranscriptomic analysis.
In addition, we also determined the relative abundance of Trichoderma spp. ITS rDNA during the time course of yellow poplar composting (Figure ), as a measure of the presence and abundance of microorganisms of genus Trichoderma. This paves the way for the profiling of functional gene expression for representative species in this genus, as described in later section.
In summary, the domain-level screening provided a strong timeline characterization for the composting process. The data from relative rDNA abundance for the microbial groups pointed to population shifts in the microbial composition during the composting process.
It is noteworthy that, in contrast to the dynamic changes in relative abundance of bacteria and fungi, archaea remain relatively stable in the amount of rDNA (1-2%) throughout the course of the composting. Archaeal mass found in present yellow poplar compost is similar to that reported in other ecosystems, such as agricultural and field soils [27
], suggesting as-yet-unknown roles for archaea in biomass decay systems.
Functional gene expression profiling
To deeply understand the dynamics of biomass composting, it is important to conduct functional expression profiling related specifically to the biomass-degrading process, namely of the known genes encoding cellulases, hemicellulases, and lignin-modification enzymes. However, such functional studies are challenging because of the vast variety in the types of cell-wall-degrading enzymes and the lack of experimentally-validated function annotations of related genes in public databases. As described above, the population dominance shifted from bacteria to fungi in later composting stage. We therefore focused on fungal genes. To this end, several model cellulolytic fungi for which genome sequences are available were selected for sequence alignment and primer design for RT-PCR of functional genes. Table shows the list of primer sequences used for genes encoding cellulases, hemicellulases and β-glucosidases in the model aerobic fungus, Trichoderma
spp., which is the dominant genus found in various biomass decay ecosystems, as well as being a common producer for most of the cellulase and hemicellulase enzymes used in industry), and for genes apparently encoding ligninase enzymes in the white-rot fungus Phanerochaete chrysosporium
Subgenus- and species-level primers used to determine the transcriptional levels of fungal cellulolytic genes in biomass compost
With the exception of those for Trichoderma
sp. ITS rRNA [31
], and cbh
1/Cel7A and bgl
1/Cel3A [adapted from literature [32
]], and xyn
1 and xyn
2 of Trichoderma
spp. [adapted from literature [33
]], the primers employed were designed in this study.
Using the approaches described in the Materials and Methods section, expression levels of functional genes were calculated with the delta-delta Ct method, using Trichoderma spp. ITS (listed in Table ) or fungal 5.8 s and ITS2 rDNA/rRNA (listed in Table ) as an internal control for Trichoderma spp. and Phanerochaete chrysosporium genes, respectively. Each individual gene's mRNA level at 3 weeks was set as the reference value to calculate the subsequent fold changes.
(i) Transcription-level profiling of fungal hemicellulases and cellulases reflects coordination of gene expression in targeting progressively degrading biomass substrates
Trichoderma is a genus of fungi that exists, and often predominates, in broad types of soils and diverse environments including composts. Most species of this genus, including the industrial cellulase producer T. reesei, are saprophytes that can degrade bio-polymeric substrates such as lignocelluloses. This prompted us to use this genus as a model group to investigate the transcriptional dynamics of hemicellulase- and cellulase-encoding genes during the composting process.
To assess the relative expression levels of hemicellulases in composted samples, primers targeting both of the two major xylanases in Trichoderma (xyn1 and xyn2; Table ) were used in real-time RT-PCR analysis. The results demonstrated that the expression levels for these xylanases steadily increased between 6 to 15 weeks of composting (Figure ), and then declined markedly after that.
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 (more ...)
Meanwhile, to assess the expression of cellulase genes of genus Trichoderma during the composting, three pairs of group primers listed in Table were used for real-time RT-PCR. These primers correspond to three main categories of the Trichoderma cellulolytic enzyme systems that include cellobiohydrolase (CBH; exo-cellulases), endoglucanase (EGL; endo-cellulases) and beta-glucosidase (BGL). The gene expression profiling of the cellulases is shown in Figure , with an increase between 6 to 18 weeks of composting, and a decrease thereafter. The expression patterns appeared to be similar among the tested genes during composting, an observation suggesting that these three types of cellulases may be expressed in a coordinated way that can enhance the overall efficiency of cellulose degradation.
Interestingly, as shown in Figure , the gene expression of hemicellulases and cellulolytic system peaked at 15 and 18 weeks, respectively, suggesting that the microbial communities produce hemicellulases earlier than cellulases.
(ii) Transcription-level profiling of fungal LiPs and MnPs
is a model fungus that can degrade lignin without "touching" the cellulose of the wood. Like other white rot fungi, P. chrysosporium
secretes an array of peroxidases and oxidases that attack lignin [30
]. We have successfully designed primers, as listed in Table , for the genes encoding manganese peroxidases (MnPs) and lignin peroxidases (LiPs). Real-time RT-PCR was used in determining their expression levels.
As shown in Figure , the maximal fold changes of MnP1 and MnP2 were relatively small, in that the peak of MnP1 expression was at 15 weeks with a 1.5-fold increase (relative to the expression level at 3 weeks), while MnP2 expression peaked later at 18 weeks with a larger (2.7-fold) increase. In contrast, the expression levels of the four LiP genes peaked at 18 weeks with more prominent changes than that of the MnP genes (Figure ). Peaks are observed in the expression levels for LiPA/B and LiPD at 18 weeks, whereas the fold-values for LiPH and LiPJ, while also maximal at 18 weeks, maintained quite high expression levels at 24 weeks, indicating a longer high plateau for their expressions.
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 (more ...)
In this study, we examined the expression patterns of a total of six P. chrysosporium
genes at seven sampling time points (from 3 weeks to 24 weeks yellow poplar composting); we glean from the expression profiling data that the two MnP
genes are likely to be regulated differently, not only between themselves but also from the LiPs
examined (Figure ). This is in agreement with the findings by Janse et al. and Orth et al. who showed that MnP
1-3 genes are genetically unlinked to each other or to any LiP
Hemicellulase and cellulase activities confirm microbial response to changes in chemical nature of exposed biomass surface
In addition to examining the expression levels of functional genes, another approach to studying the function of a microbial community is to measure the actual activities of enzymes that we are interested in (i.e., glycoside hydrolases, specifically cellulolytic and hemicellulolytic enzymes, among others). We used low-molecular-weight, soluble "model" substrates to assay activities in finely-ground samples of the total composted biomass materials, rather than in extracts. Our use of whole materials in the assays reflects our intention to conduct as comprehensive a survey as possible of the targeted glycoside hydrolase activities present in the composting material, including those activities tightly bound to the biomass as well as those readily extractable.
Using fluorogenic model substrates, we found that the cellulase activities show increasing predominance in later stages (24 weeks) of composting (Figure ). In contrast, the measured hemicellulase activities, mainly α-arabinosidase and β-galactosidase, were higher in the earlier stages (3 weeks). These results are consistent with the light and fluorescence microscope observations that showed celluloses are exposed mainly at the later stages of composting. These parallel optical and enzyme-activity surveys provide direct evidence that local microbial populations adjust their production of "harvesting" enzymes in response to the accessibility and digestibility of chemically different biomass materials (going after the more accessible and digestible materials first) and indirectly suggest that the makeup of the microbial population itself may change in response to the changes in the chemical and physical nature of the biomass as degradation proceeds.
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. (more ...)