Characterization of Trapped Lignin-Degrading Microbes in Tropical Forest Soil

Lignin is often the most difficult portion of plant biomass to degrade, with fungi generally thought to dominate during late stage decomposition. Lignin in feedstock plant material represents a barrier to more efficient plant biomass conversion and can also hinder enzymatic access to cellulose, which is critical for biofuels production. Tropical rain forest soils in Puerto Rico are characterized by frequent anoxic conditions and fluctuating redox, suggesting the presence of lignin-degrading organisms and mechanisms that are different from known fungal decomposers and oxygen-dependent enzyme activities. We explored microbial lignin-degraders by burying bio-traps containing lignin-amended and unamended biosep beads in the soil for 1, 4, 13 and 30 weeks. At each time point, phenol oxidase and peroxidase enzyme activity was found to be elevated in the lignin-amended versus the unamended beads, while cellulolytic enzyme activities were significantly depressed in lignin-amended beads. Quantitative PCR of bacterial communities showed more bacterial colonization in the lignin-amended compared to the unamended beads after one and four weeks, suggesting that the lignin supported increased bacterial abundance. The microbial community was analyzed by small subunit 16S ribosomal RNA genes using microarray (PhyloChip) and by high-throughput amplicon pyrosequencing based on universal primers targeting bacterial, archaeal, and eukaryotic communities. Community trends were significantly affected by time and the presence of lignin on the beads. Lignin-amended beads have higher relative abundances of representatives from the phyla Actinobacteria, Firmicutes, Acidobacteria and Proteobacteria compared to unamended beads. This study suggests that in low and fluctuating redox soils, bacteria could play a role in anaerobic lignin decomposition

Evaluating DNA Extraction Methods for Community Profiling of Pig Hindgut Microbial Community

Recovery of high quality PCR-amplifiable DNA has been the general minimal requirement for DNA extraction methods for bulk molecular analysis. However, modern high through-put community profiling technologies are more sensitive to representativeness and reproducibility of DNA extraction method. Here, we assess the impact of three DNA extraction methods (with different levels of extraction harshness) for assessing hindgut microbiomes from pigs fed with different diets (with different physical properties). DNA extraction from each sample was performed in three technical replicates for each extraction method and sequenced by 16S rRNA amplicon sequencing. Host was the primary driver of molecular sequencing outcomes, particularly on samples analysed by wheat based diets, but higher variability, with one failed extraction occurred on samples from a barley fed pig. Based on these results, an effective method will enable reproducible and quality outcomes on a range of samples, whereas an ineffective method will fail to generate extract, but host (rather than extraction method) remains the primary factor

Hemicellulolytic organisms in the particle-associated microbiota of the hoatzin crop

The hoatzin (Opisthocomus hoazin) is a South American herbivorous bird, that has an enlarged crop analogous to the rumen, where foregut microbes degrade the otherwise indigestible plant materials, providing energy to the host. The crop harbors an impressive array of microorganisms with potentially novel cellulolytic enzymes. Thie study describes the composition ofthe particle-associated microbiota in the hoatzin crop, combining a survey of 16S rRNA genes in 7 adult birds and metagenome sequencing of two animals. The pyrotag survey demonstrates that Prevotellaceae, are the most abundant and ubiquitous taxa, suggesting that the degradation of hemicellulose is an important activity in the crop. Nonetheless, preliminary results from the metagnome of the particle-associated microbiota of two adult birds show that the crop microbiome contains a high number of genes encoding cellulases (such as GH5) more abundant than those of the termite gut, as well as genes encoding hemicellulases. These preliminary results show that the carbohydate-active enzyme genes in the cropmetagenome could be a source of biochemical catalysts able to deconstruct plant biomass

Trapping Lignin Degrading Microbes In Tropical Forest Soil

Lignin in feedstock plant material represents a barrier to more efficient plant-to-biomass conversion and can also hinder enzymatic access to cellulose. For this reason, it is critical to develop a way to degrade recovered lignin for next generation feedstock-derived biofuels. While the best-known ligninases are fungal, bacteria are more amenable to emerging cellulosic biofuels technologies. Tropical rain forest soils in Puerto Rico are likely dominated by bacterial decomposers because of the frequent anoxic conditions and fluctuating redox characteristic of these soils, so we focused here to search for novel bacterial lignase producers. To do this, we buried bug traps containing lignin-amended and unamended biosep beads in the soil and incubated them for 1, 4, 13 and 30 weeks. At each time point, phenol oxidase and peroxidase enzyme activity was found to be elevated in the lignin-amended versus the unamended beads, while cellulolytic enzyme activities were significantly depressed in lignin-amended beads. Quantitative PCR of bacterial communities showed more colonization in the lignin-amended compared to the unamended beads after one and four weeks, which attenuated over the course of the incubation. The microbial community was analyzed by microarray (PhyloChip) and by pyrotag sequencing of the community16S ribosomal RNA genes. Community trends were strongly driven by time but also lignin-amendment to the beads. These techniques also allow us to identify which taxa were increased in lignin-amended compared to unamended beads, which included representatives from the phyla Actinobacteria, Firmicutes, Acidobacterial and Proteobacteria