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Revealing the secretome and enriching the function of newly established hyperthermophilic microbial communities with superior cellulolytic and hemicellulolytic activities

EMSL Project ID


Vast amount of organic waste materials is generated in U.S. These carbon-containing waste streams represent a potentially valuable but underutilized feedstock for production of carbon-neutral biofuels and commodity chemicals. Current technologies for waste processing have limited capability in hydrolysis of refractory fractions of waste including lignocellulosic materials. Therefore, our ongoing research efforts are focused on developing a novel biological process based on the superior hydrolytic and metabolic capabilities of the hyperthermophilic microbial communities proliferating at temperature of 70C and above. This innovative process aims to significantly improve carbon conversion efficiency and to reduce the overall processing time for minimizing capital and operating costs. Our preliminary results reproducibly confirm the enhanced capacity of the hyperthermophilic microbial communities established in our studies. However, high richness and complexity of these hyperthermophilic communities hinders our ability to characterize key species, pathways and specific enzymes responsible for improved conversion of cellulose and hemicellulose-containing biomass into bioproducts. This challenge could be addressed through high throughput generation and characterization of reduced complexity communities and isolates capable for efficient hydrolysis of recalcitrant components of biomass. Moreover, generation of a “library” of function-specific hyperthermophilic reduced complexity communities, species and enzymes will allow application of various omics’ tools as well as synthetic biology and synthetic ecology approaches for their further optimization and deployment of various industry relevant platforms. Therefore, this project aims to: in a high-throughput and enzyme function-depended approach (i) generate reduced complexity microbiomes enriched for specific hydrolytic functions to serve as model functionally active hyperthermophilic subpopulations; (ii) isolate species responsible for specific improved hydrolytic activities; (iii) identify and characterize thermostable hydrolytic enzymes expressed and secreted in these hyperthermophilic communities, which are responsible for their superior capability to de-polymerize cellulose and hemicellulose fiber, and waste biomass. First, we will generate functionally active reduced complexity subpopulations and isolate species with specific hydrolytic activities by using fluorescence-activated and function-dependent cell sorting. Second, we will probe-enhanced metaproteome profiling coupled with metatranscriptomics to identify unique secretome pools in most efficient hyper-thermophilic communities. Combining the metatranscriptome, metaproteomics data and activity profiling will enable us to significantly improve identification of novel enzymes and data reproducibility compared to metaproteome alone, and to elucidate the actual experimental response of community to given substrates extracellularly and intracellularly in terms of levels (from proteomics and transcriptomics), and activities and specificities (from ABPP and enzyme assays). This project can lead to generation of a “library” of the reduced complexity hyperthermophilic communities and isolates that can be utilized as model hyperthermophilic systems for characterization and synthetic ecology studies (bioreactor bioaugmentation experiments); identifying novel efficient thermostable enzymes for synthetic biology efforts and formulation of industry-relevant enzyme cocktails for improved degradation lignocellulose-containing materials.

Project Details

Project type
Exploratory Research
Start Date
End Date


Principal Investigator

Pavlo Bohutskyi
Pacific Northwest National Laboratory


Shulin Chen
Washington State University

Team Members

Zachary Johnson
Pacific Northwest National Laboratory

Natalie Sadler
Pacific Northwest National Laboratory