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Title: Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times

Background: Anaerobic fermentation of lignocellulose occurs in both natural and managed environments, and is an essential part of the carbon cycle as well as a promising route to sustainable production of fuels and chemicals. Lignocellulose solubilization by mixed microbiomes is important in these contexts.Results: Here in this paper, we report the development of stable switchgrass-fermenting enrichment cultures maintained at various residence times and moderately high (55 °C) temperatures. Anaerobic microbiomes derived from a digester inoculum were incubated at 55 °C and fed semi-continuously with medium containing 30 g/L mid-season harvested switchgrass to achieve residence times (RT) of 20, 10, 5, and 3.3 days. Stable, time-invariant cellulolytic methanogenic cultures with minimal accumulation of organic acids were achieved for all RTs. Fractional carbohydrate solubilization was 0.711, 0.654, 0.581 and 0.538 at RT = 20, 10, 5 and 3.3 days, respectively, and glucan solubilization was proportional to xylan solubilization at all RTs. The rate of solubilization was described well by the equation r = k(C-C 0f r), where C represents the concentration of unutilized carbohydrate, C 0 is the concentration of carbohydrate (cellulose and hemicellulose) entering the bioreactor and f r is the extrapolated fraction of entering carbohydrate that is recalcitrant at infinitemore » residence time. The 3.3 day RT is among the shortest RT reported for stable thermophilic, methanogenic digestion of a lignocellulosic feedstock. 16S rDNA phylotyping and metagenomic analyses were conducted to characterize the effect of RT on community dynamics and to infer functional roles in the switchgrass to biogas conversion to the various microbial taxa. Firmicutes were the dominant phylum, increasing in relative abundance from 54 to 96% as RT decreased. A Clostridium clariflavum strain with genetic markers for xylose metabolism was the most abundant lignocellulose-solubilizing bacterium. A Thermotogae (Defluviitoga tunisiensis) was the most abundant bacterium in switchgrass digesters at RT = 20 days but decreased in abundance at lower RTs as did multiple Chloroflexi. Synergistetes and Euryarchaeota were present at roughly constant levels over the range of RTs examined.Conclusions: A system was developed in which stable methanogenic steady-states were readily obtained with a particulate biomass feedstock, mid-season switchgrass, at laboratory (1 L) scale. Characterization of the extent and rate of carbohydrate solubilization in combination with 16S rDNA and metagenomic sequencing provides a multi-dimensional view of performance, species composition, glycoside hydrolases, and metabolic function with varying residence time. These results provide a point of reference and guidance for future studies and organism development efforts involving defined cultures.« less
Authors:
ORCiD logo [1] ; ORCiD logo [2] ;  [1] ;  [1] ;  [1] ;  [3] ;  [4] ;  [5] ; ORCiD logo [2] ;  [6] ; ORCiD logo [6] ;  [7] ; ORCiD logo [2] ; ORCiD logo [8] ;  [1]
  1. Dartmouth College, Hanover, NH (United States). Thayer School of Engineering; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Sciences Center
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Sciences Center, and Biosciences Division
  3. Taipei Medical Univ., Taipei (Taiwan). Graduate Inst. of Biomedical Informatics, College of Medical Science and Technology
  4. Aix-Marseille Univ., and CNRS, Marseille (France); French National Inst. for Agricultural Research (INRA), Marseille (France)
  5. Aix-Marseille Univ., and CNRS, Marseille (France); French National Inst. for Agricultural Research (INRA), Marseille (France); King Abdulaziz Univ., Jeddah (Saudi Arabia). Dept. of Biological Sciences
  6. BioEnergy Sciences Center, and Biosciences Division
  7. Pennsylvania State Univ., University Park, PA (United States). Dept. of Agricultural and Biological Engineering
  8. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Sciences Center, and Biosciences Division; LanzaTech, Inc., Skokie, IL (United States)
Publication Date:
Grant/Contract Number:
AC05-00OR22725; AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Research Org:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; Lignocellulose; Anaerobic; Methanogenic; Thermophilic; Solubilization; Microbial communities; Metagenomics; Clostridium clariflavum
OSTI Identifier:
1471931

Liang, Xiaoyu, Whitham, Jason M., Holwerda, Evert K., Shao, Xiongjun, Tian, Liang, Wu, Yu-Wei, Lombard, Vincent, Henrissat, Bernard, Klingeman, Dawn M., Yang, Zamin K., Podar, Mircea, Richard, Tom L., Elkins, James G., Brown, Steven D., and Lynd, Lee R.. Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times. United States: N. p., Web. doi:10.1186/s13068-018-1238-1.
Liang, Xiaoyu, Whitham, Jason M., Holwerda, Evert K., Shao, Xiongjun, Tian, Liang, Wu, Yu-Wei, Lombard, Vincent, Henrissat, Bernard, Klingeman, Dawn M., Yang, Zamin K., Podar, Mircea, Richard, Tom L., Elkins, James G., Brown, Steven D., & Lynd, Lee R.. Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times. United States. doi:10.1186/s13068-018-1238-1.
Liang, Xiaoyu, Whitham, Jason M., Holwerda, Evert K., Shao, Xiongjun, Tian, Liang, Wu, Yu-Wei, Lombard, Vincent, Henrissat, Bernard, Klingeman, Dawn M., Yang, Zamin K., Podar, Mircea, Richard, Tom L., Elkins, James G., Brown, Steven D., and Lynd, Lee R.. 2018. "Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times". United States. doi:10.1186/s13068-018-1238-1. https://www.osti.gov/servlets/purl/1471931.
@article{osti_1471931,
title = {Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times},
author = {Liang, Xiaoyu and Whitham, Jason M. and Holwerda, Evert K. and Shao, Xiongjun and Tian, Liang and Wu, Yu-Wei and Lombard, Vincent and Henrissat, Bernard and Klingeman, Dawn M. and Yang, Zamin K. and Podar, Mircea and Richard, Tom L. and Elkins, James G. and Brown, Steven D. and Lynd, Lee R.},
abstractNote = {Background: Anaerobic fermentation of lignocellulose occurs in both natural and managed environments, and is an essential part of the carbon cycle as well as a promising route to sustainable production of fuels and chemicals. Lignocellulose solubilization by mixed microbiomes is important in these contexts.Results: Here in this paper, we report the development of stable switchgrass-fermenting enrichment cultures maintained at various residence times and moderately high (55 °C) temperatures. Anaerobic microbiomes derived from a digester inoculum were incubated at 55 °C and fed semi-continuously with medium containing 30 g/L mid-season harvested switchgrass to achieve residence times (RT) of 20, 10, 5, and 3.3 days. Stable, time-invariant cellulolytic methanogenic cultures with minimal accumulation of organic acids were achieved for all RTs. Fractional carbohydrate solubilization was 0.711, 0.654, 0.581 and 0.538 at RT = 20, 10, 5 and 3.3 days, respectively, and glucan solubilization was proportional to xylan solubilization at all RTs. The rate of solubilization was described well by the equation r = k(C-C0fr), where C represents the concentration of unutilized carbohydrate, C0 is the concentration of carbohydrate (cellulose and hemicellulose) entering the bioreactor and fr is the extrapolated fraction of entering carbohydrate that is recalcitrant at infinite residence time. The 3.3 day RT is among the shortest RT reported for stable thermophilic, methanogenic digestion of a lignocellulosic feedstock. 16S rDNA phylotyping and metagenomic analyses were conducted to characterize the effect of RT on community dynamics and to infer functional roles in the switchgrass to biogas conversion to the various microbial taxa. Firmicutes were the dominant phylum, increasing in relative abundance from 54 to 96% as RT decreased. A Clostridium clariflavum strain with genetic markers for xylose metabolism was the most abundant lignocellulose-solubilizing bacterium. A Thermotogae (Defluviitoga tunisiensis) was the most abundant bacterium in switchgrass digesters at RT = 20 days but decreased in abundance at lower RTs as did multiple Chloroflexi. Synergistetes and Euryarchaeota were present at roughly constant levels over the range of RTs examined.Conclusions: A system was developed in which stable methanogenic steady-states were readily obtained with a particulate biomass feedstock, mid-season switchgrass, at laboratory (1 L) scale. Characterization of the extent and rate of carbohydrate solubilization in combination with 16S rDNA and metagenomic sequencing provides a multi-dimensional view of performance, species composition, glycoside hydrolases, and metabolic function with varying residence time. These results provide a point of reference and guidance for future studies and organism development efforts involving defined cultures.},
doi = {10.1186/s13068-018-1238-1},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 11,
place = {United States},
year = {2018},
month = {9}
}

Works referenced in this record:

Glycoside Hydrolase Activities of Thermophilic Bacterial Consortia Adapted to Switchgrass
journal, July 2011
  • Gladden, John M.; Allgaier, Martin; Miller, Christopher S.
  • Applied and Environmental Microbiology, Vol. 77, Issue 16, p. 5804-5812
  • DOI: 10.1128/AEM.00032-11

MUSCLE: multiple sequence alignment with high accuracy and high throughput
journal, March 2004
  • Edgar, R. C.
  • Nucleic Acids Research, Vol. 32, Issue 5, p. 1792-1797
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