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

X. Liang, J.M. Whitham, E.K. Holwerda, X. Shao, L. Tian, Y.-W. Wu, V. Lombard, B. Henrissat, D.M. Klingeman, Z.K. Yang, M. Podar, T.L. Richard, J.G. Elkins, S.D. Brown, L.R. Lynd

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Abstract

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, 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 0 f 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 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. © 2018 The Author(s).
Original languageEnglish
JournalBiotechnology for Biofuels
Volume11
Issue number1
DOIs
Publication statusPublished - 2018

Fingerprint

Panicum
Microbiota
Carbohydrates
residence time
solubilization
carbohydrate
Feedstocks
Bacteria
Clostridium
Xylose
Metagenomics
Organic acids
Biogas
Bioreactors
Metabolism
Fermentation
Ribosomal DNA
Cellulose
Biomass
Euryarchaeota

Keywords

  • Anaerobic
  • Clostridium clariflavum
  • Lignocellulose
  • Metagenomics
  • Methanogenic
  • Microbial communities
  • Solubilization
  • Thermophilic
  • Anaerobic digestion
  • Carbohydrates
  • Cellulose
  • Clostridium
  • Feedstocks
  • Hydrolases
  • Lignin
  • Metabolism
  • Plants (botany)
  • Pulp digesters
  • Solubility
  • Bacteria (microorganisms)
  • Chloroflexi
  • Chloroflexi (class)
  • Euryarchaeota
  • Firmicutes
  • Panicum virgatum
  • Thermotogae
  • Thermotogae (class)

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Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times. / Liang, X.; Whitham, J.M.; Holwerda, E.K.; Shao, X.; Tian, L.; Wu, Y.-W.; Lombard, V.; Henrissat, B.; Klingeman, D.M.; Yang, Z.K.; Podar, M.; Richard, T.L.; Elkins, J.G.; Brown, S.D.; Lynd, L.R.

In: Biotechnology for Biofuels, Vol. 11, No. 1, 2018.

Research output: Contribution to journalArticle

Liang, X, Whitham, JM, Holwerda, EK, Shao, X, Tian, L, Wu, Y-W, Lombard, V, Henrissat, B, Klingeman, DM, Yang, ZK, Podar, M, Richard, TL, Elkins, JG, Brown, SD & Lynd, LR 2018, 'Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times', Biotechnology for Biofuels, vol. 11, no. 1. https://doi.org/10.1186/s13068-018-1238-1
Liang, X. ; Whitham, J.M. ; Holwerda, E.K. ; Shao, X. ; Tian, L. ; Wu, Y.-W. ; Lombard, V. ; Henrissat, B. ; Klingeman, D.M. ; Yang, Z.K. ; Podar, M. ; Richard, T.L. ; Elkins, J.G. ; Brown, S.D. ; Lynd, L.R. / Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times. In: Biotechnology for Biofuels. 2018 ; Vol. 11, No. 1.
@article{b7c861d3822d46b386e120f2478e6d5a,
title = "Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times",
abstract = "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, 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 0 f 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 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. {\circledC} 2018 The Author(s).",
keywords = "Anaerobic, Clostridium clariflavum, Lignocellulose, Metagenomics, Methanogenic, Microbial communities, Solubilization, Thermophilic, Anaerobic digestion, Carbohydrates, Cellulose, Clostridium, Feedstocks, Hydrolases, Lignin, Metabolism, Plants (botany), Pulp digesters, Solubility, Bacteria (microorganisms), Chloroflexi, Chloroflexi (class), Euryarchaeota, Firmicutes, Panicum virgatum, Thermotogae, Thermotogae (class), Anaerobic, Clostridium clariflavum, Lignocellulose, Metagenomics, Methanogenic, Microbial communities, Solubilization, Thermophilic",
author = "X. Liang and J.M. Whitham and E.K. Holwerda and X. Shao and L. Tian and Y.-W. Wu and V. Lombard and B. Henrissat and D.M. Klingeman and Z.K. Yang and M. Podar and T.L. Richard and J.G. Elkins and S.D. Brown and L.R. Lynd",
note = "Export Date: 2 October 2018 通訊地址: Lynd, L.R.; Thayer School of Engineering, Dartmouth CollegeUnited States; 電子郵件: lee.r.lynd@dartmouth.edu 出資詳情: ORNL, Oak Ridge National Laboratory 出資詳情: DE‑AC02‑05CH11231 出資詳情: SC, Office of Science 出資詳情: PSU, Pennsylvania State University 出資詳情: DE‑AC05‑00OR22725 出資詳情: 2016‑10008‑25319 出資詳情: NIFA, National Institute of Food and Agriculture 出資正文: We thank the Vermont Technical College anaerobic digester team for providing samples for the initial inocula and Marvin Hall and Kay DiMarco from the Pennsylvania State University for partially preparing the feedstock for this study. We thank Sean J.L. Murphy and Robert S. Worthen for help in reactor maintenance. Chris Schadt (Bioscience Division, Oak Ridge National Laboratory) provided instructional support for building a phylogenetic tree in Geneious and Michael Robeson (Interstitial Genomics, LLC) provided scripts and consultation for 16S amplicon data processing and analysis. Metagen‑ omic data were generated and annotated by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under Contract no. DE‑AC02‑05CH11231. 出資正文: This research was sponsored primarily by the BioEnergy Science Center, a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research (Grant No. DE‑AC05‑00OR22725) in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT‑Battelle, LLC, for the US DOE under contract DE‑AC05‑00OR22725. This work was also supported by the Biomass Research and Development Initiative (Grant No. 2016‑10008‑25319) from the National Institute of Food and Agricul‑ ture, US Department of Agriculture. 參考文獻: Sawatdeenarunat, C., Surendra, K.C., Takara, D., Oechsner, H., Khanal, S.K., Anaerobic digestion of lignocellulosic biomass: Challenges and opportunities (2015) Bioresour Technol, 178, pp. 178-186; Podkaminer, K., Lin, Z., (2017) Analyzing the Impacts of A Biogas-to-electricity Purchase Incentive on Electric Vehicle Deployment with the MA3T Vehicle Choice Model, , US Department of Energy Washington, D.C; Lynd, L.R., Weimer, P.J., Van Zyl, W.H., Pretorius, I.S., Microbial cellulose utilization: Fundamentals and biotechnology (2002) Microbiol Mol Biol Rev, 66, pp. 506-577. , http://www.ncbi.nlm.nih.gov/pubmed/12209002, Accessed 23 Dec 2016; Paye, J.M.D., Guseva, A., Hammer, S.K., Gjersing, E., Davis, M.F., Davison, B.H., Biological lignocellulose solubilization: Comparative evaluation of biocatalysts and enhancement via cotreatment (2016) Biotechnol Biofuels, 9, pp. 1-13; Li, Y., Park, S.Y., Zhu, J., Solid-state anaerobic digestion for methane production from organic waste (2011) Renew Sustain Energy Rev, 15, pp. 821-826; Pohl, M., Mumme, J., Heeg, K., Nettmann, E., Thermo- and mesophilic anaerobic digestion of wheat straw by the upflow anaerobic solid-state (UASS) process (2012) Bioresour Technol, 124, pp. 321-327; Sheets, J.P., Ge, X., Li, Y., Effect of limited air exposure and comparative performance between thermophilic and mesophilic solid-state anaerobic digestion of switchgrass (2015) Bioresour Technol, 180, pp. 296-303; Holwerda, E.K., Lynd, L.R., Testing alternative kinetic models for utilization of crystalline cellulose (Avicel) by batch cultures of Clostridium thermocellum (2013) Biotechnol Bioeng, 110, pp. 2389-2394; Mata-Alvarez, J., Mac{\'e}, S., Llabr{\'e}s, P., Anaerobic digestion of organic solid wastes. 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year = "2018",
doi = "10.1186/s13068-018-1238-1",
language = "English",
volume = "11",
journal = "Biotechnology for Biofuels",
issn = "1754-6834",
publisher = "BioMed Central Ltd.",
number = "1",

}

TY - JOUR

T1 - Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times

AU - Liang, X.

AU - Whitham, J.M.

AU - Holwerda, E.K.

AU - Shao, X.

AU - Tian, L.

AU - Wu, Y.-W.

AU - Lombard, V.

AU - Henrissat, B.

AU - Klingeman, D.M.

AU - Yang, Z.K.

AU - Podar, M.

AU - Richard, T.L.

AU - Elkins, J.G.

AU - Brown, S.D.

AU - Lynd, L.R.

N1 - Export Date: 2 October 2018 通訊地址: Lynd, L.R.; Thayer School of Engineering, Dartmouth CollegeUnited States; 電子郵件: lee.r.lynd@dartmouth.edu 出資詳情: ORNL, Oak Ridge National Laboratory 出資詳情: DE‑AC02‑05CH11231 出資詳情: SC, Office of Science 出資詳情: PSU, Pennsylvania State University 出資詳情: DE‑AC05‑00OR22725 出資詳情: 2016‑10008‑25319 出資詳情: NIFA, National Institute of Food and Agriculture 出資正文: We thank the Vermont Technical College anaerobic digester team for providing samples for the initial inocula and Marvin Hall and Kay DiMarco from the Pennsylvania State University for partially preparing the feedstock for this study. We thank Sean J.L. Murphy and Robert S. Worthen for help in reactor maintenance. Chris Schadt (Bioscience Division, Oak Ridge National Laboratory) provided instructional support for building a phylogenetic tree in Geneious and Michael Robeson (Interstitial Genomics, LLC) provided scripts and consultation for 16S amplicon data processing and analysis. Metagen‑ omic data were generated and annotated by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under Contract no. DE‑AC02‑05CH11231. 出資正文: This research was sponsored primarily by the BioEnergy Science Center, a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research (Grant No. DE‑AC05‑00OR22725) in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT‑Battelle, LLC, for the US DOE under contract DE‑AC05‑00OR22725. 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PY - 2018

Y1 - 2018

N2 - 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, 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 0 f 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 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. © 2018 The Author(s).

AB - 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, 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 0 f 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 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. © 2018 The Author(s).

KW - Anaerobic

KW - Clostridium clariflavum

KW - Lignocellulose

KW - Metagenomics

KW - Methanogenic

KW - Microbial communities

KW - Solubilization

KW - Thermophilic

KW - Anaerobic digestion

KW - Carbohydrates

KW - Cellulose

KW - Clostridium

KW - Feedstocks

KW - Hydrolases

KW - Lignin

KW - Metabolism

KW - Plants (botany)

KW - Pulp digesters

KW - Solubility

KW - Bacteria (microorganisms)

KW - Chloroflexi

KW - Chloroflexi (class)

KW - Euryarchaeota

KW - Firmicutes

KW - Panicum virgatum

KW - Thermotogae

KW - Thermotogae (class)

KW - Anaerobic

KW - Clostridium clariflavum

KW - Lignocellulose

KW - Metagenomics

KW - Methanogenic

KW - Microbial communities

KW - Solubilization

KW - Thermophilic

U2 - 10.1186/s13068-018-1238-1

DO - 10.1186/s13068-018-1238-1

M3 - Article

VL - 11

JO - Biotechnology for Biofuels

JF - Biotechnology for Biofuels

SN - 1754-6834

IS - 1

ER -