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

Xiaoyu Liang; Jason M. Whitham; Evert K. Holwerda; Xiongjun Shao; Liang Tian; Yu Wei Wu; Vincent Lombard; Bernard Henrissat; Dawn M. Klingeman; Zamin K. Yang; Mircea Podar; Tom L. Richard; James G. Elkins; Steven D. Brown; Lee R. Lynd

doi:10.1186/s13068-018-1238-1

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

Xiaoyu Liang, Jason M. Whitham, Evert K. Holwerda, Xiongjun Shao, Liang Tian, Yu Wei Wu, Vincent Lombard, Bernard Henrissat, Dawn M. Klingeman, Zamin K. Yang, Mircea Podar, Tom L. Richard, James G. Elkins, Steven D. Brown, Lee R. Lynd

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26 Scopus citations

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.

Original language	English
Article number	243
Journal	Biotechnology for Biofuels
Volume	11
Issue number	1
DOIs	https://doi.org/10.1186/s13068-018-1238-1
State	Published - Sep 6 2018

Funding

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. This manuscript has been authored by Dartmouth College and UT‑Battelle, LLC, under contract DE‑AC05‑00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid‑up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downl oads/doe‑public‑access‑plan). 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.

Funders	Funder number
BioEnergy Science Center
Biomass Research and Development Initiative	2016‑10008‑25319
National Institute of Food and Agricul
Office of Biological and Environmental Research
US Department of Agriculture
US Department of Energy
US Department of Energy Bioenergy Research Center
US Department of Energy Joint Genome Institute
U.S. Department of Energy	DE‑AC05‑00OR22725
Office of Science

Keywords

Anaerobic
Clostridium clariflavum
Lignocellulose
Metagenomics
Methanogenic
Microbial communities
Solubilization
Thermophilic

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1186/s13068-018-1238-1

Cite this

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. (2018). Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times. Biotechnology for Biofuels, 11(1), Article 243. https://doi.org/10.1186/s13068-018-1238-1

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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.",

keywords = "Anaerobic, Clostridium clariflavum, Lignocellulose, Metagenomics, Methanogenic, Microbial communities, Solubilization, Thermophilic",

author = "Xiaoyu Liang and Whitham, {Jason M.} and Holwerda, {Evert K.} and Xiongjun Shao and Liang Tian and Wu, {Yu Wei} and Vincent Lombard and Bernard Henrissat and Klingeman, {Dawn M.} and Yang, {Zamin K.} and Mircea Podar and Richard, {Tom L.} and Elkins, {James G.} and Brown, {Steven D.} and Lynd, {Lee R.}",

note = "Publisher Copyright: {\textcopyright} 2018 The Author(s).",

year = "2018",

month = sep,

day = "6",

doi = "10.1186/s13068-018-1238-1",

language = "English",

volume = "11",

journal = "Biotechnology for Biofuels",

issn = "1754-6834",

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Liang, X, Whitham, JM, Holwerda, EK, Shao, X, Tian, L, Wu, YW, 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, 243. https://doi.org/10.1186/s13068-018-1238-1

TY - JOUR

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

AU - Liang, Xiaoyu

AU - Whitham, Jason M.

AU - Holwerda, Evert K.

AU - Shao, Xiongjun

AU - Tian, Liang

AU - Wu, Yu Wei

AU - Lombard, Vincent

AU - Henrissat, Bernard

AU - Klingeman, Dawn M.

AU - Yang, Zamin K.

AU - Podar, Mircea

AU - Richard, Tom L.

AU - Elkins, James G.

AU - Brown, Steven D.

AU - Lynd, Lee R.

PY - 2018/9/6

Y1 - 2018/9/6

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.

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.

KW - Anaerobic

KW - Clostridium clariflavum

KW - Lignocellulose

KW - Metagenomics

KW - Methanogenic

KW - Microbial communities

KW - Solubilization

KW - Thermophilic

UR - http://www.scopus.com/inward/record.url?scp=85053104286&partnerID=8YFLogxK

U2 - 10.1186/s13068-018-1238-1

DO - 10.1186/s13068-018-1238-1

M3 - Article

AN - SCOPUS:85053104286

SN - 1754-6834

VL - 11

JO - Biotechnology for Biofuels

JF - Biotechnology for Biofuels

IS - 1

M1 - 243

ER -

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

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Keywords

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