Contact for DOE Bioenergy Research Centers

N. Kent Peters

U.S. Department of Energy Office of Science

Office of Biological and Environmental Research

kent.peters@science.doe.gov

Websites for DOE Bioenergy Research Centers

BioEnergy Science Center (BESC)

bioenergycenter.org

Great Lakes Bioenergy 

Research Center (GLBRC)

glbrc.org

Joint BioEnergy Institute (JBEI)

jbei.org

Suggested citation: U.S. DOE. 2018. U.S. Department of Energy Bioenergy Research Centers: 10-Year Retrospective; 

Breakthroughs and Impacts. DOE/SC-0193. U.S. Department of Energy Office of Science (genomicscience.energy.

gov/centers/brcretrospective.pdf).

Cover images: Switchgrass courtesy Great Lakes Bioenergy Research Center. Microbe Clostridium thermocellum 

growing on poplar biomass courtesy Jennifer Morrell-Falvey, BioEnergy Science Center. Poplar courtesy Joint 

BioEnergy Institute.
September 2018 BRCs: Breakthroughs and Impacts i

DOE/SC-0193

U.S. Department of Energy

Bioenergy Research Centers

10-Year Retrospective

Breakthroughs and Impacts 2007–2017

Prepared for the

U.S. Department of Energy Office of Science

Office of Biological and Environmental Research

September 2018

Web address for this document: 

genomicscience.energy.gov/centers/brcretrospective.pdf

Prepared by

Biological and Environmental Research Information System

Oak Ridge National Laboratory

Oak Ridge, TN 37830

Managed by UT-Battelle, LLC

For the U.S. Department of Energy

Under contract DE-AC05-00OR22725
ii U.S. Department of Energy Office of Science September 2018
September 2018 BRCs: Breakthroughs and Impacts iii

Contents

DOE Bioenergy Research Centers by the Numbers: 2007 to 2017............................................ iv

DOE Bioenergy Research Centers.........................................................................................................1

Ten-Year Retrospective .........................................................................................................................1

Complementary and Collaborative Approach................................................................................2

Sidebar: Accelerating Deployment of Biofuels Science and Commercialization...................2

Sidebar: Startup Companies Stemming from the BRCs..............................................................3

BRC Breakthroughs for Lignocellulosic Biofuels Production...................................................4

Top Accomplishments in Biomass Supply and Sustainability....................................................4

Sidebar: At a Glance: BRC Accomplishments from 2007 to 2017 ...........................................5

Top Accomplishments in Biomass Development..........................................................................7

Top Accomplishments in Biomass Deconstruction and Separation...................................... 10

Top Accomplishments in Conversion of Biomass to Biofuels................................................. 12

Sidebar: Non–Bioenergy Benefits of BRC Research ................................................................. 15

Top Accomplishments in Enabling Technologies...................................................................... 15

DOE Bioenergy Research Centers: Next Iteration ...................................................................... 17

References Cited in This Document.................................................................................................. 19

DOE Bioenergy Research Centers and Partners 

Spanning 2007 to 2017 (Map)............................................................................................Back Cover
iv U.S. Department of Energy Office of Science September 2018

• Biomass Supply and Sustainability 

• Biomass Development

• Biomass Deconstruction and Separation

• Conversion of Biomass to Biofuels

• Enabling Technologies

SCIENTIFIC STAFF*

BIOENERGY 

RESEARCH 

CENTERS (BRCS)

5 RESEARCH 

FOCUS 

AREAS

2,696

Publications

BIOENERGY RESEARCH CENTERS 

BY THE NUMBERS: 2007 TO 2017

EDUCATION, OUTREACH

BRC RESEARCH SPANNED

35

Academic, 

Nonprofit, 

and Industrial 

Institutions

7

DOE 

National 

Laboratories

25

States

TECHNOLOGICAL OUTPUT

Invention Disclosures ............................ 619

Patent Applications .............................. 397

Patents .................................................101

Licenses/Options ................................... 199

Company Startups .................................... 15

MULTIDISCIPLINARY TRAINING PROVIDED FOR MORE THAN 970 ALUMNI

From 2007 to 2017, more than 970 graduate 

students and postdoctoral researchers participated 

in the BRCs before moving on to other positions 

in academia, national laboratories, industry, and 

nonprofit research organizations.

* Total number of scientists and research laboratory technicians (excluding graduate students and postdoctoral researchers) supported 

over the 10-year period at the Great Lakes Bioenergy Research Center (GLBRC), BioEnergy Science Center (BESC), and Joint BioEnergy 

Institute (JBEI).

CITATIONS AS OF 

DECEMBER 2017

>113,000

IMPACT ON FUTURE SCIENCE

GLBRC

~325

BESC

~250

JBEI

~150 EDUCATORS

U.S. DEPARTMENT OF ENERGY

>1,000 >650,000

STUDENTS

and increasing

GLBRC

60% Academia

25% Industry

15% Nonprofit

Research 

Organizations

BESC

45% Industry

40% Academia

15% National

Laboratories

JBEI

40% Academia

40% Industry

10% National

Laboratories

10% Other
September 2018 BRCs: Breakthroughs and Impacts 1

Ten-Year Retrospective

The Genomic Science program of the 

Office of Biological and Environmental 

Research (BER) within the U.S. Department of Energy (DOE) Office of Science focuses 

on understanding microbes, microbial communities, and plants as integrated systems of relevance 

to DOE’s energy and environmental missions. 

One aspect of this program seeks to develop the 

fundamental science, research technologies, and 

knowledgebase necessary to enable the costeffective, sustainable production of advanced 

biofuels and bioproducts from plant biomass. 

Lignocellulosic materials from plants provide 

the largest reservoir of raw materials for biofuels 

and biobased products, yet cost-effective use 

of these materials is hampered by their complexity and resistance to breakdown. Cellulose is contained within plant cell walls in the 

form of long, tightly bound chains of sugars 

(polysaccharides) that can be converted into 

biofuels and bioproducts by microbes and 

enzymes. Physically accessing these sugars, 

however, is difficult because the cellulose 

is embedded within a matrix of other polymers including hemicellulose and lignin, 

making it resistant to degradation (see Fig. 1. 

Three-Dimensional Illustration of Lignocellulose Meshwork, this page). This resistance, 

called recalcitrance, and a lack of efficient methods to convert lignocellulose to useful products 

are major impediments to the cost-effective 

production of biofuels and bioproducts from 

plant biomass.

To understand the challenges associated with 

lignocellulosic usage for biofuels, BER supported the initial three Bioenergy Research 

Centers (BRCs) from 2007 to 2017. The 

purpose of forming centers was to promote 

large, multidisciplinary, integrative efforts 

capable of addressing biofuel research from 

multiple angles and to accelerate transformational breakthroughs in the basic sciences 

needed to develop cost-effective and sustainable 

U.S. Department of Energy 

Bioenergy Research Centers

Fig. 1. ThreeDimensional Illustration of Lignocellulose 

Meshwork. Researchers are using computational modeling to 

gain a molecular-level 

understanding of the 

plant cell wall and its 

major components, 

including cellulose 

fibers (green), lignin 

molecules (brown 

wooden texture), and 

hemicellulose (light 

green). [Image courtesy 

Thomas Splettstoesser, 

www.scistyle.com, for 

Oak Ridge National 

Laboratory]
2 U.S. Department of Energy Office of Science September 2018

commercial production of cellulosic biofuels on 

a national scale (U.S. DOE 2006). 

The three BRCs were based in the geographically diverse Southeast, Midwest, and West 

Coast regions and included the BioEnergy 

Science Center (BESC), led by Oak Ridge 

National Laboratory; the Great Lakes Bioenergy Research Center (GLBRC), led by the 

University of Wisconsin–Madison in close 

partnership with Michigan State University; 

and the Joint BioEnergy Institute ( JBEI), led by 

Lawrence Berkeley National Laboratory. Center 

partners included experts from many areas of 

science and engineering, as well as economics, 

government, and industry.

Complementary and 

Collaborative Approach

The mission of these BRCs, broadly stated, was 

to advance science, engineering, and technology 

to support conversion of lignocellulosic biomass 

into renewable liquid transportation biofuels. 

Working toward this mission, the BRCs 

developed complementary and collaborative 

research portfolios to address the key technical 

and economic challenges in large-scale biofuel 

production from lignocellulosic biomass. 

In this endeavor, the BRCs coordinated sustainable biofuels research along the entire pathway, 

from creating new energy crops and new methods for deconstructing lignocellulosic material 

into chemical building blocks to creating new 

metabolic pathways inserted into microbial 

hosts to produce ethanol or even hydrocarbon fuels. This center-scale approach allowed 

technology development specialists to design 

automated pipelines that streamlined workflows 

and increased research efficiencies, enabled the 

testing of research ideas from proof of concept 

to field trials, and allowed research breakthroughs in one area to immediately inform 

research direction in other areas (see sidebars, 

Accelerating Deployment of Biofuels Science 

and Commercialization, this page; and Startup 

Companies Stemming from the BRCs, p. 3). 

Summary of Bioenergy Research Center (BRC) Scientific and Technological Output 

Through Fiscal Year 2017. The initial three BRCs collectively produced a portfolio of diverse 

and complementary scientific strategies that address the challenges of biomass conversion 

to biofuels on a scale far greater than any effort to date. The resulting knowledgebase is providing new insights to help industry meet the broad challenges of reducing the cost of and 

meeting the demand for advanced biofuels.

Accelerating 

Deployment of 

Biofuels Science and 

Commercialization

Scientific advances made by the 

Department of Energy’s Bioenergy Research Centers (BRCs) 

have provided crucial knowledge 

needed to develop new biobased 

fuels and products, methods, and 

tools that the emerging biofuel 

industry can use. Through intellectual property licensing agreements, partnerships, and targeted 

collaborative affiliations, the BRCs 

helped to speed the translation of 

basic research results to industry, 

contributing to clean energy (see 

figure at right).
September 2018 BRCs: Breakthroughs and Impacts 3

Startup Companies Stemming from the BRCs

Some examples of the transfer of Bioenergy Research Center (BRC) research to industry are included in the following list of 

startup companies. Each of these companies brings new technologies and products to the marketplace, and they all have 

roots in research initially conducted at the BRCs.

BioEnergy Science Center

• Gate Fuels Inc. is developing a low-cost platform for 

producing methyl ethyl ketone directly from pretreated 

cellulosic biomass.

• Enchi Corporation has licensed a custom-engineered 

microbe, Clostridium thermocellum, to digest and convert 

biomass into ethanol.

• Vertimass LLC is commercializing a “green” catalyst technology that converts ethanol into gasoline, diesel, and jet 

fuel–blend stocks that are compatible with the current 

transportation fuel infrastructure.

Great Lakes Bioenergy Research Center

• Glucan Biorenewables LLC is using gammavalerolactone, a plant-based solvent, to help convert 

biomass into three streams of high-value industrial 

chemicals.

• Hyrax Energy Inc., which existed from 2011 to 2018,

used an improved method for extracting fermentable 

sugars from biomass to improve the economics of the 

cellulosic biofuel pipeline.

• Kopess Biomass Solutions is working to unlock the 

genetic regulators of cellulose biosynthesis in plants.

• Lactic Solutions LLC (purchased by Lallemand Inc.) 

genetically engineers lactic acid bacteria, the organisms 

that contaminate ethanol fermentations, to produce 

ethanol rather than lactic acid.

• Xylome uses newly developed yeast strains to transform hard-to-digest sugars into commercially viable, 

second-generation ethanol and biodiesel precursors and 

to synthesize a host of advanced biorenewable products. 

Joint BioEnergy Institute

• Afingen develops and commercializes emerging 

synthetic biology technologies expected to have enormous impact on economics and environmental sustainability through new biological products and improved 

biological capabilities.

• Cascade Fluidics Inc. builds laboratory automation systems for high-throughput biological sample 

preparation.

• Constructive Biology develops engineered microbes to 

convert inexpensive, renewable sugars into high-value 

specialty chemicals. 

• Demetrix develops engineered microbes to convert 

inexpensive, renewable sugars into high-value 

specialty chemicals. 

• Illium Technologies produces ionic liquid solvents 

from lignin. The ionic liquids can be used for biomass 

pretreatment to produce cellulose and glucose for 

biomanufacturing.

• Lygos produces “bio-advantaged” chemicals, where 

petrochemistry cannot compete with biochemistry.

• TeselaGen Biotechnology, Inc. is developing a fully integrated solution for synthetic biology. Its DNA design and 

assembly platform makes genetic engineering cheaper 

and faster. 

Companies that Have Licensed BRC Technologies

Great Lakes Bioenergy 

Research Center

• Glucan Biorenewables LLC

• Hyrax Energy Inc.

• Xylome

• Lactic Solutions LLC

• Amphora

• FuturaGene

Joint BioEnergy 

Institute

• DuPont

• Novozymes

• POET

BioEnergy Science Center

• Forage Genetics International

• GreenWood Resources, Inc.

• Mascoma Corporation

• Enchi Corporation

• Aspen Machining and 

Molding, Inc.

• DuPont

• Dow AgroSciences

• ArborGen, Inc.

• Ceres, Inc.

• Kerafast, Inc.

• Glydia Biotech LLC

• Monsanto Company
4 U.S. Department of Energy Office of Science September 2018

The BRCs also trained scores of multidisciplinary scientists in the bioenergy field, and 

BRC outreach efforts have communicated the 

value of sustainable bioenergy to the public.

BRC Breakthroughs for 

Lignocellulosic Biofuels 

Production

From 2007 to 2017, the original three DOE 

BRCs made a number of important breakthroughs toward overcoming critical challenges 

to cost-effective production of biofuels from 

biomass (see sidebar, At a Glance: BRC Accomplishments from 2007 to 2017, p. 5). These 

challenges, identified by the BRCs in their first 

years, included biomass supply and sustainability, biomass development, biomass deconstruction and separation, conversion of biomass to 

biofuels, and enabling technologies.

The BRCs built their research portfolios around 

these challenges, and select advances from their 

collective efforts are highlighted in the following sections.

Top Accomplishments 

in Biomass Supply and 

Sustainability

A key challenge when the BRCs began their 

research efforts was that the environmental, 

social, and economic consequences of growing 

biomass for biofuels needed to be thoroughly 

evaluated to ensure minimal, or even positive, 

impacts on food production and the environment (see Fig. 2. Marginal Lands, this page). For 

example, how far could biomass be transported 

from a field to a biorefinery before the fuel used 

in its transport exceeded the amount of fuel that 

could be obtained in the conversion to biofuels? 

The BRCs determined an analysis of the entire 

supply chain was necessary to understand the 

economic feasibility and logistics for collecting, 

transporting, and delivering this biomass to 

biorefineries. The BRCs also developed technoeconomic modeling approaches to study how 

Fig. 2. Marginal Lands. 

Flower-dotted marginal 

land in the foreground 

contrasts sharply with 

more uniform traditional 

croplands of corn and 

alfalfa in the distance. 

Great Lakes Bioenergy 

Research Center (GLBRC) 

scientists have shown that 

perennial crops grown 

on marginal lands not 

currently in use for food 

production could provide 

large quantities of biomass 

and major ecological and 

environmental benefits. 

[Courtesy GLBRC]
September 2018 BRCs: Breakthroughs and Impacts 5

Great Lakes Bioenergy 

Research Center

The Great Lakes Bioenergy Research Center (GLBRC) developed a 

fundamental understanding of nitrogen and carbon cycling, which 

is essential for creating sustainable biofuel landscapes. GLBRC also 

pursued economic sustainability via biological and chemical routes 

to low-cost sugars, developing unique pretreatment methods 

that release lignin for potential conversion to fuel precursors and 

value-added coproducts (see figure at right). Producing biofuels 

and chemicals from both the sugar and lignin components of plant 

biomass has the potential to provide added value and increase the 

profitability of cellulosic biofuels. 

Biomass Pretreatment for Cutting Enzyme Use, 

Boosting Biofuel Production. A liquid ammonia–

based pretreatment, called extractive ammonia (EA), 

processes biomass such as switchgrass in a substance 

that helps unlock the plant’s sugars. Simultaneously, 

EA separates out up to 45% of the lignin, while largely 

keeping its carbohydrates intact. [Courtesy Matthew 

Wisniewski, Great Lakes Bioenergy Research Center]

At a Glance: BRC Accomplishments from 2007 to 2017

BioEnergy Science Center

The BioEnergy Science Center (BESC) made crucial progress toward 

understanding, manipulating, and managing plant cell wall recalcitrance and conversion. Notably, the BESC team proved that multiple 

genes control plant cell wall recalcitrance and that manipulation 

of these genes could yield perennial bioenergy feedstocks with 

enhanced deconstruction properties. Furthermore, BESC successfully demonstrated the ability to combine the processes of cellulose 

digestion and fermentation of released sugars into biofuel in a 

single microbial organism (see figure at left). These discoveries not 

only represent significant progress toward the goal of developing 

improved feedstocks and microbial deconstruction technologies 

for advanced biofuel production, but they also have redefined the 

scientific basis of the phenomenon of biomass recalcitrance. 

Clostridium thermocellum. A model of C. thermocellum engineered for greater ethanol tolerance during 

bioprocessing and licensed by the BioEnergy Science 

Center to Enchi Corporation. [Courtesy Bryon Donohoe, National Renewable Energy Laboratory]

Joint BioEnergy Institute

The Joint BioEnergy Institute (JBEI) used the latest tools in synthetic 

biology and chemical engineering, including computational and 

automated high-throughput technologies, to transform biomass 

sugars into energy-rich fuels. JBEI successfully altered biomass 

composition in model plants and bioenergy crops, reducing inhibitors that impact downstream processing and making lignin more 

readily depolymerized. JBEI research has shown that new solvents, 

known as ionic liquids, permit near-complete dissolution and 

fractionation of plant biomass, thereby facilitating its enzymatic 

conversion to sugars at near-theoretical yields. JBEI’s pioneering 

work in synthetic biology has enabled the realization of microbes 

that produce a variety of molecules from these sugars that can 

serve as renewable jet, diesel, and gasoline “drop-in” blendstocks 

(see figure at right). 

Synthetic Biology. Joint BioEnergy Institute researchers are using the tools of synthetic biology to engineer 

new microbes as alternatives to yeast that can quickly 

and efficiently ferment complex sugars into advanced 

biofuels. [Courtesy Lawrence Berkeley National 

Laboratory]
6 U.S. Department of Energy Office of Science September 2018

their fundamental, early-stage research findings 

might operate as mature technologies at commercial scale. Additionally, this modeling helped to 

pinpoint the areas of highest cost and thus where 

new advances would have the greatest impact.

Demonstrated Potential of Marginal Lands. 

Biomass production on marginal or nonagricultural lands can avoid the negative impacts 

of diverting farmland or crops for biofuels 

production, namely the food versus fuel conflict 

and indirect greenhouse gas emissions. Field 

experiments and modeling demonstrated that 

sufficient marginal land exists in the U.S. Midwest to grow a significant fraction of biomass 

for expected biofuel needs. These analyses were 

extended to a wider geographic region, incorporating techno-economic modeling to show 

reasonable potential configurations for future 

feedstock supplies (Gelfand et al. 2013).

Increased Understanding of Potential Economic Impacts of BRC Discoveries. Researchers modeled the economic impact of newly 

developed ionic liquid deconstruction processes 

in the context of a virtual, commercial-scale 

biorefinery to understand the capital and 

operating costs, net energy needs, and material 

balance. Subsequent modeling considered the 

entire supply chain to determine cradle-to-grave 

greenhouse gas emissions and water use. One of 

these techno-economic simulations compared 

the performance of biorefineries using newly 

developed ionic liquid–based pretreatment 

technologies, including a one-pot process (see 

research accomplishment, Developed and 

Demonstrated a One-Pot Ionic Liquid Deconstruction and Conversion Technology, p. 12), 

which dramatically reduces the need for water 

washing and energy-intensive separations. Calculations indicated that combining this process 

with the use of protic ionic liquids clears the way 

to producing cellulosic biofuels at a lower cost 

and cuts greenhouse gas emissions to less than 

one-tenth that of the previous state of technology 

for ionic liquids based on a water-wash process. 

Compared with gasoline, the one-pot process 

combined with a high-performing ionic liquid 

can reduce greenhouse gas emissions by 85%. 

These results highlight the dramatic impact that 

increasing biomass loading and minimizing water 

use in the biorefinery can have on the cost and 

environmental footprint of advanced biorefineries (Klein-Marcuschamer et al. 2011; Konda et 

al. 2014; Sun et al. 2017; Neupane et al. 2017).

Examined Feedstock Variability and Effects 

on Deconstruction and Microbial Conversion. Variation in environmental conditions 

Fig. 3. “Super Yeast”: 

Potential Improvement 

for Biofuel Economics.

Great Lakes Bioenergy 

Research Center (GLBRC) 

scientists have found a 

way to nearly double the 

efficiency with which 

Saccharomyces cerevisiae, a 

commonly used industrial 

yeast strain, converts plant 

sugars to biofuel. The engineered super yeast (being 

examined by a GLBRC 

researcher) could boost 

the economics of producing ethanol and specialty 

biofuels and bioproducts. 

[Courtesy James Runde, 

GLBRC]
September 2018 BRCs: Breakthroughs and Impacts 7

where bioenergy crops are grown can have 

significant detrimental effects on microbes’ 

ability to ferment sugars to biofuels. In a fieldto-fuel pipeline analysis, researchers studied 

the effects of variable precipitation on two 

bioenergy feedstocks—corn stover and switchgrass—with sampling from a major drought 

year and 2 years with average precipitation. The 

study examined effects on hydrolysate chemical 

composition and the ability of yeast (Saccharomyces cerevisiae) or bacteria (Zymomonas 

mobilis) to ferment the hydrolysates to ethanol 

(see Fig. 3. Super Yeast: Potential Improvement for Biofuel Economics, p. 6). Most were 

readily fermented to biofuel, but the growth of 

yeast was completely inhibited in hydrolysate 

generated from drought-stressed switchgrass. 

Biomass and compositional analysis revealed 

that drought-year switchgrass accumulated 

greater concentrations of soluble sugars, likely a 

stress response to drought, that were degraded 

to inhibitory compounds during pretreatment. 

These findings indicate that environmental 

variation can have significant effects on biomass 

hydrolysate properties and subsequent fermentation to biofuels. Integrating the effects of environmental conditions and microbial molecular 

signatures into a predictive model can guide 

pretreatment and biocatalyst choices to mitigate 

inhibitory effects (Ong et al. 2016).

Top Accomplishments 

in Biomass Development

In contrast to crops used for food and fiber, no 

crops had been developed specifically for use 

as cellulosic biofuel feedstocks when the BRCs 

began in 2007. Consequently, a key challenge 

was to identify optimal agronomic properties 

and develop fundamental knowledge of plant 

cell wall synthesis and structure to enable production of high-yield, sustainable feedstocks 

such as switchgrass and poplar that are easier 

to deconstruct and convert into biofuels. Part 

of this challenge involves lignin, which is one 

of the most recalcitrant biopolymers present in 

lignocellulose.

Demonstrated Potential for Doubling 

Biofuel Production with Transgenic Switchgrass. Researchers demonstrated the potential 

of developing dedicated bioenergy crops with 

specific engineered traits capable of thriving 

in the environment. Switchgrass engineered 

to express an altered lignin structure thrived 

in field trials over a 3-year period relative to 

controls (see Fig. 4. Real-World Performance of 

Low-Lignin Switchgrass, this page). The crops 

retained the reduced recalcitrance phenotype 

and yielded 25% to 35% more sugar release 

upon deconstruction, demonstrating the viability of a specifically designed bioenergy crop 

(Dumitrache et al. 2017). 

Engineered and Field-Tested Plants with 

Modified Lignin. Using the bacterial enzyme 

3-dehydroshikimate dehydratase (QsuB) in 

crops such as sorghum, tobacco, poplar, and 

switchgrass, researchers found that QsuB can 

reduce the recalcitrance of lignin and improve 

the energy yield of biomass in two ways: 

(1) The expression of QsuB reduces the amount 

of lignin in plant cell walls, enhancing breakdown into soluble sugars. (2) QsuB catalyzes 

the synthesis of protocatechuic acid, which 

can be converted into the platform chemical 

muconic acid, a precursor for several valuable 

bioplastics. These results highlight the potential 

Fig. 4. Real-World Performance of Low-Lignin 

Switchgrass. Lignin in the 

cell walls of switchgrass 

(pictured) and other bioenergy feedstocks severely 

limits the accessibility of 

cell wall carbohydrates to 

enzymatic breakdown into 

sugars and their subsequent fermentation into 

biofuels. This first reported 

study of its kind evaluated 

the biofuel potential of 

transgenic switchgrass 

with reduced lignin 

content over several growing seasons. [Courtesy 

BioEnergy Science Center]
8 U.S. Department of Energy Office of Science September 2018

impact of tailored bioenergy crops to decrease 

costs associated with biofuel production (Eudes 

et al. 2015, 2017; Wu et al. 2017).

Elucidated Genes Controlling Plant Cell Wall 

Recalcitrance. Significant advances were made 

in understanding, manipulating, and managing 

plant cell wall recalcitrance and conversion in 

studies of Populus and switchgrass (Panicum 

virgatum). Both are high-yield perennials and 

recognized as potential domestic biofeedstocks. 

Researchers showed that multiple plant genes 

control cell wall recalcitrance and that manipulation of these genes can yield lower recalcitrance 

in perennial biofeedstocks. These feedstocks 

were analyzed by a series of chemical, biochemical, molecular, and systems biology approaches. 

Findings included increased understanding of 

cell wall structure and biosynthetic pathways for 

four components—lignin, xylan, cellulose, and 

pectin—and that each could be manipulated 

to reduce recalcitrance. For lignin biosynthesis, 

researchers manipulated every gene in the pathway, demonstrating that it is a key influencer of 

biomass recalcitrance. The most surprising effect 

was from pectin, which, though small in quantity, also proved to be an important contributor 

to recalcitrance. The results provide mechanistic 

understanding of the molecular bases of recalcitrance, enabling a path for improving feedstocks 

by cisgenic manipulations, selection of the best 

natural variants, or genetically assisted breeding 

(Shen et al. 2013; Zhao et al. 2013; Barros et al. 

2016; Peña et al. 2016; Urbanowicz et al. 2012, 

2014; Mazumder et al. 2012; Mazumder and 

York 2010; Pattathil et al. 2010; Bar-Peled et 

al. 2012; Bali et al. 2016; Vandavasi et al. 2015; 

Payyavula et al. 2014; Tan et al. 2013; Atmodjo 

et al. 2013; Xie et al. 2018; Biswal et al. 2018a, 

2018b; Kalluri et al. 2014).

Identified Nucleotide Sugar Transporters. 

Nucleotide sugars are the substrates for biosynthesis of cell wall polysaccharides. Most 

of the nucleotide sugars are produced in the 

cytoplasm and need to be transported in the 

Golgi lumen to be used for glycan biosynthesis. 

A novel method was developed to determine 

the transport activity of nucleotide sugars, and 

researchers identified the substrates for 22 of 

44 transporters in Arabidopsis (see Fig. 5. Assay 

Tool for Characterizing Plant Sugar Transporters, this page). By comparison, only eight of the 

32 human-mammalian transporters have been 

characterized at this level of detail. Identification of nucleotide sugar transporters is important for basic understanding of cell wall biology 

and provides powerful tools for engineering 

plants with modified biomass composition 

optimized for production of biofuels and 

bioproducts. Using this knowledge, researchers engineered expression of both uridine 

Fig. 5. Assay Tool for 

Characterizing Plant 

Sugar Transporters. A 

family of six nucleotide 

sugar transporters never 

before described were 

characterized in Arabidopsis (pictured), a model 

plant for research in 

advanced biofuels. [Courtesy Lawrence Berkeley 

National Laboratory]
September 2018 BRCs: Breakthroughs and Impacts 9

diphosphate (UDP)-galactose and UDP-xylose 

transporters and increased the glucose-xylose 

ratio in the engineered Arabidopsis plants. The 

sugar stream produced from these plants is 

preferable for most of the microbes currently 

used in biorefineries that prefer glucose over 

xylose (Rautengarten et al. 2014, 2017; Ebert 

et al. 2015).

Identified Biomass Recalcitrance Regulators 

with Genome-Wide Association Studies 

(GWAS). To quickly identify the genes that 

control cell wall recalcitrance (resistance to 

sugar release), researchers conducted largescale studies to understand natural variation 

in both switchgrass and Populus. These studies 

included high-throughput recalcitrance 

phenotyping and sequencing, along with 

other omics studies, for these natural variants 

as well as generated transgenics. Sequencing 

identified millions of unique variations called 

single-nucleotide polymorphisms (SNPs) in 

specific genes and in noncoding regions of the 

genome. Using a GWAS approach, many measurements (including growth, composition, and 

biomass convertibility) of the broad variation 

across the switchgrass and Populus populations 

were correlated with specific SNPs (see Fig. 6. 

Largest-Ever Dataset of Genetic Variations 

in Poplar Trees, this page). The GWAS mapping enabled researchers to discover a genetic 

regulator that modulates carbon flow among 

four major metabolic pathways in plants. 

Manipulation of the regulator leads to reduced 

lignin content, increased biofuels production, 

and increased digestibility. These results laid 

the foundation for using breeding and selection 

to identify better plant feedstocks (Serba et al. 

2015; Muchero et al. 2015; Evans et al. 2014).

Greenhouse and field trials were conducted for 

a limited number of natural variant Populus and 

switchgrass lines with reduced recalcitrance and 

those arising from directed transgenic engineering. Researchers used multiple phenotypic 

characterization assays including sugar release, 

sugar and lignin composition, ethanol production, and crystallinity. A key discovery was the 

ability to achieve both lower recalcitrance and 

higher biomass simultaneously in certain lines 

(Biswal et al. 2015; Baxter et al. 2014, 2015; 

Dumitrache et al. 2017). 

Fig. 6. Largest-Ever Dataset of Genetic Variations 

in Poplar Trees. The 

BioEnergy Science Center 

(BESC) genome-wide association study dataset comprises more than 28 million single-nucleotide 

polymorphisms, or SNPs, 

derived from approximately 900 resequenced 

poplar genotypes. The 

information is proving 

useful to researchers in the 

fields of biofuels, materials 

science, and secondary 

plant metabolism. [Courtesy BESC]
10 U.S. Department of Energy Office of Science September 2018

Used Genomic Approaches to Engineer Lignin. Redesigning lignin, the aromatic polymer 

fortifying plant cell walls, to be more amenable 

to chemical depolymerization can lower the 

energy required for industrial processing and 

increase sugar yields. Poplar trees were engineered to introduce ester linkages (“zips”) in 

place of ether linkages into the lignin backbone 

by augmenting the monomer pool with monolignol ferulate conjugates (see Fig. 7. Zip-LigninTM, 

this page). This approach incorporates weak 

bonds that are easier to break apart, improving digestibility, but retains lignin’s structural 

value to the plant. The plants also appear to 

grow normally compared with nontransgenic 

controls. Tailoring plants to use such conjugates 

during cell wall biosynthesis holds promise for 

producing plants designed for more efficient 

deconstruction (Wilkerson et al. 2014). 

Additional genetic engineering approaches 

were identified for improved plant processing. 

For example, augmentation of available pools of 

soluble sucrose in poplar yields reduced lignin 

and altered cell wall structure, while mutation 

of a gene encoding a lignin biosynthetic enzyme 

increased zip-lignin levels in maize (Unda et al. 

2017; Smith et al. 2017).

Top Accomplishments 

in Biomass Deconstruction 

and Separation

The BRCs identified deconstruction of lignocellulosic feedstocks into their constituent sugars, 

while minimizing the formation of inhibitors 

and using as few chemicals as possible, as the 

primary technical barrier to economical liquid 

biofuels. Scalable, sustainable, and affordable 

processes needed to be developed to handle 

diverse feedstocks at low cost. 

Developed a Next-Generation Deconstruction Strategy. An innovative, next-generation 

biomass deconstruction method was developed 

that works with a wide variety of energy crops 

and avoids the use of expensive chemicals. The 

method is based on gamma-valerolactone, an 

organic solvent produced from plants, that can 

break down biomass into cellulose, hemicellulose, and lignin. These components can be 

Fig. 7. Zip-LigninTM. 

The Great Lakes Bioenergy Research Center’s 

Zip-LigninTM technology 

allows for easier degradation of lignin, the most 

difficult part of the plant 

to break down. Here, 

the feruloyl-coenzyme A 

monolignol transferase is 

expressed following introduction of weak bonds 

into the lignin of poplar 

tissue. [Courtesy Shawn 

Mansfield, University of 

British Columbia]
September 2018 BRCs: Breakthroughs and Impacts 11

further converted to useful biofuels, specialty 

chemicals, and lignin-based carbon foam, all 

commercially valuable products, as a basis for 

an integrated biorefinery process producing 

biofuels and chemicals from renewable biomass 

(Luterbacher et al. 2014; Alonso et al. 2017).

Discovered New Enzymes, Microbes for 

Breaking Down Lignocellulose. Microbes 

such as fungi and bacteria produce enzymes, 

called glycoside hydrolases, to acquire nutrients 

through the degradation of cellulose. Some of 

these enzymes can break down the polysaccharides and lignin that constitute plant cell walls 

and, therefore, could be especially effective at 

generating the intermediates needed for microbial production of biofuels and bioproducts. 

Researchers studied two distinct ecosystems 

known to be very efficient in breaking down 

cellulose—composts in California (see Fig. 8. 

Cellulose-Degrading Enzymes, this page) and 

rainforest soils in Puerto Rico. From these studies, they discovered (1) a glycoside hydrolase 

family 12 enzyme, produced by the bacterium 

Thermobispora bispora, that plays a previously 

underappreciated role in efficiently breaking 

down crystalline cellulose into glucose; (2) a 

complex of cellulose-degrading enzymes that 

performs well at higher temperatures than 

current commercial enzymes can tolerate; and 

(3) a previously unknown organism, Enterobacter lignolyticus, that can efficiently break 

down and grow on lignin. Researchers isolated 

a single gene from this organism that confers 

tolerance to the presence of ionic liquids to 

biofuel-producing hosts, such as Escherichia 

coli, thus paving the way to new consolidated 

approaches to biomass conversion (Khudyakov 

et al. 2012; Ruegg et al. 2014; Hiras et al. 2016).

Developed an Innovative Pretreatment 

Method that Reduces Costly Enzyme 

Loadings and Increases Biofuel Yields.

The recalcitrance of lignocellulosic biomass 

requires energy-intensive pretreatment and 

large amounts of expensive enzymes to release 

sugars for viable biochemical conversion to 

biofuels. Co-Solvent Enhanced Lignocellulosic 

Fractionation (CELF) employs renewable 

tetrahydrofuran as a co-solvent to greatly 

enhance traditional dilute acid pretreatment. 

Testing showed that CELF dissolved a large 

portion of plant lignin, removed and recovered 

most of the hemicellulose sugars, and increased 

enzymatic digestibility of biomass. Overall, 

CELF pretreatment achieved about a 10-fold 

reduction in required enzyme loadings compared with traditional dilute acid pretreatment. 

It also removed a majority of biomass lignin 

(over 90% in wood) that could be precipitated 

as a high-purity product suitable for conversion 

to high-value chemicals and materials (Nguyen 

et al. 2015).

Developed a Feedstock-Agnostic Biomass 

Pretreatment Method. Lignocellulosic biomass is a natural resource distributed across the 

United States with significant variations as a 

function of genotype and geographical region. 

Most of the pretreatment technologies that 

have been studied and developed to date are 

highly effective in handling a specific range of 

feedstocks, but there are very few conversion 

technologies with a demonstrated ability to 

handle a wide range of feedstocks with minimal 

negative impact on efficiency and sugar yields. 

The ionic liquid pretreatment process is unique 

Fig. 8. CelluloseDegrading Enzymes. 

This 50-ml flask contains a 

symbiotic mix of bacteria 

derived from compost that 

was maintained for 3 years. 

[Courtesy Joint BioEnergy 

Institute]
12 U.S. Department of Energy Office of Science September 2018

in that it efficiently handles softwoods, hardwoods, herbaceous materials, and agricultural 

residues as single and mixed feedstocks and 

generates high yields of sugars and biofuels 

(Shi et al. 2013; Torr et al. 2012; Li et al. 2010; 

Singh et al. 2009; Dibble et al. 2011).

Top Accomplishments 

in Conversion of Biomass 

to Biofuels

Using lignocellulosic biomass as a sustainable 

feedstock for producing biofuels and bioproducts requires a cost-effective, efficient process 

for transforming the polysaccharides in lignocellulose into sugars for bioconversion. These 

sugars are then fed to microorganisms that 

convert them into biofuels and bioproducts. 

A key challenge for the BRCs was developing 

more energy-efficient and economical conversion processes, along with synthesis processes 

for new fuels in addition to ethanol.

The BRCs also targeted lignin, which constitutes 

up to a third of typical biomass, as a key focus 

area. By concentrating on the structure of lignin, 

its modification, and potential use, the BRCs 

studied ways to mitigate lignin’s effect on recalcitrance while also identifying opportunities to 

effectively use lignin for chemicals or fuels.

Developed and Demonstrated a One-Pot 

Ionic Liquid Deconstruction and Conversion Technology. Similar to the advantages of 

“one-stop” shopping in the retail and services 

industry, a “one-pot” processing system in 

which sugars could be extracted from biomass 

and turned into biofuels in a single reactor 

vessel could produce significant advantages for 

the biofuels industry. Researchers made a major 

step toward this goal with the development and 

demonstration of a one-pot, wash-free process 

for ionic liquid pretreatment and saccharification of switchgrass (see Fig. 9. Jet Fuel Brewed 

in One-Pot Recipe, this page). Ionic liquids 

are a unique and powerful class of solvents 

that are superior to conventional pretreatment 

approaches (e.g., dilute acid, ammonia, organic 

solvent, and hydrothermal). Researchers also 

discovered enzymes, microbes, and microbial 

communities that can tolerate ionic liquids used 

for pretreatment, with the goal of consolidating 

Fig. 9. Jet Fuel Brewed 

in One-Pot Recipe. 

Joint BioEnergy Institute 

scientists have engineered 

a strain of bacteria that 

enables a one-pot method 

for producing advanced 

biofuels from a slurry of 

pretreated plant material. 

[Courtesy Lawrence Berkeley National Laboratory]
September 2018 BRCs: Breakthroughs and Impacts 13

the multiple reactors currently required into a 

single reactor, known as a one-pot configuration. A demonstration of this process at high 

solid loading levels resulted in unprecedented 

biofuel yields (~90%), while minimizing water 

use and waste disposal. This new one-pot 

process uses a combination of ionic liquid pretreatment, enzymatic saccharification, and yeast 

fermentation; and it eliminates the need for 

separations, recoveries, and other operational 

steps thereby generating significant capital and 

operating cost savings (Xu, F., et al. 2016; Sun, 

J., et al. 2016, 2017).

Identified and Validated Key Microbial 

Gene and Enzyme Targets for Consolidated 

Bioprocessing (CBP). CBP is a one-step 

approach by which microbes simultaneously 

digest biomass and convert it to biofuels and 

bioproducts without the need to add enzymes. 

This approach has the potential to greatly 

reduce capital costs by eliminating separate 

enzyme production and decreasing pretreatment severity (Lynd et al. 2017; Olson et al. 

2012). A key focus was on improving product 

formation in thermophilic cellulolytic bacteria 

(e.g., Clostridium thermocellum and Caldicellulosiruptor bescii). Major advances include:

• In comprehensive comparative evaluations 

of biomass deconstruction, those microbes 

achieving the highest carbohydrate solubilization yields (several-fold higher than the 

industry standard fungal cellulose) were 

thermophilic anaerobes and C. thermocellum

in particular (see Fig. 10. Study of Nature’s 

Best Biocatalysts for Biofuels Production, 

this page; Paye et al. 2016; Lynd et al. 2016; 

Chung et al. 2014).

• Genetic tools for thermophiles were developed and used to initiate metabolic engineering of C. thermocellum and C. bescii (Cha 

et al. 2013; Guss et al. 2012; Olson and Lynd 

2012; Lipscomb et al. 2016).

• Substantial advances were made in understanding and manipulating the metabolism 

of target CBP microbes. (1) Zhou et al. 

(2013) described nonstandard glycolysis 

Fig. 10. Study of Nature’s 

Best Biocatalysts for 

Biofuels Production. 

The microbe Clostridium

thermocellum (stained 

green), seen growing on a 

piece of poplar biomass, 

was among several 

microorganisms evaluated 

in a comparative study 

analyzing their ability to 

solubilize potential bioenergy feedstocks. [Courtesy 

Jennifer Morrell-Falvey, 

BioEnergy Science Center]
14 U.S. Department of Energy Office of Science September 2018

in C. thermocellum. (2) Thermoanaerobacter 

saccharolyticum was improved to produce 

economically recoverable ethanol concentrations at near-theoretical yield in hemicellulose fermenting (Herring et al. 2016). 

(3) Isobutanol was produced by adding key 

pathway enzymes in modified C. thermocellum at unprecedented yields and titers (Lin 

et al. 2015). (4) Ethanol titer and yield were 

increased in C. thermocellum by eliminating 

side products (Papanek et al. 2015; Tian et al. 

2016; Biswas et al. 2016).

• Deconstruction enzymes targeting the major 

biopolymers of lignocellulosic biomass were 

identified. Fundamental enzymology found 

multifunctional cellulases in Caldicellulosiruptor species and C. thermocellum (Xu, Q., et al. 

2016). Additionally, CelA, a multifunctional 

glycosyl hydrolase from C. bescii, was shown to 

be a particularly powerful hydrolytic enzyme 

(Brunecky et al. 2013; Kim et al. 2016).

• With co-treatment, C. thermocellum was able 

to achieve greater than 85% carbohydrate solubilization for Populus and switchgrass in the 

absence of added enzymes and thermochemical pretreatment, implying that C. thermocellum can attack all the major chemical linkages 

in representative woody and herbaceous 

lignocellulose crops when given sufficient 

physical access (Paye et al. 2016; Lynd et al. 

2017; Holwerda et al. 2017).

Developed a Genome-Enabled Platform to 

Identify Valuable Coproducts from Biomass Hydrolysates. Processing plant biomass 

releases materials that have a negative effect 

on the ability of biofuel microbes to produce 

products. Consequently, researchers developed a genome-enabled platform to identify 

toxic compounds within biomass hydrolysates, 

determine their mode of action, and develop 

ways to bypass their negative impact on biofuel microbes. An unexpected result was the 

discovery of a compound (poacic acid) within 

biomass hydrolysates that is a potent antifungal 

agent (see sidebar, Non–Bioenergy Benefits 

of BRC Research, p. 15). Kilogram quantities 

of this compound have been prepared for field 

trials as a natural fungicide against agricultural pests. Poacic acid is a prime example of 

how genome- and systems biology–enabled 

advances can provide fuels and valuable products that can be produced by lignocellulosic 

biorefineries (Piotrowski et al. 2015). 

Discovered and Optimized Metabolic 

Pathways and Hosts to Produce Advanced 

Biofuels. Researchers built on previous metabolic engineering advances to produce advanced 

jet-fuel, diesel, and gasoline-blending agents 

from cellulosic sugars. A fatty acid biosynthetic 

pathway was harnessed to produce diesel substitutes, fatty acid ethyl esters, and methyl ketones. 

Isoprenoid pathways were engineered in E. coli

and S. cerevisiae to produce biodiesel (bisabolene), jet fuels (limonene, eucalyptol, and 

pinenes), and gasoline (isopentenol). Researchers used systems biology and host engineering to 

discover and develop mechanisms that improve 

host tolerance to final products and inhibitors 

from pretreated biomass. The research is a 

culmination of a series of metabolic engineering advances, laying a foundation for a broader 

biotechnology industry that produces biofuels 

and chemicals from renewable biomass (Steen et 

al. 2010; George et al. 2015; Ghosh et al. 2016; 

Yuzawa et al. 2016). 

Developed Techniques to Valorize Lignin.

Researchers found that lignin plays a key role in 

biomass recalcitrance (Ziebell et al. 2010, 2016; 

Li et al. 2016). Lignin removal would allow its 

valorization (Ragauskas et al. 2014), whether into 

fiber (Sun, Q., et al. 2016) or into value-added 

intermediates (Beckham et al. 2016).

Converting lignin into aromatics and other 

valuable products has the potential to improve 

the sustainability and economic viability of 

lignocellulosic biorefineries. Researchers took 

several approaches to enable lignin valorization, 

including (1) structural and biochemical analyses of bacterial enzymes that cleave lignin bonds 

(Helmich et al. 2016; Pereira et al. 2016); 
September 2018 BRCs: Breakthroughs and Impacts 15

(2) chemical analysis of dimeric degradation 

products released by lignin depolymerization to 

verify and quantify components used in lignification (Yue et al. 2017); (3) utilization of formaldehyde to facilitate lignin monomer release 

during biomass depolymerization (Shuai et al. 

2016); (4) identification of valuable extractable 

“clip-off ” bioproducts like the flavonoid tricin, 

shown to be involved in lignification in monocots (Lan et al. 2015); and (5) identification of 

new and complex lignin compositions in palm 

species, including polyphenolic compounds 

and hydroxystilbenes, incorporated into lignin, 

and an unprecedented range of monolignol 

conjugates not previously known to be involved 

in lignification (del Rio et al. 2017). All these 

approaches help to inform understanding of 

lignification and enable lignin valorization, 

both through extraction of naturally occurring 

components and of compounds that have been 

engineered into bioenergy crops.

Top Accomplishments 

in Enabling Technologies

New technologies were needed to facilitate and 

accelerate BRC research, including, but not 

limited to, high-throughput laboratory technologies and computational and information 

systems, several of which have applications to 

biological research as a whole.

Developed Software Tools for Synthetic 

Biology and Metabolic Engineering. A powerful suite of synthetic biology approaches and 

computational tools was developed to facilitate 

Non–Bioenergy Benefits of BRC Research

Some of the discoveries made by the Bioenergy Research Centers also have important potential applications in other 

sectors, including sustainable agriculture, biomanufacturing, biodefense, and human health. Examples follow.

Plant-Derived 

Antifungal Agent

Great Lakes Bioenergy Research 

Center scientists discovered poacic 

acid through research focused 

on the small-molecule inhibitors 

present when cellulosic biomass is 

broken down for biofuel production. 

These inhibitors curb the growth of 

biofuel-producing microbes and are 

considered obstacles in the process 

of converting biomass to biofuels. 

Screening them for bioactivity 

against yeast revealed that one of 

the compounds, poacic acid, caused 

considerable cell death. Further testing against some common fungal 

pathogens found that a single 

application substantially reduced 

pathogen growth compared with 

control applications. If field tests are 

successful, the chemical compound 

eventually could be used as a natural fungicide in both sustainable and 

conventional agriculture.

Higher-Value Forage Plants

Working with a population of 

poplar trees, researchers at the 

BioEnergy Science Center identified a gene that regulates the 

production of lignin, the material that imparts plant rigidity. 

By altering lignin synthesis, the 

gene can decrease lignin content, 

making the plant easier to break 

down and convert into biofuels. 

The researchers also found that the 

reduced lignin content increases 

desirable flavonoids, improving the 

digestibility and nutritional value 

of animal feedstocks such as alfalfa, 

corn, and sorghum. Forage Genetics International, a major supplier 

of alfalfa seed, has licensed this 

plant gene and is evaluating it for 

commercial use in animal feed.

High-Throughput Microfluidics

Scientists and engineers working at 

the Joint BioEnergy Institute developed microfluidic devices for rapid, 

precise, and high-throughput characterization of the polysaccharides 

and lignin present in lignocellulosic 

biomass. This research included 

assay development at the interface of droplet microfluidics and 

mass spectrometry to streamline 

the discovery and optimization of 

biofuel metabolic pathways. These 

devices and assays have vastly 

superior performance compared 

to conventional approaches such 

as standard chromatography and 

colorimetric assays. They also 

have applications beyond bioenergy, such as biomanufacturing, 

agriculture, biodefense, and 

human health.
16 U.S. Department of Energy Office of Science September 2018

the engineering of microbes to produce 

biofuels from sugars generated from lignocellulose deconstruction. This suite includes the 

establishment of a parts-based approach to 

DNA construction and quantitative tools to 

facilitate and improve the design of engineered 

pathways, such as the identification of metabolic flux–diverting side reactions as targets for 

metabolic engineering. Other newly developed 

resources include an open-source, web-based 

digital repository platform for microbial 

strains, sequences, and parts and plant seeds, 

as well as a web-based canvas for biological 

computer-aided design that enables users to 

visually design combinatorial DNA constructs 

(Chen et al. 2012; Hillson et al. 2012; Ham et 

al. 2012; Birkel et al. 2017).

Developed High-Throughput Methods 

for Rapid Analysis. The development of 

high-throughput methods for rapid analysis of 

pretreatment and enzymatic hydrolysis allowed 

for rapid identification of low-recalcitrance plant 

lines from thousands of natural and transgenic 

variants. These low-recalcitrance plant lines 

then could be characterized using multiple analytical and omics approaches, quickly advancing 

deeper understanding of the recalcitrance pheonotype (Studer et al. 2011; Decker et al. 2015; 

Selig et al. 2011). Increased understanding of 

recalcitrance mechanisms also was supported 

by developing techniques such as glycome 

profiling (Pattathil et al. 2012) and improving 

the use of nuclear magnetic resonance spectroscopy for biomass (Foston and Ragauskas 2012; 

Peña et al. 2016; Pu et al. 2013, 2016; Kataeva 

et al. 2013).

Integrated Nanostructure-Initiator Mass 

Spectrometry (NIMS) with Microfluidics. 

Glycoside hydrolases are natively used by 

microbes to degrade cellulosic biomass, and 

they also are used in industrial production 

of biofuels from cellulosic biomass. These 

proteins are incredibly diverse, and relatively 

few examples from their more than 150,000 

unique domain arrangements have been 

functionally characterized. The integration 

of in vitro expression and acoustic printing 

with NIMS technologies has proven to be a 

powerful combination for screening enzyme 

and microbial activities on lignocellulosic 

substrates. This analysis platform was used to 

characterize native plant biomass under a range 

of conditions. Product detection is achieved by 

coupling a unique chemical probe to the oligosaccharides using a stable oxime linkage; this 

process was used to characterize the functional 

diversity of glycoside hydrolases secreted by C. 

thermocellum, a model cellulolytic organism. 

New enzyme specificities were identified, and 

differences in individual enzyme activities 

were demonstrated in reactions with biomass. 

Analysis of the data suggested that use of multifunctional glycoside hydrolases can decrease 

the complexity of enzyme cocktails needed for 

liberating sugars from plant biomass during 

biofuel production. The NIMS method has 

been integrated with microfluidics technology 

to reduce sample volumes and greatly increase 

the number of assays that can be performed 

for enzyme cocktail optimization and strain 

optimization experiments (Reindl et al. 2011; 

Heinemann et al. 2017).

Developed New Cell Wall Chemical Imaging 

Approaches. New methods were developed 

to image the chemical components composing the cell wall. (1) Raman spectroscopy was 

used to image hemicellulose for the first time 

(Zeng et al. 2016). (2) Modified atomic force 

microscopy (AFM) techniques were used to 

chemically image the cell wall at the submicron 

level (Tetard et al. 2011). (3) Quantitative 

fluorescence confocal laser scanning microscopy (CLSM) and surface spectroscopy by 

time-of-flight secondary ion mass spectroscopy 

(TOF-SIMS) showed that, following microbial 

digestion, surface cellulose decreased while 

surface lignin increased, indicating that biomass 

recalcitrance may be controlled by surface characteristics (Dumitrache et al. 2017).

Integrated Omics and Software Tools. Key 

to advancing metabolic engineering beyond 

trial-and-error research are genetic parts with 
September 2018 BRCs: Breakthroughs and Impacts 17

well-defined performance metrics that can be 

readily applied in a predictable way. Optimization of engineered microbes depends greatly 

on analytical tools that screen for target biofuel 

production across many thousands of samples 

because the number of design possibilities 

is so large. To help find the optimal microbe 

rapidly, researchers developed technologies 

to measure proteins and metabolites across 

different microbes and biofuels pathways. This 

analysis provides a systems-level view of the 

cell factory to yield quantitative information 

of dynamic processes between parts and the 

microbe that drive the next engineering steps 

to reach the highest biofuel production level. 

These methods have significantly reduced 

the time needed for optimizing microbes to 

produce large amounts of biofuel molecules by 

identifying bottlenecks resulting from low protein levels or metabolites that are toxic to the 

cell. Complementing the omics technologies is 

a suite of software tools developed to standardize procedures and data-type reporting 

for metabolic engineering research as well as 

to encourage sharing with the greater biofuels 

community. With this integrated toolbox, the 

large datasets, statistical analyses, and rulebased approaches help increase success rates 

for achieving high biofuel production (Morrell 

et al. 2017).

Integrated Omics Data for Key Processes.

Integrated omics of microbial growth on complex lignocellulosic biomass over time provided 

a detailed view of the molecular machinery 

(metabolites and enzymes) and revealed temporal adaptation to a complex, lignocellulose 

substrate (Poudel et al. 2017). For Populus, 

profiling genotype-specific proteomes derived 

from ribonucleic acid sequencing data better 

defined the link between genotypes and phenotypes (Abraham et al. 2015).

DOE Bioenergy 

Research Centers: 

Next Iteration

Collectively, the original BRCs provided 

multiple proofs of concept for further capability improvements with the next generation of 

lignocellulosic biofuels and bioproducts, but 

remaining basic science challenges continue 

to limit the cost-effective conversion of plant 

biomass to advanced biofuels and bioproducts 

(U.S. DOE 2015). 

Significant advances in plant breeding, molecular genetics, and genomic technologies 

provide unique opportunities to build on 

existing knowledge of plant biology and more 

confidently predict and manipulate functional 

properties of biomass feedstock crops. Similarly, continuing advances in omics-enabled 

technologies and synthetic biology approaches 

for microorganisms provide opportunities to 

further develop nonmodel microorganisms 

for applications in industrial biotechnology 

and for conversion of biomass into biofuels 

and bioproducts. Most importantly, integrating plant and microbial systems biology with 

cutting-edge research in chemical engineering, 

synthetic biology, and computational biology 

facilitates the scientific breakthroughs needed 

to foster the development of a sustainable 

bioeconomy.

Leveraging these opportunities in the next 

phase of DOE bioenergy research are four 

BRCs: (1) Center for Advanced Bioenergy and 

Bioproducts Innovation, led by the University 

of Illinois at Urbana-Champaign; (2) Center 

for Bioenergy Innovation, led by Oak Ridge 

National Laboratory in Oak Ridge, Tennessee; 

(3) Great Lakes Bioenergy Research Center, 

led by the University of Wisconsin–Madison in 

close partnership with Michigan State University; and (4) Joint BioEnergy Institute, led by 

Lawrence Berkeley National Laboratory.
18 U.S. Department of Energy Office of Science September 2018
September 2018 BRCs: Breakthroughs and Impacts 19

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Protein Sequencing Enables the Detection and Quantitative Profiling of Natural Protein Sequence Variants 

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