February 2023 - Summary slides of Joint BioEnergy Institute (JBEI) research on biofuels and bioproducts.

1. Office of Biological and Environmental Research
Background
• Mycelium-based composite materials are currently produced for packaging
applications by growing fungi on a scaffold made of hemp biomass.
• We hypothesized that fungal growth in the composite material can facilitate its
deconstruction and further valorization at the end of its life cycle.
Approach
Hemp hurd and the mycelium-based packaging material were tested as biomass
feedstocks for production of the jet-fuel precursor bisabolene using a one-pot ionic
liquid deconstruction process and microbial conversion.
Results
Hydrolysates obtained from the packaging materials resulted in higher glucose and
xylose concentrations, sugar yields, and bisabolene concentration after conversion by
an engineered strain of Rhodosporidium toruloides.
Significance/Impacts
This study demonstrated that a hemp hurd-based packaging material can be valorized
a second time as feedstock to produce biofuels.
Choi, J., et al. Molecules, DOI: 10.3390/molecules28031427
Figure 1: Sugar yields (top) and growth and bisabolene production (bottom)
obtained in hydrolysates prepared from hemp hurd or the packaging material.
Valorization of hemp-based packaging waste
with one-pot ionic liquid technology

2. Office of Biological and Environmental Research
Scale-Up of the Ionic Liquid-Based
Biomass Conversion Processes
Background/Objective
• Significant progress has been made with ionic liquid (IL)
pretreatment due to their unique solvation properties
• One critical aspect of IL pretreatment is how the technologies
perform as a function of scale-up
Approach
This entry discusses the experimental outcomes of pilot scale studies
related to IL pretreatment, in consideration of various process
configurations and different feedstocks
Results
The use of bioderived ILs (e.g., cholinium lysinate ([Ch][Lys]) in a
one-pot process has been shown to be an effective biomass
conversion process eliminating the water-washing step) at scale
Significance/Impacts
IL is a promising pretreatment technology with advantages
including feedstock agnostic, efficient sugar conversion, fast
kinetics, and consolidated process configuration
Papa G., et al. (2022) In: Zhang, S. (eds) Encyclopedia of Ionic Liquids. DOI: 10.1007/978-981-33-4221-7_49

3. Office of Biological and Environmental Research
Prediction of Carbon Dioxide Capture by Deep Eutectic
Solvents using Quantum Chemistry and a Neural Network
Background/Objective
• Fermentative production of biofuels generates significant
amounts of CO2 that should be captured at the biorefinery.
• Deep eutectic solvents (DESs) are a promising CO2 capture
solvent but there are no effective predictive screening tools.
Approach
Established a database containing 1973 experimental data points for
CO2 solubility in 132 DESs at different temperatures and pressures.
Results
The database was used for verification and development of
COSMO-RS models and ML models.
Significance/Impacts
The ML model developed here more accurately predicts CO2
solubilities in DESs and can therefore be a useful tool for designing
and selecting a DES for CO2 capture.
Mohan, M, et al. Green Chemistry, 2023, DOI: 10.1039/D2GC04425K
Development of new DESs combination for improving CO2 solubilities using the machine learning
model, DES composed of (a) menthol as HBA and decanoic acid and dodecanoic acid as HBDs,
(b) [ATTP]Br HBA and EA, DECA, MDEA, and ECH are HBDs, and (c) [TBP]Br, [TOA]Br, and
[TOMA]Br are HBAs with TEG HBD.

4. Office of Biological and Environmental Research
Structural and biochemical insight into a modular β-
1,4-galactan synthase in plants
Background/Objective
• Galactan synthase 1 (GalS1) transfers galactose and arabinose to
either extend or cap the β-1,4-galactan side chains of
Rhamnogalacturonan I (RGI) present in plant cell walls.
• Unlike other natural polymers, such as DNA, RNA and proteins,
far less is known about the synthesis and essential biology of the
carbohydrates that constitute plant cell walls.
Results
The crystal structure of GalS1 from Populus trichocarpa, showing a
modular protein consisting of an N-terminal domain that represents
the founding member of a new family of carbohydrate-binding
module, CBM95, and a C-terminal glycosyltransferase family92
(GT92) catalytic domain that adopts a GT-A fold.
Significance/Impacts
Understanding the enzymatic mechanism of GalS1 allowed to
propose a new model for pectic galactan side-chain addition during
the cell wall biosynthesis.
Prabhakar, et al. 2023. Nature plants. DOI: 10.1038/s41477-023-01358-4
Fig. 1 - The structure of GalS1 obtained by X-ray crystallography. a) Monomer of GalS1
highlighting the stem domain (blue), CBM95 (magenta) and core GT-A domain (grey). b) Cartoon
representation of a GalS1 homodimer. c) Experimental (black) and theoretical (coloured as indicated)
SAXS profiles for the solution-state models fitting of GalS1. Average SAXS envelop (grey
transparent) is superimposed onto the atomic model of the solution-state dimer (red and blue).

5. Office of Biological and Environmental Research
Co-Processing Agricultural Residues and Wet Organic Waste
Can Produce Lower-Cost Carbon-Negative Fuels and Bioplastics
Background/Objective
• Biorefineries are often located in rural agricultural areas where limited
municipal infrastructure exists.
• Co-processing manure and other wastes to generate energy or other value-
added products can benefit local communities and improve economics.
Approach
The study uses technoeconomic analysis and life-cycle assessment to explore
multi-product biorefineries that convert lignocellulosic biomass and manure
and/or food waste to liquid fuel, electricity, compressed renewable natural gas,
biopolymers, and aquaculture feed.
Results
Converting biogas from on-site wastewater treatment to compressed biomethane
achieves the lowest cost per tonne CO2e mitigated.
Significance/Impacts
The study suggests a shift in how biorefineries are viewed, away from single-
purpose facilities to multi-purpose facilities that co-process biomass with local
wastes and generate fuels, polymers and other co-products.
Wang, Y. et al Environmental Science & Technology DOI: 10.1021/acs.est.2c06674

6. Office of Biological and Environmental Research
Plant glycosyltransferases for expanding
bioactive glycoside diversity
Background/Objective
• Glycosylation is a successful strategy to alter the pharmacological
properties of small molecules, and it has emerged as a unique approach
to expand the chemical space of natural products that can be explored
in drug discovery.
• Traditionally, most glycosylation events have been carried out
chemically, often requiring many protection and deprotection steps to
achieve a target molecule.
• Enzymatic glycosylation by glycosyltransferases could provide an
alternative strategy for producing new glycosides.
Significance/Impacts
Many relevant plant bioproducts require glycosylation; thus,
understanding the enzymes that may biosynthesize such compounds may
be relevant in producing value-adding co-products in bioenergy feedstock
crops.
Sirirungruang S., et al. Natural product reports. DOI: 10.1039/d2np00077f
Glycosyltransferases
Nu
O
UDP
(OH)n
O
O
O
O
O
O
O

7. Office of Biological and Environmental Research
Funneled Depolymerization of Ionic Liquid-Based Biorefinery
“Heterogeneous” Lignin into Guaiacols over Reusable Palladium Catalyst
Background/Objective
Low yields of products, high char formation, and limited to no
recycling of transition-metal-based catalyst involved in lignin
depolymerization demands attention to enable practical-scale
lignocellulosic biorefineries
Approach
Utilize Pd/ZrP catalyst for lignin hydrogenolysis - Robust ZrP with a
tunable Bronsted/Lewis acid site can facilitate higher conversion,
low char formation, and easy recycling of the catalyst
Results
Up to 200 mg guaiacols per g of lignin were achieved with minimum
char formation (~15%). Extensive C-O bond cleavage over the acidity
of ZrP followed by hydrogenation using Pd in the presence of
hydrogen source
Significance/Impacts
Commercial biorefineries must be able to convert the majority of
components, including sugars and lignin. This study enables lignin
conversion over reusable Pd/ZrP catalyst without compromising
fermentable sugar yields
Choudhary, H., et al. Chemistry, DOI: doi.org/10.1002/chem.202300330
Figure 1: Solvent effect (left) and Pd/ZrP reusaibility (right) in lignin hydrogenolysis.

8. Office of Biological and Environmental Research
Production of fatty acid-derived chemicals
from CO2 in cyanobacteria
Background/Objective
• Cyanobacteria can convert CO2 into various fatty acid-derived chemicals
• Various strategies have been demonstrated to produce those chemicals in
cyanobacteria
Approach
We provide an extensive literature review on recent advancements in metabolic
engineering of cyanobacteria and discuss commercial opportunities to
manufacture those chemicals
Results
Substantial progress in engineering cyanobacteria for the production of a wide
range of fatty acids and derived products has been demonstrated in research
laboratories across the world. However, strict regulations govern the use of
GMOs, and more research is needed before commercialization can occur.
Significance/Impacts
Metabolic engineering of cyanobacteria has the potential to enable
sustainable production of fatty acid-derived products.
Sattayawat, P., et al. Advances in Biochemical Engineering/Biotechnology. DOI: 10.1007/10_2022_213
Figure 1: (A) Simplified schematic diagram of roduction of renewable
chemicals from different organisms, (B) Synthetic pathways for
production of fatty acid-derived products
A
B

9. Office of Biological and Environmental Research
Review of Biosystems Design for Corynebacterium
glutamicum as a bioconversion host
Background
• Corynebacterium glutamicum, an established industrial
workhorse for amino acid production, is seeing extensive
interest for bulk chemicals and biofuels
• We review the recent advances with a focus on
biosystem design principles, metabolic, modeling and
omics analysis
• We review emerging CRISPR tools and biosensors for
Corynebacterium and novel strains of C. glutamicum
that were optimized in JBEI
Results and Significance
• C. glutamicum offers superior biosynthesis for diverse compounds, including bulk chemicals and biofuels
• Natural aromatic-degrading pathway and resistance to aromatic inhibitors make C. glutamicum ideal for
lignocellulose conversion
• Scale-up and bio-separation for C. glutamicum bioproduction is advanced
• Development of omics analyses and high-throughput workflows are speeding strain development
• Future steps to optimize additional products, improve catabolic capabilities, use of advanced metabolic modeling
and emerging AI technologies may greatly accelerate C. glutamicum biomanufacturing of valuable products.
Zha et al, Current Opinion in Biotechnology, 2023, DOI: doi.org/10.1016/j.copbio.2022.102870
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10. Office of Biological and Environmental Research
JBEI-Enabled Publica1ons

11. Office of Biological and Environmental Research
Impacts of cover crops on soil organic carbon
in US Midwestern agroecosystems
Background/Objective
• Impacts of cover crops on soil organic carbon (SOC) are soil and
site-specific.
• Assess the impacts of winter cover crops on SOC sequestration
under different environment and management conditions.
Approach
We combined observations from field experiments with ecosys
process-based agroecosystem model to project the cover crop yields
and SOC sequestration rates across six sites in Illinois.
Results
• Cover crops can sequester SOC by 0.33 ± 0.06 MgC ha−1 year−1, and SOC
benefits are specific to legume and non-legume cover crops.
• SOC benefits from cover crops can be maximized by selecting cover crop
types and controlling growth period in US Midwest.
Significance/Impacts
This study provides practical tools and insights for practitioners and policy-
makers to design effective cover crop policies and programs.
Qin, Z., et al. 2023. Global Change Biology, DOI: 10.1111/gcb.16632
Figure 1: Ecosys model simulated and field measured
average SOC change in 0 - 0.75 m depth interval
Figure 2: Processes involved in SOC change in the cover crop
agroecosystems

12. Office of Biological and Environmental Research
A Membrane-Associated Light-Harvesting Model is Enabled by
Functionalized Assemblies of Gene-Doubled TMV Proteins
Background/Objective
• Photosynthetic light harvesting requires efficient energy transfer
within dynamic networks of light-harvesting complexes
embedded within phospholipid membranes.
• The protein model consists of the tobacco mosaic viral capsid
proteins that are gene-doubled to create a tandem dimer (dTMV)
Approach
A single reactive lysine residue is incorporated into the dTMV
assemblies for the site-selective attachment of chromophores for
light absorption.
Results
A crystal structure of the dTMV assembly was obtained and refined
to 2.8 Å resolution.
Significance/Impacts
Artificial light-harvesting models are valuable tools for
understanding the structural features underpinning energy absorption
and transfer within chromophore arrays.
Dai et al., 2023. Small, 2207805. DOI: 10.1002/smll.202207805
Figure 1. Structure of the dTMV double-disk assembly. Based on the crystal structure, dTMV forms an
A–A ring pair of the double-disk assembly containing a total of 17 subunits. a)The side view of the
assembly. b) The cross-section view reveals that the two disks are not perfectly C2-symmetric as in
cpTMV and wtTMV, but instead contain one disk puckering inward (colored grey) relative to the other
(colored purple) as determined by x-ray crystallography. The monomers in the concave disk have a
more ordered structure in the low-radius region. c) The top view of the assembly is shown, with a
single monomer colored in green. d) The inter-chain interactions between the flat and concave disks are
illustrated. The hydrogen bonds between e) Glu50 and Gln47′ as well as f) Arg46 and Asn33′ are
shown together with the 2mFO-DFC map contoured at the 1.2σ level (shown as mesh line). g) The
inter-chain interactions in the low-radius regions of two adjacent monomers are illustrated. h) The
hydrogen bonding network among Gln36′, Asp115′, and Arg113′ and i) the hydrophobic interactions
between Val114′ and Ile93′, as well as Ala110′ and Ile94′ are shown together with the 2mFO-DFC map
contoured at the 1.2σ level (shown as mesh line).