Research summaries for papers published by the Joint BioEnergy Institute in November 2021

1. High temperature acclimation of leaf gas
exchange, photochemistry, and metabolomic
profiles in Populus trichocarpa
Dewhirst et al. (2021) ACS Earth and Space Chemistry, doi: 10.1021/acsearthspacechem.0c00299
Background
• Temperature acclimation of poplar trees is important to
understand in the face of climate change-induced increase of
surface temperatures and the corresponding impact on tree
productivity for biofuel and bioproducts.
• Application of methanol has the potential to mitigate effects of
high temperature stress.
Approach
• Potted poplar saplings were incubated with or without methanol
at 25 °C or 35°C
• Leaf gas exchange and chlorophyll fluorescence were monitored
with a portable photosynthesis systems and a fluorometer
• Metabolomic profiles were determined using LC-MS.
Outcomes and Impacts
• High growth temperature plants showed suppression of leaf
water use, photosynthesis and transpiration, but an upregulation
of isoprene emissions and an increased optimum temperature of
electron transport rate.
• Metabolomics analysis revealed a substantial number of
metabolites with altered abundance in heat stress, and distinct
subsets altered by methanol treatment at both temperatures.
• We found large physiological and biochemical impacts of high
growth temperature and we highlight the enhancement of the
optimum temperature of electron transport rate as a rapid
thermal acclimation mechanism
Poplar sapling were grown in high or low growth temperatures
with and without additions of methanol to the soil. Leaf gas
exchange, metabolomic profiles and photochemistry were
assessed throughout an 8 week incubation period.

2. Are Methanol-Derived Foliar Methyl Acetate Emissions
a Tracer of Acetate-Mediated Drought Survival in Plants?
Background
• Increased acetate fermentation is a recently described
plant drought survival strategy
• Destructive measurements are usually required to
evaluate acetate-linked drought responses
• This limits the spatial/temporal scales that can be
investigated
Approach
• Here we used 13
C-labelling studies with poplar
branches, and whole trees, and measured volatile
emissions.
Outcomes and Impacts
• Methyl acetate emissions from detached leaves were
strongly stimulated during desiccation
• Diurnal methyl acetate emissions from whole
physiologically active poplar branches increased as a
function of temperature, and light-dark transitions
resulted in significant emission bursts lasting several
hours
• During drought treatments of potted poplar saplings
strong enhancements in methyl acetate emissions
lasting > 6 days were observed, with their initiation
coinciding with the suppression of transpiration and
photosynthesis.
• We suggest that methyl acetate emissions represent a
novel non-invasive tracer of acetate-mediated
temperature and drought survival response in plants.
Dewhirst et al. (2021) Plants, doi: 10.3390/plants10020411
Project led by Kolby Jardine, as part of his
EC award
Figure 1. Schematic of acetate fermentation pathway
and methyl acetate production. The addition of
13
C methanol and 13
C2 during these experiments are
indicated in the orange box. Labeled
13
C atoms of the exogenous 13
C methanol and acetate
are shown in orange. The four isotopologues
(13C0–3) of methyl acetate formed are shown..

3. Cell Wall Compositions of Sorghum bicolor Leaves and
Roots Remain Relatively Constant Under Drought Conditions
Background
• Plants in the field often experience drought. With changing
climate this is expected to be an increasing challenge for
bioenergy crops grown with low inputs.
• Drought could affect the biomass composition of plants such as
sorghum with effects on downstream processing. Past
transcriptomic studies have suggested that impacts could be
large, but few studies have looked at the actual biomass.
Approach
• Sorghum were grown in the field and exposed to pre- or post-
flowering drought. At different timepoints, various tissues were
harvested and analyzed for composition. The data was compared
with extensive transcriptomic data from the same plants.
Outcomes and Impacts
• Cell wall changes due to drought were observed but they were
minor
• Changes in saccharification of cell wall polysaccharides due to
drought were not observed
• Drought-treated plants showed many changes in transcript
abundance of cell wall related genes, but these changes did not
generally correlate with changes in biomass composition
• This study indicate that at least in sorghum changes in biomass
composition or conversion in response to drought is not a major
concern
• The study suggest that a given sorghum genotype may have a
consistent biomass composition and conversion in different
environments, but further studies are required to investigate that.
Scavuzzo-Duggan et al. (2021) Frontiers Plant Sci, doi: 10.3389/fpls.2021.747225
C D
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Figure 2
Week 7 Control Week 7 Pre-Flowering Week 14 Control Week 14 Recovery Week 14 Post-Flowering
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Figure 5
Week 7 Control Week 7 Pre-Flowering Week 14 Control Week 14 Recovery Week 14 Post-Flowering
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BTx642
Cell wall monosaccharide composition of shoots from sorghum
harvested at weeks 7 and 14. Pre-flowering droughted plants had
no irrigation in weeks 3-8, while post flowering droughted plants
had no irrigation in weeks 10-17. Small changes in wall
composition were detected in RTx430, but no significant changes
were found in BTx642. Data are means ± SD (n=3), asterisks
indicate p < 0.05.
Saccharification of biomass from sorghum harvested at weeks 7
and 14. Samples were treated with hot water at 120 C for 1 hr
prior to digestion with a polysaccharide hydrolase cocktail. No
significant changes were found in response to drought (p > 0.05).
Data are means ± SD (n=3).

4. Overexpression of the rice BAHD acyltransferase AT10
increases xylan-bound p-coumarate and reduces lignin in
Sorghum bicolor
Background
• The development of bioenergy crops with reduced recalcitrance
to enzymatic degradation is an important challenge to enable the
sustainable production of advanced biofuels and bioproducts.
• This study focuses on the engineering of sorghum to alter the
amount of aromatics involved in cell wall crosslinks and reduce
cell wall recalcitrance.
Approach
• Overexpression of the rice acyltransferase OsAT10 was
previously shown to be effective at modifying cell-wall-bound
aromatics (i.e. p-coumarate and ferulate) in rice and switchgrass.
• Here, we overexpressed OsAT10 in sorghum and analyzed stem
biomass from eight independent transgenic lines and wild-type
controls (WT) grown in the greenhouse.
Outcomes and Impacts
• Sorghum lines engineered with OsAT10 show increased amount
of p-coumarate esters bound to xylan chains (hemicellulose).
• OsAT10 sorghum lines have reduced amount of lignin (6-17%).
• Biomass from engineered sorghum yields higher amount of
fermentable sugars after ionic liquid pretreatment and enzymatic
saccharification.
• We validate in sorghum the OsAT10 engineering strategy to
reduce biomass recalcitrance.
• Transgenic lines will be field-tested to evaluate crop yield and
biomass quality under natural environment towards conversion
into biofuels and bioproducts
Tian et al. (2021) Biotechnol Biofuels, doi: 10.1186/s13068-021-02068-9
*P<0.05, **P<0.01
Increased xylan-linked p-coumarate Reduced lignin content
Higher sugar yields in OsAT10 transgenics
*P<0.05, **P<0.01

5. One-pot ethanol production from agave bagasse at
high solids loading using protic ionic liquids
Background
• Agave bagasse (AG) is an attractive bioenergy
feedstock due to the high biomass productivity of
agave (up to 44 tons/ha*year).
• Prior work demonstrated that certain ionic liquids are
an effective pretreatment system for AG.
• Biocompatible protic ionic liquids (PILs) are a
promising low-cost alternative to conventional ionic
liquids.
Approach
• We evaluated the efficacy of the PIL 2-
hydroxyethylammonium acetate ([2-HEA][OAc]) in a
one-pot configuration.
• Pretreatment was followed by enzymatic
saccharification and ethanol fermentation using S.
cerevisiae in the same vessel.
• Pretreatment conditions were optimized by a central
composite design using a response surface
methodology.
Outcomes and Impacts
• Achieved glucan and xylan yields of 95.2% and
41.3%, respectively.
• Achieved ethanol yields of 132kg/ton of biomass, a
significant improvement over previous results.
• Results from [2-HEA][OAc] demonstrate the potential
of converting AG using a one-pot process
configuration with high sugar and biofuel yields.
Perez-Pimienta et al. (2021) Green Chemistry, doi: 10.1039/D1GC03774A

6. Plant single-cell solutions for energy and the
environment
Background
• Single-cell transcriptomics has led to fundamental new insights
into animal biology, such as the discovery of new cell types
and cell type-specific disease processes
• The application of single-cell approaches to plants, fungi,
algae, or bacteria (environmental organisms) has been far
more limited, largely due to the challenges posed by
polysaccharide walls surrounding these species’ cells
Approach
• In this perspective, we discuss opportunities afforded by
single-cell technologies for energy and environmental science
and grand challenges that must be tackled to apply these
approaches to plants, fungi and algae
• Highlight the need to develop better and more comprehensive
single-cell technologies, analysis and visualization tools, and
tissue preparation methods
Outcomes and Impacts
• advocate for the creation of a centralized, open-access
database to house plant single-cell data.
• These efforts should balance the need for deep
characterization of select model species while still capturing
the diversity in the plant kingdom. Investments into the
development of methods, their application to relevant species,
and the creation of resources to support data dissemination
will enable groundbreaking insights to propel energy and
environmental science forward
Cole et al. (2021) Commun Biol. doi: 10.1038/s42003-021-02477-4
Using single-cell methods in bioproducts
and biomaterials applications.

7. Cooperative Brønsted-Lewis acid sites in encapsulated
metal-organic frameworks for selective glucose
conversion to 5-hydroxymethylfurfural
Background
• Hydroxymethylfurfural (HMF) is a versatile platform chemical
that can be produced by the catalytic conversion of glucose
• HMF can be transformed into biofuels and bioproducts, but a
viable commercial route has not been identified.
• There is a need to develop heterogeneous chrome-free acid
catalytic systems with both Brønsted and Lewis acid sites
that regulate HMF selectivity on glucose dehydration.
Approach
• We encapsulated phosphotungstic acid (PTA) in the pores of
the metal organic framework MIL-101(Al)-NH2 to form
PTA_MIL-101(Al)-NH2.
• Evaluated the effect of PTA encapsulation on catalytic
performance in glucose dehydration with the ionic liquid
[C4C1im]Cl as the solvent.
Outcomes and Impacts
• Observed that the highest HMF selectivity of 58% at 44%
glucose conversion at 120 oC was based on the synergistic
effect of Brønsted and Lewis acid sites in the
phosphotungstic acid encapsulated MIL-101(Al)-NH2
catalyst
• Matching Lewis acid and Brønsted acid species was critical
in maximizing HMF selectivity in glucose dehydration.
• This encapsulated metal-organic framework catalyst is
applicable to other acid-catalyzed biomass transformations
into biofuels and bioproducts.
Rahaman et al. (2021) Fuel, doi: 10.1016/j.fuel.2021.122459
Reaction network of HMF production (BA =
Brønsted acid, LA = Lewis acid)
HMF selectivity of encapsulated PTA_MIL-101(Al)-NH2
catalysts at similar glucose conversions.