Lay highlights of the research papers published by the Joint BioEnergy Institute in September 2021 (FY21)

1. Engineered Sorghum Bagasse Enables a Sustainable Biorefinery
with p-Hydroxybenzoic Acid-Based Deep Eutectic Solvent
Background
• Bioenergy crops represent an important feedstock for the
manufacturing of biofuels and bioproducts
• p-Hydroxybenzoic acid (PB) can be used for the synthesis
of deep eutectic solvents (DES) for biomass pretreatment
• Enriching bioenergy crops with PB is an attractive strategy
to add value to biomass and supply PB at low cost for
biomass pretreatment
Approach
• Sorghum was engineered to overproduce PB
• Two PB-based DES were synthesized for the pretreatment
of engineered sorghum biomass: NDES (choline
chloride:PB at a molar ratio of 3:2) and AQDES (choline
chloride:PB:water at a molar ratio of 3:2:5)
• An hydrothermal treatment was tested for the release of PB
from lignin isolated from engineered sorghum after AQDES
pretreatment.
Outcomes and Impacts
• Lignin from engineered PB-rich sorghum contains PB esters
• PB-based DES are effective at pretreating sorghum
biomass and enable increased sugar yields after
saccharification
• PB is the main product obtained from hydrothermal
treatment of lignin isolated from engineered sorghum
• These results suggest that combining PB-based DES with
engineered PB-rich biomass is a promising strategy to
achieve a sustainable closed-loop biorefinery
Wang et al. (2021) ChemSusChem, doi: 10.1002/cssc.202101492
Gas chromatogram of products from hydrothermal
depolymerization of lignin recovered from
engineered sorghum pretreated by AQDES.
PB is the major product.
PB
Sugar yields from wildtype (WT)
and engineered (Eng-2) sorghum
biomass with and without NDES
and AQDES pretreatments

2. A predictive tool-set for the identification of effective
lignocellulosic pretreatment solvents: A case study
of solvents tailored for lignin extraction
Achinivu et.al (2021) Green Chemistry, doi: 10.1039/D1GC01186C
Systematic approach for predicting lignin extraction and
studying mechanistic effects using computational chemistry and
experimental correlations.
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Energy
(kJ/mol)
Lignin-Solvent
Total
Dispersion
Induction
Exchange-repulsion
Electrostatic
Background
• Pretreatment of lignocellulosic biomass (primarily based on
lignin removal) is essential for efficient biomass
deconstruction and conversion into biofuels and bioproducts.
• The present study develops a predictive toolset to
computationally identify solvents that can efficiently dissolve
lignin and therefore can be used to extract it from
lignocellulose during pretreatment.
Approach
• Hansen solubility parameters (HSP) and activity coefficients
and excess enthalpies of solvent/lignin mixtures predicted by
COSMO-RS model to screen the potential solvents followed
by experimental validation.
• Quantum theory of atoms in molecules (QTAIM) and Quantum
chemical (QC) simulations were performed to study the
mechanism of amines with lignin.
Outcomes and Impacts
• The initial screening revealed that diethylenetriamine was the
most effective solvent, promoting the highest lignin removal
(79.2%) and fermentable sugar yields (>72%) leading to
further studies on diamines/polyamines.
• Next, amines with different chemical functionalities were
tested and shown to promote higher lignin removal and
fermentable sugar yields >82% of lignin from biomass and
enabling >88% yields of fermentable sugars.
• QC and QTAIM analysis was performed and suggest that
amines exhibit strong electrostatic interactions and hydrogen
bonding strengths with lignin leading to higher lignin removal
(a) (FI-SAPT) decomposition of the non-bonded interaction energies
between lignin and molecular solvents, (b) COSMO cavity (surface
polarity) diagram of diethylenetriamine, (c) Optimized geometries for
lignin guaiacyl glycerol‒β‒guaiacyl ether (GGE)–spermidine
(a)
(c)
(b)

3. Bacterial diversity dynamics in microbial
consortia selected for lignin utilization
Background
• Lignin is nature’s largest source of phenolic compounds. Its
recalcitrance to enzymatic conversion is still a limiting step to
increase the value of lignin.
• Although bacteria are able to degrade lignin in nature, most
studies have focused on lignin degradation by fungi.
Approach
• To understand which bacteria are able to use lignin as the sole
carbon source, natural selection over time was used to obtain
enriched microbial consortia over a 12-week period.
• The source of microorganisms to establish these microbial
consortia were commercial and backyard compost soils.
• Cultivation occurred at two different temperatures, 30˚C and
37˚C, in defined culture media containing either Kraft lignin or
alkaline-extracted lignin as carbon source.
Outcomes and Impacts
• iTag DNA sequencing of bacterial 16S rDNA gene was
performed for each of the consortia at six timepoints (passages).
• Bacterial consortia composition tended to stabilize from the
fourth passage on.
• After the enrichment protocol, Firmicutes phylum bacteria were
predominant when grown on alkaline lignin, whereas
Proteobacteria were predominant on Kraft lignin.
• Bacteria from these genera can be of particular interest for
studying lignindegradation and utilization, as well as for lignin-
related biotechnology.
Mendez et al. (2021) PLOS ONE, doi: https://doi.org/10.1371/journal.pone.0255083
Schematic of enrichment experiment strategy. Eight microbial
consortia enriched for microorganisms able to
utilize lignin (i.e., base-extracted or Kraft) as carbon source.
Four of the consortia had back yard soil and other four had
MG soil as the as the original source of microorganisms. The
enrichment experiments were conducted in aerobic
conditions at 30˚C and 37˚C. Samples were taken for each
passage, DNA was extracted and the ribosomal gene 16S rDNA
was amplified and sequenced.

4. Utilizing plant synthetic biology for to improve human
health and wellness
Background
• Plants produce a wealth of therapeutic compounds; however, they
are often produced in small amounts by plant species that are
difficult to cultivate
• This review examines the past successes, current challenges, and
potential of plant synthetic biology to generate plants that can
create a diverse set of therapeutic molecules capable of adding
value to bioenergy feedstock crops
Approach
• This review examines the benefits and challenges of utilizing plants
as a platform for synthetic biology. Additionally, it highlights current
and potential target compounds that can be generated in planta.
Outcomes and Impacts
• As multicellular organisms with various tissue types, plants can
facilitate the expression of complex metabolic pathways that may
not be tenable in microbial systems
• The ability of plants to generate multiple products simultaneously
(biofuels, pharmaceuticals, etc.) makes them ideal for coproduction
of valuable chemicals
• The principles of microbial host engineering should be applied to
plants to improve yields of valuable products
• Further improvements in plant transformation methods are needed
to increase the speed at which researchers can generate and test
new plant lines
Barnum et al. (2021) Frontiers in Plant Science, doi: https://doi.org/10.3389/fpls.2021.691462
Plants as a platform for synthetic biology. Improvements in
tissue-specific promoters, tools for compartmentalization,
plant host engineering, and genome editing will aid in the
development of plants as a platform for the production of
health promoting small molecules and the enrichment of
crops with nutritive

5. Evaluation of bacterial hosts for conversion
of lignin-derived p-coumaric acid to 4-vinylphenol
Background
• Hydroxycinnamic acids such as p-coumaric acid (CA) are chemically
linked to lignin in grassy biomass with labile ester bonds and
represent an opportunity to extract and valorize lignin components.
Approach
• We investigated the enzymatic conversion of CA extracted from
lignocellulose to 4-vinylphenol (4VP) by expressing a microbial
phenolic acid decarboxylase (PAD) in Corynebacterium glutamicum,
Escherichia coli, and Bacillus subtilis.
• The performance of the recombinant strains was evaluated in
response to the substrate concentration in rich medium or a lignin
liquor and the addition of an organic overlay to perform a continuous
product extraction.
Outcomes and Impacts
• The use of undecanol as an overlay enhanced the 4VP titers under
high substrate concentrations, while extracting > 97% of the product
from the aqueous phase.
• C. glutamicum showed the highest tolerance to CA and resulted in
the accumulation of up to 187 g/L of 4VP from pure CA in the overlay
with a 90% yield when using rich media, or 17 g/L of 4VP with a 73%
yield from CA extracted from lignin.
• These results indicate that C. glutamicum is a suitable host for the
high-level production of 4VP and that further bioprocess engineering
strategies should be explored to optimize the production, extraction,
and purification of 4VP from lignin.
Rodriguez et al. (2021), Microbial Cell Factories, doi: 10.1186/s12934-021-01670-8

6. JBEI Enabled Publications

7. Conservation agriculture for food security and
climate resilience in Nepal
Background
• Innovations in agriculture to meet the sustainable development goals of
the United Nations require climate-smart and economically feasible
approaches for smallholder farmers in developing countries.
• This paper reviews existing literature and provides an overview of
farming practices in Nepal, highlights near-term challenges associated
with climate change and food security, and discusses the role of CA as a
climate-smart strategy to minimize soil degradation and improve food
security.
Historical Climate trends
• Over the last 116 years period, temperature has increased and
precipitation has decreased in Nepal.
• Such climate trends could enhance glacier melt associated flooding and
delayed monsoon rainfalls, negatively impacting the agricultural
production.
Outcomes and Impacts
• Increased variability in temperature and more frequent extreme weather
events has increased the vulnerability of crops to biotic and abiotic
stresses and altered the timing of agricultural operations, affecting the
crop production.
• Conservation agricultural techniques of no-tillage, use of mulch and
cover cropping can reduce soil erosion, improve soil aggregate structure,
support microbial growth, increase soil organic matter, and reduce soil
erosion.
• Government policies should prioritize and promote conservation
agriculture technologies to maintain agricultural productivity and buffer
resilience negative impacts of climate change.
Joshi et al. (2021) Agronomy Journal, doi: 10.1002/agj2.20830
Figure: A conceptual model for increasing food security and
climate resilience in agriculture through conservation agriculture

8. Tree-Based Automated Machine Learning to
Predict Biogas Production for Anaerobic Co-
digestion of Organic Waste
Background
• dynamics of microbial communities involved in anaerobic digestion
of mixed organic waste are notoriously complex and difficult to
model
• Yet successful operation of anaerobic digestion is critical to the
goals of diverting high-moisture organic waste from landfills
Approach
• This study uses 8 years of data collected from an industrial-scale
anaerobic co-digestion (AcoD) operation at a municipal
wastewater treatment plant in Oakland, California, combined with a
powerful automated ML method, Tree-based Pipeline Optimization
Tool, to develop an improved understanding of how different waste
inputs and operating conditions impact biogas yield.
• The model inputs included daily input volumes of 31 waste
streams and 5 operating parameters. Because different wastes are
broken down at varying rates, the model explored a range of time
lags ascribed to each waste input ranging from 0 to 30 days.
Outcomes and Impacts
• Using the TPOT model gave an R2
value of 0.79, which is similar
to the performance demonstrated by artificial neural networks
(ANN) used in prior studies
• Results indicated that ML-based predictions can inform operational
decision-making at AD facilities
• The results suggest that the waste types (including rendering
waste, lactose, poultry waste, and fats, oils, and greases) differ
considerably in their impact on biogas yield on both a per-gallon
basis and a mass of volatile solids basis, while operating
parameters were not good predictors of yield at this facility.
Wang et al. (2021) ACS Sustain. Chem. Eng., doi: 10.1021/acssuschemeng.1c04612
Figure 2. Partial dependence (PD) plots depicting the quantitative
relationships between (a) daily input volume (gallon/day) or (b)
daily volatile solid (VS) load (metric ton/day) of the 8 most
influential waste types and the resulting change in biogas yield
(scfm). The table provides the numeric values of line slopes
determined by linear curve fitting.
Figure 1. Process flow diagram for anaerobic digestion facility